Identification of a Vitamin D Response Element in the Proximal Promoter of the Chicken Carbonic Anhydrase II Gene*

Isabelle Quélo, Irma Machuca, and Pierre JurdicDagger

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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 beta 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 alpha  or gamma  isoform of RXR.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

Oligonucleotides containing 3-base pair mutations within the -62/-29 sequence were inserted into the XbaI site of pBLCAT2 to create mutant plasmids. The mutants are as follows: in m1tkCat, nucleotides -56 to -54 (AGA) were changed into CCC; in m2tkCat, nucleotides -53 to -51 (AGG) into TTA; in m3tkCat, -50 to -48 (GCA) into CTG; in m4tkCat, -47 to -45 (TGG) into CCC; in m5tkCat, -44 to -42 (AGT) into TTA; in m6tkCat, -41 to -39 (TCG) into CAC; in m7tkCat, -38 to -36 (CGG) into AAA. Two oligonucleotides containing point mutations of the second base of the 3'-hexameric motif of the putative DR1 or DR3 were cloned as above. This gave the sequences AGGGCAtGCAGTT for DR1 (mutant m8) and AGGGACtggACTTCG for DR3 (mutant m9) (underlined nucleotides represent mutations).

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-beta -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-beta -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 beta -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 beta -galactosidase activity.

Electrophoretic Mobility Shift Assays (EMSAs)-- The macrophage nuclear extracts were prepared from the HD11 cells treated for 2 days with 10-8 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, RXRalpha , RARalpha , beta , and gamma , and chicken RXRgamma 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).

EMSAs and antibody supershifts were performed as described elsewhere (48). Briefly, 2 µl of in vitro translated proteins or 4 µl of nuclear extracts was incubated with gamma -32P-labeled probe (0.5 ng) in 15 µl of binding buffer containing 1 µg of poly(dI-dC) (Amersham Pharmacia Biotech) for 20 min at 4 °C. The antibodies and 1,25-(OH)2D3 were incubated for 10 min at 4 °C before the addition of the labeled probe. Unlabeled probes used in competition (100-fold excess) were added together with labeled probes. The complexes were resolved on 4% nondenaturing polyacrylamide gel in 0.2 × TBE.

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 [gamma -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 RXRalpha 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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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-beta -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 beta -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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Fig. 1.   Identification of a potential VDRE within the CAII promoter. Scheme of the CAII promoter constructs and their corresponding 1,25-(OH)2D3 induction. The pHHcaCat reporter plasmid contains the CAII promoter (-1321 to +31) in front of the CAT gene. The pPstCat, pPvuCat, and pApaCat deleted mutants were named with respect to the restriction enzymes used to perform the progressive deletions. 10 µg of each construct was transfected in the HD11 cell line, and the cells were cultured for 48 h in the absence or presence of 10-8 M 1,25-(OH)2D3. CAT activities from three averaged experiments are represented as ratios of 1,25-(OH)2D3 to no 1,25-(OH)2D3 relative to the corresponding reporter.

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 p-177-101tkCat, 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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Fig. 2.   1,25-(OH)2D3 induction of the CAII promoter mutants. The deleted fragments of the CAII proximal promoter were obtained from the pApaCat plasmid (-178/+31) and cloned into pBLCAT2. 10 µg of the reporter constructs was transfected into HD11 cells. pBLCAT2 and pApaCat constructs were used as negative and positive controls, respectively. After transfection, HD11 cells were cultured in the presence or absence of 10-8 M 1,25-(OH)2D3 for 48 h.

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 (alpha  or gamma  isoforms), or RAR (alpha , beta , gamma  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 RXRalpha or gamma  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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Fig. 3.   The VDR·RXR heterodimer specifically binds to the -62/-29 DNA fragment of the CAII promoter. Panel A, VDR·RXR heterodimer binds to the -62/-29 region. The -62/-29 fragment containing the putative 1,25-(OH)2D3 response domain was used as a probe for EMSAs and incubated with 2 µl of the reticulocyte lysate containing in vitro translated recombinant receptors (VDR, RXRalpha , RARalpha , beta  or gamma  of human origin and chicken RXRgamma , and luciferase as a negative control). The only detectable specific complexes were composed of VDR·RXRalpha or VDR·RXRgamma heterodimers. Panel B, the VDR·RXRalpha binding to the -62/-29 DNA fragment is specific. EMSAs were performed using recombinant VDR and RXRalpha incubated with the -62/-29-labeled probe. Left, DR3 is an artificial VDRE containing two directly repeated AGGTCA motifs spaced by 3 bp. AP1 is the DNA binding site for the AP-1 complex. -62, -90, and -120 correspond to the -62/-29, -90/-55, and -120/-83 regions of the CAII promoter, respectively. The unlabeled competitors (50 ng) are indicated above each lane. Right, supershift experiments were performed using antibodies directed against either chicken VDR (alpha -VDR, dilution 1:15), human RXR (alpha -RXR, dilution 1:40), or unrelated chicken Mi transcription factor (alpha -Mi, dilution 1:15).

-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, TRalpha , 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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Fig. 4.   Binding of macrophage endogenous nuclear proteins on the -62/-29 domain. EMSAs were performed with 4 µl of nuclear extracts obtained from 1,25-(OH)2D3-treated HD11 cells. The -62/-29 fragment, containing the 1,25-(OH)2D3 response domain, was used as a probe. Two specific complexes, C1 and C2, were detected (lanes 2 and 8). To characterize the nature of the protein complex bound to DNA, antibodies directed against avian VDR (alpha -VDR, dilution 1:15), human RXR (alpha -RXR, dilution 1:40), human RAR (alpha -RAR, dilution 1:40), avian T3Ralpha (alpha -T3R, dilution 1:15), avian Mi (alpha -Mi, dilution 1:15), or avian P19gag (alpha -P19, dilution 1:15) were added 10 min before the addition of the labeled probe. The two complexes are both composed of VDR and RXR proteins as shown by the supershift obtained with the anti-RXR antibody (lane 5) and its disruption with the anti-VDR antibody (lane 4).

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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Fig. 5.   Comparison between the complexes formed on -62/-29 probe with 1,25-(OH)2D3-treated or untreated nuclear extracts. Effects of the in vitro addition of 1,25-(OH)2D3 on complex formation are shown. The -62/-29-labeled probe was incubated with nuclear extracts obtained from 1,25-(OH)2D3-treated HD11 cells (lanes 2-5) or vehicle-treated (lanes 6-9). 10-7 M (lanes 3 and 7) or 10-6 M (lanes 4 and 8) 1,25-(OH)2D3 was added to the reaction mixture and incubated for 10 min before adding the probe. Two complexes (C1 and C2) were obtained with 1,25-(OH)2D3-treated nuclear extracts (lanes 2-4), whereas only the C1 complex was observed with untreated extracts (lanes 6-8). Both C1 and C2 complexes were competed by an excess of cold probe (lanes 5 and 9). No change in the protein-DNA binding pattern was observed in the presence of in vitro added 1,25-(OH)2D3.

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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Fig. 6.   Methylation interference experiments. Panel A, methylation interference assays were performed with methylated -62/-29 probes radiolabeled on the 5'-end of the sense or antisense strands. The probes were incubated with VDR and RXRalpha recombinant proteins or 1,25-(OH)2D3-treated HD11 cell nuclear extracts for EMSAs. The free and retarded probes were then sequenced and compared with the G+A sequence obtained with the nonmethylated -62/-29-labeled probes. The G residues implicated in protein-DNA interactions were identified by a disappearance of the signal or a weaker signal. Panel B, summary of the interactions between VDR·RXR proteins and the -62-29 domain of the CAII promoter. The guanines identified by methylation interference assays are presented on a double-stranded DNA fragment.

To discriminate between these two possibilities, we cloned oligonucleotides containing 3-bp mutations between positions -56 and -36 (Fig. 7) upstream from the tk promoter fused to the CAT gene. These constructs were transfected into HD11 cells and tested for putative activation by 1,25-(OH)2D3. Compared with the p-62-29tkCat construct (Fig. 7, row 1), we observed that the 1,25-(OH)2D3 stimulation obtained with the m1tkCat and m7tkCat constructs with mutations localized outside the interfering area was not affected (Fig. 7, rows 2 and 8). Stimulations with m2, m3, m5, and m6 constructs carrying mutations within the interfering domain were abolished completely (Fig. 7, rows 3, 4, 6, and 7, respectively). Stimulation obtained with the m4tkCat construct was only slightly decreased in response to 1,25-(OH)2D3 (Fig. 7, row 5). These results indicate that only mutations between positions -53 and -39 altered the 1,25-(OH)2D3 responsiveness, defining a more precise putative VDRE. Among the mutants showing a loss of induction, m4tkCat is the less effective. It appears that the mutations fall upon the three spacing nucleotides of the putative DR3 element.


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Fig. 7.   Functional analysis of the serial -62/-29 region mutants. The serial mutations introduced into the -62/-29 region are boxed and in bold. 10 µg of the reporter plasmids, containing the mutants fused to the tk promoter and the CAT reporter gene, was transfected into HD11 cells, and the cells were subsequently treated for 2 days with 10-8 M 1,25-(OH)2D3. The results are the means ± S.D. calculated from the CAT activities obtained from at least six experiments and normalized with beta -galactosidase activities.

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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Fig. 8.   DNA binding abilities of the various -62/-29 mutants. Panel A, mutant promoter elements were used as competitors for the binding of 1,25-(OH)2D3-treated nuclear extracts on the -62/-29 probe. The -62/-29 sequence was used as a probe for EMSAs with nuclear extracts from 1,25-(OH)2D3-treated HD11 cells, and competitions were made with 50 ng of each mutant. Unlabeled competitors are indicated above each lane. -62 corresponds to the -62/-29 region and AP1 to an AP-1 binding site. Panel B, identification of the proteins bound to the mutated probes. m1, m2, and m3 mutated oligonucleotides were used as a probe for EMSAs with nuclear extracts from 1,25-(OH)2D3-treated HD11 cells. Lanes 1, 7, and 13 correspond to the free probes. The signals obtained with 1,25-(OH)2D3-treated nuclear extracts (lanes 2, 8, and 14) were analyzed by competition with unlabeled probe or -62/-29 fragment (-62) (50 ng) and by using antibodies directed against avian VDR (alpha -VDR, dilution 1/15) or human RXR (alpha -RXR, dilution 1/40). Representative results obtained with m1, m2, and m3 mutants are shown.

Therefore, the ability of these different mutated sequences to bind nuclear proteins was tested directly. To identify the complexes generated, we used these mutants as probes for EMSAs with 1,25-(OH)2D3-treated nuclear extracts. One probe representative of each of the three groups (i.e. m1, m2, m3) was used in the EMSAs that are shown in Fig. 8B. As expected from the previous results, m1 and m3 probes (Fig. 8A) as well as m1 and m7 (data not shown) were able to form complexes with VDR and RXR as shown by treatment with both anti-VDR and anti-RXR antibodies, and this binding is competed by an excess of cold-related probes (Fig. 8B, lanes 1-6 and 13-18, respectively). The m2 mutant, in one element of the two putative hexameric motifs of the VDRE, failed to generate any significant complex with HD11-derived nuclear proteins (Fig. 8B, lanes 7-12); the same results were observed using m5 and m6 as probes (data not shown). These results correlate with the abrogation of the functional response obtained with these mutants. So those mutations (m2, m5, and m6) inhibit the transcription activation by preventing the DNA binding of the transcription factors. In conclusion, the oligonucleotides containing mutations in the second hexameric core motif of the putative DR3-type element (m5 and m6 mutants) were not able to bind nuclear proteins nor to respond to 1,25-(OH)2D3. In contrast, mutations in the 5'-binding site AGGGCA differ in their effects: the first three nucleotides (m2 mutant) are essential for both binding and transactivation, whereas the last three (m3 mutant) are essential for transactivation but not for binding.

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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Fig. 9.   Point mutations analysis of the CAII VDRE. Panel A, functional analyses of the point mutants of the CAII VDRE. -62/-29 sequences containing point mutations were cloned into pBLCAT2 plasmid, and the constructs were transfected into HD11 cells treated for 2 days with 10-8 M 1,25-(OH)2D3. The p-62-29tkCat construct was used as a positive control. The m9 mutant is unable to respond to 1,25-(OH)2D3 treatment, whereas the m8 mutant acts as the -62/-29 sequence. Panel B, mobility shift experiments with radiolabeled point mutant VDREs. -62/-29, m8, and m9 probes were radiolabeled and used for EMSAs with nuclear extracts from 1,25-(OH)2D3-treated HD11 cells. The m8 mutant can bind equivalently to the native -62/-29 sequence, whereas the m9 mutant cannot compete or bind to the VDR·RXR heterodimer.

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-beta -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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Fig. 10.   Functional analysis of the CAII proximal VDRE in ROS 17/2.8 cell line. The ROS 17/2.8 cells were transfected with the p-62-29tkCat construct or tkCat constructs containing mutated forms of the -62/-29 DNA fragment together with the CMV-beta -galactosidase plasmid as an internal control. pBLCAT2 was used as a negative control. After 2 days of treatment with 10-8 M 1,25-(OH)2D3, CAT activities were calculated and normalized with the beta -galactosidase activities. The results show that the CAII proximal VDRE is functional in ROS 17/2.8 cells.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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), NCoA-62 (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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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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Abstract
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Discussion
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