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Molecular Endocrinology, doi:10.1210/me.2006-0231
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Molecular Endocrinology 21 (2): 334-342
Copyright © 2007 by The Endocrine Society

1{alpha},25(OH)2D3-Induced Transrepression by Vitamin D Receptor through E-Box-Type Elements in the Human Parathyroid Hormone Gene Promoter

Mi-sun Kim, Ryoji Fujiki, Akiko Murayama, Hirochika Kitagawa, Kazuyoshi Yamaoka, Yoko Yamamoto, Masatomo Mihara, Ken-ichi Takeyama and Shigeaki Kato

Institute of Molecular and Cellular Biosciences (M.-s.K., R.F., A.M., H.K., Y.Y., M.M., K.-i.T., S.K.), University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan; Teijin Institute for Biomedical Research (K.Y.), Teijin Pharma Limited, Hino, Tokyo 191-8512, Japan; Graduate School of Life and Environmental Sciences (A.M.), University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan; and Exploratory Research for Advanced Technology (K.Y., S.K.), Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan

Address all correspondence and requests for reprints to: Shigeaki Kato, The Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. E-mail: uskato{at}mail.ecc.u-tokyo.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although transactivation by the liganded vitamin D receptor (VDR) is well described at the molecular level, the precise molecular mechanism of negative regulation by the liganded VDR remains to be elucidated. We have previously reported a novel class of negative vitamin D response element (nVDRE) called 1{alpha}nVDRE in the human 25(OH)D31{alpha}-hydroxylase [1{alpha}(OH)ase] gene by 1{alpha},25(OH)2D3-bound VDR. This element was composed of two E-box-type motifs that bound to VDIR for transactivation, which was attenuated by liganded VDR. Here, we explore the possible functions of VDIR and E-box motifs in the human (h) PTH and hPTHrP gene promoters. Functional mapping of the hPTH and hPTHrP promoters identified E-box-type elements acting as nVDREs in both the hPTH promoter (hPTHnVDRE; –87 to –60 bp) and in the hPTHrP promoter (hPTHrPnVDRE; –850 to –600 bp; –463 to –104 bp) in a mouse renal tubule cell line. The hPTHnVDRE alone was enough to direct ligand-induced transrepression mediated through VDR/retinoid X receptor and VDIR. Direct DNA binding of hPTHnVDRE to VDIR, but not VDR/retinoid X receptor, was observed and ligand-induced transrepression was coupled with recruitment of VDR and histone deacetylase 2 (HDAC2) to the hPTH promoter. These results suggest that negative regulation of the hPTH gene by liganded VDR is mediated by VDIR directly binding to the E-box-type nVDRE at the promoter, together with recruitment of an HDAC corepressor for ligand-induced transrepression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
1{alpha},25-DIHYDROXYVITAMIN D3 [1{alpha},25(OH)2D3] and PTH play pivotal roles in maintaining calcium homeostasis and supporting bone metabolism (1, 2, 3). In the kidney, PTH is secreted from the parathyroid, and induces gene expression of 25(OH)D3 1{alpha}-hydroxylase [1{alpha}(OH)ase] at the transcriptional level, encoding the critical enzyme that converts 25(OH)2D3 into 1{alpha},25(OH)2D3 (4, 5, 6). In turn, an excess of 1{alpha},25(OH)2D3 leads to transcriptional repression of both 1{alpha}(OH)ase in the kidney and PTH in the parathyroid (7, 8, 9, 10). Although the molecular mechanisms of transactivation by 1{alpha},25(OH)2D3 through the vitamin D receptor (VDR) have been well illustrated (11, 12), the negative response to 1{alpha},25(OH)2D3 in VDR-mediated gene expression still remains to be investigated at the molecular level.

1{alpha},25(OH)2D3 is thought to exert its physiological effect via the VDR leading to transcriptional regulation of target gene promoters. VDR dimerizes with the retinoid X receptor (RXR) and stably binds vitamin D receptor response elements (VDREs) for ligand-induced transactivation (13, 14). Such positive VDREs are believed to contain a direct repeat of two consensus core motifs (AGGTCA) with a 3-bp spacer (DR3) (15). Ligand-induced transactivation by VDR couples with coregulator switching, in that coactivator recruitment is coupled to corepressor dissociation (16, 17, 18). VDR is also known to transcriptionally associate with other elements (nVDREs) for ligand-dependent transrepression. Many nuclear receptors have been shown to physically interact with other classes of DNA binding activators for ligand-induced transrepression (19, 20). In such cases, nuclear receptors do not bind their cognate DNA binding sequences for transactivation.

One class of nVDRE in the human (h) PTH and hPTHrP gene promoters was reported to comprise a single DR3-like motif (AGGTCA) as a direct DNA binding element of the VDR monomer (7, 21, 22). Recently, we identified an E-box (CANNTG)-like motif as another class of nVDRE in the human 1{alpha}(OH)ase promoter (23, 24). In sharp contrast to the previously reported DR3-like motif in the hPTH gene promoter, a basic helix-loop-helix factor, designated VDR interacting repressor (VDIR), transactivates through direct binding to this E-box-type element (1{alpha}nVDRE). However, the VDIR transactivation function is transrepressed through ligand-induced protein-protein interaction of VDIR with VDR/RXR (24). This ligand-induced transrepression, stimulated when liganded VDR is not bound to DNA, is coupled with coregulator switching, with an opposite effect to ligand-induced transactivation. Namely, in the absence of 1{alpha},25(OH)2D3, VDIR appears to bind to 1{alpha}nVDRE for transactivation through the histone acetylase (HAT) coactivator, p300/CBP. Binding of 1{alpha},25(OH)2D3 to VDR induces interaction with VDIR and dissociation of the HAT coactivator, resulting in recruitment of histone deacetylase (HDAC) corepressor for ligand-induced transrepression (24). Although such a model of ligand-induced transrepression through VDR has been proposed for the human 1{alpha}(OH)ase gene promoter (9, 23, 24), it is unclear whether this model of transrepression is applicable to other negative target gene promoters for VDR.

To address this issue, in the present study we analyzed the human PTH and PTHrP gene promoters and identified E-box-type elements as negative VDRE (hPTHnVDRE and hPTHrPnVDRE). We observed similar behavior in the human PTH gene promoter as previously observed for 1{alpha}nVDRE (24). Direct DNA binding of VDIR to the E-box-type element (hPTHnVDRE) caused transactivation, and 1{alpha},25(OH)2D3-induced association of VDIR with DNA-unbound VDR-induced coregulator switching for transrepression. Thus, the E-box-type elements may serve as one class of nVDRE in the promoters of at least some negative target genes for VDR.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Ligand-Induced Transrepression of the PTH and PTHrP Genes Requires VDR
PTH and PTHrP gene expression are well known to be under negative control by 1{alpha},25(OH)2D3 in both intact animals and several cell lines (21, 25). To verify whether VDR function is indeed required for ligand-induced repression of PTH and PTHrP genes in intact animals, we analyzed their expression levels by semiquantitative RT-PCR in mice deficient of VDR (VDR–/–) (26). As expected, 1{alpha},25(OH)2D3 repressed endogenous PTH gene expression in the parathyroid glands of wild-type (VDR+/+) mice (Fig. 1AGo). In the parathyroid gland of VDR–/– mice, PTH gene expression was significantly up-regulated, but 1{alpha},25(OH)2D3 treatment failed to suppress its expression (Fig. 1AGo). Similarly, renal expression of both PTHrP as well as 1{alpha}(OH)ase genes were also up-regulated in VDR–/– mice and the negative response to 1{alpha},25(OH)2D3 was again abolished, whereas the expected repression of these genes by 1{alpha},25(OH)2D3 treatment was seen in VDR+/+ mice (Fig. 1BGo). These results supported previous findings that VDR mediates the negative response to 1{alpha},25(OH)2D3 (7, 21, 25).


Figure 1
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  		Fig. 1. VDR Is Indispensable for Transrepression of PTH and PTHrP Gene by 1{alpha},25(OH)2D3

A and B, Expression of the mPTH gene in the parathyroid gland, and mPTHrP genes, 1{alpha}-hydroxylase genes in the kidney was analyzed by RT-PCR. The total RNA was extracted from tissues of VDR+/+ mice and VDR–/– mice. gapdh, Glyceraldehyde-3-phosphate dehydrogenase.

 
These results raised the possibility that VDR-mediated transrepression of the PTH and PTHrP genes is via E-box motifs present in their promoters because such E-box motifs are also present in the promoters of other mammals (Table 1Go). To address this issue, a transient expression assay using a luciferase reporter plasmid driven by a series of deletion mutants of hPTH promoter and hPTHrP promoter was performed. The hPTH promoter, 700 bp upstream region from the transcription start site, conferred a weak negative response to 1{alpha},25(OH)2D3 in mouse cortical tubular (MCT) cells (Fig. 2AGo, lanes 1 and 2). The effect of 1{alpha},25(OH)2D3 on transrepression was significantly enhanced by transfection of VDR/RXR (Fig. 2AGo, lanes 3 and 4). When the hPTH promoter was deleted up to –60 bp upstream, the negative response was completely abolished even in the presence of VDR/RXR (Fig. 2AGo, lanes 15 and 16). A region –87 to –60 bp containing an E-box motif in hPTH promoter was mapped as an hPTHnVDRE. Using a similar approach on the hPTHrP promoter, an upstream region (–463 to –104 bp) containing E-box motifs was mapped as nVDRE, presumably as well as the more upstream region (–850 to –600 bp) (Fig. 2BGo). The E-box-type elements in promoters of negative target genes for VDR are illustrated in Table 1Go. The two mapped E-box motifs in the hPTHrP gene promoters are identical in sequence to those of the 1{alpha}nVDRE in the 1{alpha}(OH)ase promoter, although the space between the two E-box motifs is different (Table 1Go).


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  		Table 1. Comparison of E-Box-Like Motifs in 1{alpha}(OH)ase Gene, PTH Gene, and PTHrP Gen

 

Figure 2
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  		Fig. 2. Functional Analysis of nVDRE in hPTH and hPTHrP Gene Promoters

A and B, Luciferase (LUC) reporter plasmids driven by the deleted promoters were transfected with expression vectors of pSG5-ratVDR (0.05 µg), pSG5-rat RXR{alpha} (0.05 µg) in MCT cells. After transfection, the cells were incubated with/without 1{alpha},25(OH)2D3 (1 x 10–8 M) for 24 h. The promoter activity was measured at 24 h by the luciferase activity. All values are shown as means ± SD for at least three independent experiments.

 
An E-Box-Type Element in the hPTH Promoter Functions as a Negative VDRE through the VDR/RXR Heterodimer
We previously identified direct repeats of E-box motifs (CANNTG) as nVDRE (1{alpha}nVDRE) in the 1{alpha}(OH)ase promoter (9, 24). We examined whether the mapped E-box-type element (–87 to –60 bp) in the hPTH promoter alone serves as an nVDRE using a reporter assay in MCT cells. A clear negative response was only observed with the intact promoter (hPTH460) when compared with a positive control hPTH promoter (hPTH460) (Fig. 3BGo, lanes 3 and 4). An hPTH promoter mutant lacking the E-box-type element (hPTH{Delta}E-box) was unable to confer the negative response to 1{alpha},25(OH)2D3 (Fig. 3BGo, lanes 5–8), suggesting this motif functions as an nVDRE. Indeed, synthetic short elements (hPTHnVDRE) alone were sufficient to confer an expected negative response to 1{alpha},25(OH)2D3 (Fig. 3BGo, lanes 11 and 12). Therefore, we concluded that these elements are the nVDRE for the hPTH gene promoter.


Figure 3
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  		Fig. 3. 1{alpha},25(OH)2D3-Induced Transrepression of hPTH Gene by VDR Mediates an E-Box-Type nVDRE in the Promoter

A, Schematic representation of an hPTH promoter and a mutant deleted E-box. The previously reported DR3-core motif and an E-box motif are located in the –460-bp region. B, A mutant promoter deleted E-box motif in hPTH promoter (hPTH{Delta}E-box) lost the negative response to 1{alpha},25(OH)2D3. The luciferase (LUC) activity was measured as described in Fig. 2AGo. C, Transrepression of VDIR via VDR mutants measured by luciferase assay. E42A mutant inhibits DNA binding affinity, and I260R and I384R mutants lack heterodimerization of VDR and RXR. Luciferase assay was performed with either hPTH460 or DR3 reporter after cotransfection of wild-type VDR or mutant VDRs into MCT cells.

 
To directly test DNA binding and the requirement for heterodimerization of VDR with RXR on ligand-induced transrepression of the E-box-like motif in the hPTH promoter, two classes of VDR mutants were applied. One class of the two VDR mutants lacked dimerization ability (VDR I260R and I384R) (27). These mutants lost their transrepressive function upon the hPTHnVDRE (Fig. 3CGo, lanes 4 and 6). The second class of VDR mutant lacked DNA binding ability for DR3-type-positive VDRE (VDR E42A) (24), and indeed impaired ligand-induced transactivation was observed (Fig. 3CGo, lanes 7 and 8). However, this mutant was still potent to transrepress the hPTH gene promoter (Fig. 3CGo, lanes 1 and 2). Thus, these findings suggest that transrepression of the hPTH gene promoter by VDR requires heterodimerization, but not necessarily direct DNA binding of VDR/RXR heterodimer.

VDIR Is Indispensable for the Transrepression Function by VDR/RXR through the E-Box-Type Element in the hPTH Promoter
Because we have previously shown that VDIR transactivates through physical interaction with 1{alpha}nVDRE (24), we then assessed whether VDIR directly binds to the identified hPTHnVDRE by EMSA. No direct DNA binding of recombinant VDR/RXR heterodimer to the hPTHnVDRE as well as the reported DR3-like nVDRE was observed (Fig. 4AGo, lanes 5–8). However, clear DNA binding was detected for positive DR3 VDRE as a positive control (Fig. 4AGo, lanes 1 and 2). A recombinant VDIR bound to the hPTHnVDRE, similar to 1{alpha}nVDRE (Fig. 4BGo, lanes 4 and 8). The specificity of DNA binding of VDIR to the hPTHnVDRE was further confirmed by competition with a large excess of cold 1{alpha}nVDRE oligomers or vice versa (Fig. 4BGo, lanes 5, 6, 9, and 10). In fact, a ligand-induced transrepression by VDR/RXR on hPTH promoter (hPTH460) was enhanced by overexpression of VDIR (Fig. 5AGo, lanes 1 and 2) when compared with conditions without VDIR overexpression (Fig. 3BGo, lanes 3 and 4). Conversely, when an endogenous VDIR protein was knocked down using RNA interference (RNAi) (see Fig. 5CGo, right panel), no transrepression by VDR/RXR through both of the hPTH460 and hPTHrP850 was observed (Fig. 5BGo), and indeed VDIR RNAi abrogated the transrepression of the endogenous mouse (m) PTHrP gene expression by activated VDR in MCT cells (left panel in Fig. 5CGo). Moreover, in our preliminary experiment using primary cultured cells of parathyroid glands, we also observed that VDIR ablation by RNAi derepressed the endogenous mPTH expression by activated VDR (data not shown). These results suggest that ligand-induced transrepression by VDR/RXR heterodimer requires VDIR upon the hPTHnVDRE. This idea is supported by the result that a VDR-point mutation (P61T) failed to transrepress through hPTHnVDRE but still retained ligand-induced transactivation through the positive VDRE (DR3 pVDRE) (Fig. 5AGo, lanes 4 and 8). This mutant where proline is replaced with threonine at position 62 in the DNA binding domain was unable to interact with VDIR in vitro (Fig. 5DGo). Thus, this functional abrogation of VDR by the P61T mutation verified a significant role of VDIR in the transrepression.


Figure 4
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  		Fig. 4. E-Box-Type nVDREs Physically Interact with VDIR, But Not VDR/RXR

A and B, EMSA was performed with 1{alpha}nVDRE, hPTHnVDRE as a designed probe, and direct repeat (DR) 3 oligomers as a positive VDRE. VDR and RXR recombinant proteins were expressed as GST fusions. Specific representative cold-hPTHnVDRE or cold-1{alpha}nVDRE oligomers were used at a 50-fold molar excess. F, Free probe.

 

Figure 5
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  		Fig. 5. Direct DNA Binding of VDIR to the hPTH nVDRE Is Essential for Ligand-Dependent Transrepression by VDR

A, A P61T mutant abolished 1{alpha},25(OH)2D3-induced transrepression activity using a luciferase assay. Luciferase activity was assessed as shown in Fig. 2AGo. B, VDIR is required for 1{alpha},25(OH)2D3-induced transrepression by VDR in the hPTH460 and hPTHrP850. For VDIR-specific knockdown, whole-cell extracts were prepared from MCT cells transfected with 100 nM siRNA. C, Endogenous PTHrP gene was analyzed by RT-PCR. To assess gene-knockdown effects by siRNA of VDIR, Western blots were performed using {alpha}-VDIR, {alpha}-VDR and {alpha}-actin (as a control). D, P61T mutant loses binding activity with VDR. GST-alone, GST-VDR wild-type, or GST-P61T mutant was incubated with 35S-labeled VDIR, which was translated in vitro, in the presence of 1{alpha},25(OH)2D3 (1 x 10–6 M) or not. DR, Direct repeat; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WB, Western blot.

 
Previously, we demonstrated that VDIR acted as an activator on the 1{alpha}nVDRE by recruiting p300 HAT coactivator complexes in response to activated-protein kinase A signaling. Also, activation of VDR-induced recruitment of HDAC corepressor complex to the VDIR for transrepression (24). We therefore examined whether the transactivation function of VDIR bound on the hPTHnVDRE was transrepressed by liganded VDR/RXR through recruitment of an HDAC corepressor. As expected, VDIR clearly activated transcription through hPTH nVDRE (Fig. 6AGo, lane 6), but activated VDR attenuated this transactivation by VDIR (Fig. 6AGo, lane 8). An HDAC inhibitor, trichostatin A (TSA) abrogated ligand-induced transrepression through VDIR and VDR/RXR (Fig. 6AGo, lanes 9 and 10), confirming that HDAC activity is required for transrepression. We then tested coregulator recruitment to the nVDREs by an avidin-biotin complex DNA binding assay. As previously reported (24), treatment with forskolin, a protein kinase A activator to stimulate VDIR function, induced p300 HAT recruitment to nVDREs (see upper panel of Fig. 6BGo). In contrast, when the cells were treated with 1{alpha},25(OH)2D3, HDAC2, but neither HDAC1 nor 3 (data not shown), was recruited to VDR/VDIR bound upon hPTHnVDRE as well as 1{alpha}nVDRE (Fig. 6BGo, lower panel). Altogether, these findings demonstrated that ligand-induced transrepression by VDR/RXR with VDIR upon hPTHnVDRE also mediates HDAC2 recruitment, presumably coupled to HAT coactivator dissociation. Such ligand-induced coregulator switching through VDR/RXR and VDIR upon the hPTHnVDRE appears similar to that reported for 1{alpha}nVDRE (24).


Figure 6
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  		Fig. 6. The 1{alpha},25(OH)2D3-Induced Transrepression by VDR upon the hPTHnVDRE Requires HDAC2

A, The transfected cells were treated with TSA (10–7 M) for 24 h before harvest. B, A ligand-induced HDAC2 recruitment to VDR/RXR and VDIR bound upon the hPTHnVDRE. Two copies of hPTHnVDRE and h1{alpha}nVDRE oligomers (as a control) were conjugated to the beads and incubated with whole-cell extracts from MCT cells treated in the presence and absence of 1{alpha},25(OH)2D3 and forskolin. The interactants with oligo-beads were detected by Western blots using individual antibody. IP, Immunoprecipitation.

 
E-Box-Type Elements Act as a Class of nVDRE
Considering that vitamin D plays a number of roles in various target tissues, it is likely that there are a number of as-yet-unidentified target genes whose expression is under negative control by VDR. The present model might explain at the molecular level the negative response to 1{alpha},25(OH)2D3 in the expression of at least some VDR-negative target genes. E-box type elements in the h1{alpha}(OH)ase and hPTH gene promoters may be representative of those in other gene promoters. In this respect, it would be intriguing to identify the genes negatively regulated by VDR and analyze the negative VDREs in those gene promoters. There may be other classes of nVDRE because modes of ligand-induced transrepression generally appear diverse among nuclear receptors (18, 28).

A Cell Type-Specific Coregulator Complex Is Mediated by Ligand-Induced Transrepression by VDR
Although the enzyme is expressed in many nonrenal tissues besides kidney, including skin, the regulation of 1{alpha}(OH)ase gene expression is different between kidney and skin cells (29). In the previous report, we revealed that ligand-induced transrepression by VDR through E-box type nVDRE in the h1{alpha}(OH)ase gene promoter is reproducible only in the renal tubule cell line (MCT) that endogenously expresses 1{alpha}(OH)ase, VDIR and VDR genes (24). Such ligand-induced transrepression was undetectable in other cell lines, even other kidney-derived cell lines (data not shown). These results suggest transrepression by liganded VDR may require a cell type-specific coregulator (16, 17). Similarly, because the PTH gene is expressed only in the parathyroid and thymus, a tissue-specific factor may also be indispensable there for transrepression of the PTH gene by liganded VDR. It remains unclear whether such a tissue cell type-specific coregulator is identical or not in both parathyroid and renal tubule cells. Even though HDAC appears principally responsible for transrepression, it was unexpected that TSA, even at much higher concentrations than the normally applied range, could not fully abrogate 1{alpha},25(OH)2D3-induced transrepression by VDR. This may imply that other histone modifiers such as histone methylase/demethylase are involved in this transrepression. In this sense, biochemical identification of complex(es) containing both VDR/RXR and VDIR clearly requires identification of tissue-specific factor(s) involved in 1{alpha},25(OH)2D3-induced transrepression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
RT-PCR
The male VDR KO mice have been described previously (26). 1{alpha},25(OH)2D3 (10 µg/kg) or vehicle (medium chain triglyceride) were administered orally three times a week. After the final 24 h, total RNA was extracted from several mice (12-wk-old mice) using an ISOGEN Kit (Nippon Gene). One microgram of total RNA was reverse-transcribed by Manuscript (Invitrogen, Carlsbad, CA). The PCR was performed for 35 cycles consisting of 40 sec of denaturing at 95 C, 40 sec annealing at 59 C (mPTH/mPTHrP) or 57 C [m1{alpha}(OH)ase], and then 40 sec extension at 72 C. For RT-PCR, the following primers were used: mPTH 5'-ATGATGTCTGCAAACACCGTGG-3' and 5'-ATCAGCTTTGTCTCCCTCACCAG-3'; mPTHrP 5'-CTGGTTCAGCAGTGGAGCTC-3' and 5'-GTTAGGGGACACCTCCGAGGT-3'; mouse 1{alpha}(OH)ase 5'-AAACACAGACATGACCCAGG-3' and 5'-GCAGCGCCATGCACCTGCAG-3'; mouse glyceraldehyde-3-phosphate dehydrogenase 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3' (30).

Plasmid and RNAi
Deletion mutants of the hPTH and hPTHrP promoters were generated by PCR and cloned into the pGL3-TK Luciferase vector (Promega, Madison, WI). The two hPTH VDRE sequences (5'-CATCATCTGTAA-3') were inserted into the pGL3-Luciferase vector driven by a thymidine kinase (TK) and TATA promoter, respectively. The small interfering RNA (siRNA) sequences of VDIR were designed by Dharmacon (Lafayette, CO) (23).

Cell Culture and Transient Transfection
MCT cells (derived from proximal tubules of mouse kidney) were maintained in DMEM supplemented with 5% fetal bovine serum (FBS) (Invitrogen) at 37 C, 5% CO2. For transfection, cells were cultured in DMEM supplemented with 5% charcoal-stripped FBS. Cells were transfected with expression vector using Lipofectamin Plus (Invitrogen) according to the manufacturer’s instructions. After treatment with vehicle or ligand (1 x 10–8 M), cells were further cultured for 24 h. Luciferase assays were performed as described (31). siRNA transfection were as described (23).

EMSA
Recombinant VDR, RXRß, and VDIR were expressed in E. coli as glutathione-S-transferase (GST)-fusion proteins and purified by digestion with thrombin after affinity column chromatography. Double-stranded oligonucleotide probes were end-labeled using [{gamma}-32P]ATP and T4 polynucleotide kinase. For reaction, 10 ng of recombinant proteins were preincubated in binding buffer [10 mM Tris-HCl (pH 7.5), 75 mM KCl, 5 mM EDTA, 1 mM MgCl2, 4% glycerol, 1 mM dithiothreitol] with/without 10–8 M 1{alpha},25(OH)2D3 for 20 min on ice. Ten microliters of labeled probes were added in each sample. After incubation for 30 min at room temperature, the samples were resolved on 5% polyacrylamide gels and run in 0.5x Tris-EDTA buffer. The gel was dried and subjected to autoradiography. The following sequences of double-stranded oligonucleotides were used as probes: [1{alpha}nVDRE; 5'-CCATTAACCCACCTGCCATCTGCC x 2–3', hPTHnVDRE: 5'-CATCATCTGTAAC x 2–3', DR3-like motif; 5'-GTGTGTATGTGCTGCTTT x 2–3']. Assays were performed as described (24).

Template-Pull-Down Assay (Avidin-Biotin Complex DNA Binding Assay)
3'-End biotin conjugated sense and antisense DNA were incubated at 100 C to anneal and cooled slowly to room temperature. The following sequences of double-stranded oligonucleotides were used as probes: [1{alpha}nVDRE; 5'-CCATTAACCCACCTGCCATCTGCC x 2–3', hPTHnVDRE: 5'-CATCATCTGTAAC x 2–3', distal regin (negative control); 5'-GGCATGCATC x 2–3']. To prepare bead-DNA complex, biotin-conjugated double-strand DNA was mixed with 50% slurry avidin-beads. Cells were cultured in DMEM supplemented with 5% charcoal-stripped FBS for 24 h, after treatment with vehicle or ligand (1 x 10–8 M). Cells were lysed in lysis buffer [10 mM Tris-HCl (pH 7.8), 1 mM EDTA, 0.15 M NaCl, 0.1% Nonidet P40]. Whole cell lysates were immunoprecipitated by bead-DNA complex containing poly (deoxyinosine-deoxycytosine) (32). Proteins were resolved on SDS-PAGE and Western blots were visualized with {alpha}-E47 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), {alpha}-VDR antibody (NeoMarkers; Lab Vision, Fremont, CA), {alpha}-HDAC2 antibody (Affinity BioReagents, Golden, CO), and {alpha}-p300 antibody (Santa Cruz Biotechnology) (24).


   ACKNOWLEDGMENTS
 
We thank I. Takada for helpful discussions and Chugai Pharmaceutical Co. Ltd. for the 1{alpha},25(OH)2D3 relative compound. We also thank H. Higuchi for preparation of the manuscript.


   FOOTNOTES
 
This work was supported in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and priority areas from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to S.K.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 9, 2006

Abbreviations: FBS, Fetal bovine serum; GST, glutathione-S-transferase; h, human; HAT, histone acetyltransferase; HDAC, histone deacetylase; MCT, mouse cortical tubular; nVDRE, negative VDRE; 1{alpha},25(OH)2D3, 1{alpha},25-dihydroxyvitaminD3; 1{alpha}(OH)ase, 25(OH) D31{alpha}-hydroxylase; RNAi, RNA interference; RXR, retinoid X receptor; siRNA, small interfering RNA; TK, thymidine kinase; TSA, trichostatin A; VDIR, VDR interacting repressor; VDR, vitamin D receptor; VDRE, vitamin D response element.

Received for publication May 26, 2006. Accepted for publication November 1, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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NURSA Molecule Pages Link:

Nuclear Receptors:   VDR
Coregulators:   HDAC2
Ligands:   Calcitriol



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