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.

	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 (p177101tkCat), 120 and 31 (p12031tkCat), and 24 and +31 (p24+31tkCat), respectively. For analysis of the 1,25-(OH)₂D₃ response domain, three overlapping oligonucleotides of the 12031 fragment (p120-83tkCat, p90-55tkCat, and p6229tkCat) 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 10⁸ 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. 10⁸ 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 P19^gag 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 p6229tkCat construct as a probe. p6229tkCat 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.

	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 10⁸ 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.

View larger version (18K): [in this window] [in a new window]   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 108 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 p177101tkCat, p12031tkCat, p24+31tkCat, p120-83tkCAT, p9055ptkCAT, and p6229ptkCAT 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 p12031tkCat and p6229ptkCAT 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.

View larger version (32K): [in this window] [in a new window]   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 108 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 ( 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 the62/29 region of the CAII gene. This signal was not affected by the addition of 10⁶ 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.

View larger version (49K): [in this window] [in a new window]   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, RXR, RAR,  or  of human origin and chicken RXR, and luciferase as a negative control). The only detectable specific complexes were composed of VDR·RXR or VDR·RXR heterodimers. Panel B, the VDR·RXR binding to the 62/29 DNA fragment is specific. EMSAs were performed using recombinant VDR and RXR 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 (-VDR, dilution 1:15), human RXR (-RXR, dilution 1:40), or unrelated chicken Mi transcription factor (-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, 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.

View larger version (101K): [in this window] [in a new window]   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 (-VDR, dilution 1:15), human RXR (-RXR, dilution 1:40), human RAR (-RAR, dilution 1:40), avian T3R (-T3R, dilution 1:15), avian Mi (-Mi, dilution 1:15), or avian P19gag (-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.

View larger version (93K): [in this window] [in a new window]   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). 107 M (lanes 3 and 7) or 106 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 p6229tkCat 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.

View larger version (30K): [in this window] [in a new window]   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 RXR 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 6229 domain of the CAII promoter. The guanines identified by methylation interference assays are presented on a double-stranded DNA fragment.

View larger version (28K): [in this window] [in a new window]   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 108 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 -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).

View larger version (72K): [in this window] [in a new window]   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 (-VDR, dilution 1/15) or human RXR (-RXR, dilution 1/40). Representative results obtained with m1, m2, and m3 mutants are shown.

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 p6229tkCat 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.

View larger version (38K): [in this window] [in a new window]   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 108 M 1,25-(OH)2D3. The p6229tkCat 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--galactosidase as an internal control and were then treated for 2 days with 10⁸ 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.

View larger version (19K): [in this window] [in a new window]   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 p6229tkCat construct or tkCat constructs containing mutated forms of the 62/29 DNA fragment together with the CMV--galactosidase plasmid as an internal control. pBLCAT2 was used as a negative control. After 2 days of treatment with 108 M 1,25-(OH)2D3, CAT activities were calculated and normalized with the -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), 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.