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Molecular Endocrinology, doi:10.1210/me.2006-0554
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Molecular Endocrinology 21 (7): 1513-1525
Copyright © 2007 by The Endocrine Society

Cellular Expression Levels of the Vitamin D Receptor Are Critical to Its Transcriptional Regulation by the Pituitary Transcription Factor Pit-1

Samuel Seoane, Isabel Ben, Viviana Centeno and Roman Perez-Fernandez

Department of Physiology, School of Medicine, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain

Address all correspondence and requests for reprints to: Roman Perez-Fernandez, Departamento de Fisiología, Facultad de Medicina, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain. E-mail: fsropefe{at}usc.es.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The biological role of 1,25-dihydroxyvitamin D3 has generally been related to calcium homeostasis, but this hormone also has fundamental effects on processes of cellular proliferation and differentiation. The genomic actions of 1,25-dihydroxyvitamin D3 are mediated by the vitamin D receptor (VDR) present in target cells. However, VDR transcriptional regulation is not well understood, probably attributable to the complexity of the VDR gene and its promoter. In the present study, it is demonstrated that administration of the pituitary transcription factor Pit-1 (originally found in the pituitary gland but also present in other nonpituitary cell types and tissues) to the MCF-7 (human breast adenocarcinoma) cell line induces a significant increase in VDR mRNA and protein levels. Conversely, Pit-1-targeted small interference RNA markedly reduced expression of VDR in MCF-7 cells. Reporter gene assays demonstrated that the effect of Pit-1 is mediated by its binding to a region located between –254 and –246 bp from the VDR transcription start site. Selective mutations of this site completely abolished VDR transcription. Chromatin immunoprecipitation analysis showed that binding of Pit-1 to the VDR promoter leads additionally to recruitment of cAMP response element-binding protein binding protein, acetylated histone H4, and RNA polymerase II. Surprisingly, Pit-1 binding also recruits VDR protein to the VDR promoter. Using several cell lines with different levels of VDR expression, it was demonstrated that up-regulation of VDR transcription by Pit-1 is dependent on the presence of VDR protein, suggesting that transcriptional expression of VDR in a given cell type is dependent on, among other factors, its own expression levels.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
VITAMIN D RECEPTOR (VDR) is a ligand-inducible transcription factor that mediates the biological actions of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], a hormone classically considered to be related to calcium and bone homeostasis, but also involved in many other cell processes, such as proliferation and differentiation (1, 2). During binding hormone, VDR binds to specific DNA response elements in the promoter regions of target genes, leading to the recruitment and/or dissociation of additional coregulatory proteins and thus inducing or repressing their transcriptional activity (3, 4). Thus, the genomic actions of 1,25-(OH)2D3 on a given target cell are dependent on the presence of VDR in that cell, so that regulation of VDR levels has important effects on vitamin D-mediated actions. VDR levels are regulated by several factors, including hormones, growth factors, and calcium (5, 6, 7, 8, 9), and some of these actions may be mediated by cAMP-activated protein kinase A, which increases VDR gene expression, or by protein kinase C, which inhibits VDR expression (10, 11). In addition, VDR protein levels are modulated posttranslationally by its ligand 1,25-(OH)2D3, which induces receptor stabilization and increases the half-life of the protein (12).

The structural organization of the human (h) VDR chromosomal gene and its promoter were reported by Miyamoto et al. (13), demonstrating that, at its noncoding 5' end, the hVDR gene is composed of exons 1A, 1B, and 1C, whereas eight additional exons (exons 2–9) encode the structural portion of the VDR gene product (13). Subsequent characterization by Crofts et al. (14) identified three more exons (1D, 1E, and 1F), making a total of 14 exons, and at least three differentially used promoters. Identification and cloning of VDR promoter gave researchers the opportunity to better evaluate transcriptional regulation of its expression. However, and probably attributable to the complexity of the VDR gene, little information has been reported to date about its transcriptional regulation. Using reporter gene assays, it has been demonstrated that, in MCF-7 cells, a TATA-containing promoter upstream of exon 1C of the hVDR is up-regulated by 17ß-estradiol, retinoic acid, and forskolin, which seems to indicate that these agents act directly on the VDR promoter (15). Jehan and DeLuca (16) found that the mouse VDR is mainly expressed through an specificity protein 1 promoter in vivo, and recently Wietzke et al. (17), using luciferase reporter constructs containing the –800- to +31-bp segment of exon 1C of the VDR promoter, found that VDR is transcriptionally regulated at specificity protein 1 consensus sites by estrogens, resveratrol, and 1,25-(OH)2D3 (17). VDR expression can also be modulated at the transcriptional level by various factors, including the caudal-related homeodomain protein Cdx-2 (18), Wilms’ tumor suppressor protein (19), the transcription factors SNAIL (20), p63 (21), and ZEB (22), and the adaptor protein ß-catenin (23). In addition, a direct autoregulatory mechanism has been demonstrated recently, whereby 1,25-(OH)2D3 induces transcriptional regulation of the mouse VDR gene through an enhancer located within two introns downstream of the transcription start site of this gene (24).

The homeobox POU family of DNA-binding proteins is a large family of transcription factors, including the pituitary transcription factor Pit-1, that have a critical role in development, either repressing or activating the expression of specific genes (25). Pit-1 is essential in cell development and in the maintenance of somatotrophs, lactotrophs, and thyrotrophs, as well as in pituitary cell proliferation (26, 27, 28). However, Pit-1 is also expressed in nonpituitary tissues and cell lines, including normal and tumoral human breast tissue and the human breast adenocarcinoma cell line MCF-7 (29, 30). In this cell line, as demonstrated previously in other lines (26, 31), Pit-1 induces cell proliferation (29). Interactions between Pit-1 and nuclear receptors, including VDR, have been reported (32, 33). In addition, it has been demonstrated recently that the VDR inhibits Pit-1 gene expression at the transcription level, suggesting that Pit-1 inhibition by VDR may be related to the antiproliferative effects of vitamin D (34). In the present study, to evaluate the relationships between VDR and Pit-1, MCF-7 cells were transfected with either Pit-1 expression vector or Pit-1 small interference RNA (siRNA), and VDR mRNA and protein levels were then evaluated. VDR gene transcription activity was also assessed, using reporter gene assays. Our results indicate that VDR gene expression is repressed by Pit-1 knockdown and up-regulated by Pit-1 overexpression. This up-regulation of the VDR gene by Pit-1 is mediated by Pit-1 binding to a region located between –254 and –246 bp upstream of the VDR transcription start site. Binding of Pit-1 to this Pit-1 response element in the VDR promoter, demonstrated by gel retardation assays and chromatin immunoprecipitation (ChIP) analysis, induces recruitment of CREB (cAMP response element binding protein) binding protein (CBP), acetylated histone H4 (Ac-H4), and RNA polymerase II (Pol II). Surprisingly, VDR protein was also recruited to this complex. To evaluate the role of VDR protein in this complex, additional experiments were performed. Specifically, using several cell lines with different levels of VDR expression (EA.hy926, COS-7, and HeLA), it was demonstrated by RT-PCR, Western blot, reporter gene assays, and ChIP analysis that endogenous VDR protein levels are critical for inducing Pit-1-dependent VDR transcriptional regulation, suggesting a mechanism in which the rate of expression of VDR may be dependent on its own levels in the cell.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pit-1 Overexpression Raises, and Knockdown Decreases, VDR mRNA and Protein Levels
To evaluate whether Pit-1 influences VDR expression, we performed a real-time PCR amplification of cDNA from MCF-7 human adenocarcinoma cells transfected with pRSV-hPit-1 48 h previously, using primers for VDR. The results of this analysis indicate a significant (P < 0.05) increase in VDR mRNA expression (relative to 18S mRNA expression) (5.04 ± 1.10) with respect to untransfected MCF-7 cells (2.67 ± 0.13) (Fig. 1AGo). Pit-1 mRNA was also quantified by Northern blotting. Transfection of MCF-7 cells with pRSV-hPit-1 (Fig. 1BGo, lanes 4–6) or pCMV-hVDR (used as positive control) (Fig. 1BGo, lanes 7–9) in both cases induced a significant increase in VDR mRNA levels with respect to control cells (Fig. 1BGo, lanes 1–3).


Figure 1
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  		Fig. 1. Pit-1 Increases VDR mRNA and Protein Expression in MCF-7 Cells

A, VDR mRNA expression, with respect to 18S mRNA levels, evaluated in MCF-7 cells transfected with pRSV-hPit-1 for 48 h (+Pit-1) and in untransfected control (C) MCF-7 cells by real-time RT-PCR (*, P < 0.05 with respect to control cells). Bars represent the mean and SD of triplicate samples from four separate experiments. B, Representative Northern blot of VDR (top) and 18S (bottom) mRNA in MCF-7 cells. Lanes 1–3, Nontreated control cells; lanes 4–6, cells transfected with 4 µg pRSV-hPit-1 construct for 48 h; and lanes 7–9, cells transfected with 4 µg pCMV-hVDR construct for 48 h (positive control). C, Western blots of Pit-1, VDR, and ß-actin (used as loading control). Lane 1, Untreated (control) cells; lane 2, cells transfected with 4 µg pRc/RSV plasmid for 48 h; lane 3, cells transfected with 4 µg pRSV-hPit-1 construct for 48 h. D, Western blots of Pit-1, VDR, and ß-actin (used as loading control). Lane 1, Untreated (control) cells; lane 2, cells transfected with 20 nM missense siRNA (negative control) for 48 h; lane 3, cells transfected with Pit-1 siRNA (20 nM) for 48 h.

 
Western blotting of Pit-1 and VDR in MCF-7 cells transfected with pRSV-hPit-1 is shown in Fig. 1CGo. VDR was detectable in untransfected control cells (lane 1) and pRc/RSV-transfected MCF-7 cells (lane 2). Significantly increased Pit-1 and VDR levels, with respect to untransfected or pRc/RSV-transfected cells, were observed in MCF-7 cells transfected with the pRSV-hPit-1 construct (lane 3).

To evaluate whether Pit-1 knockdown induces changes in VDR protein expression levels, we used siRNA. MCF-7 cells were transfected with a missense siRNA or Pit-1 siRNA. As control, Pit-1 protein expression was also evaluated. As shown in Fig. 1DGo, transfection of MCF-7 cells with 20 nM missense siRNA (lane 2) did not modify Pit-1 or VDR expression levels with respect to the untransfected control cells (lane 1). However, knockdown of Pit-1 by transfection with 20 nM Pit-1 siRNA resulted in markedly reduced expression of both Pit-1 and VDR (lane 3).

Pit-1 Increases VDR Expression at the Transcriptional Level by Binding to a Response Element in the VDR Promoter
To investigate the effect of Pit-1 on the transcriptional activity of the VDR promoter in the MCF-7 cell line, cells were cotransfected with pRSV-hPit-1 and a construct linking 600 bp of the VDR gene promoter to a luciferase reporter vector (pGL3B-hVDR623) (Fig. 2AGo). At 48 h after transfection, cells were harvested for measurement of luciferase activity. As shown in Fig. 2BGo, cotransfection with the control plasmid (pGL3-basic) and pRSV-hPit-1 had a negligible effect on luciferase activity, whereas cotransfection with pGL3B-hVDR623 and pRSV-hPit-1 led to a significant increase in luciferase activity (P < 0.001).


Figure 2
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  		Fig. 2. Transactivation of the hVDR Gene by Pit-1 Is Mediated by a –254/–246-bp Fragment in the VDR Promoter

A, Diagram of the hVDR gene promoter construct (pGL3B-hVDR623) and the mutated construct (pGL3B-hVDR623mut), showing the location of the putative Pit-1 response element. B, MCF-7 cells were transfected with the empty vectors (pGL3Basic and/or pRc/RSV), the pRSV-hPit-1 construct, the pGL3B-hVDR623 construct, or the VDR promoter fused to pGL3Basic with 5-bp mutations in the –254/–246 region (pGL3B-hVDR623mut) and then cultured for 48 h. Transcription of the wild-type VDR was significantly (***, P < 0.001) increased after overexpression of Pit-1, whereas the VDR mutant showed no signs of regulation by Pit-1. Normalized relative luciferase units (RLU) were calculated as the ratio of luciferase activity between cells transfected with the empty pRc/RSV vector or the pRSV-hPit-1 vector, and the values represent means ± SD from three independent determinations.

 
Using the TESS program [Transcription Element Search Software, Computational Biology and Informatics Laboratory, School of Medicine, University of Pennsylvania, Philadelphia, PA (http://www.cbil.upenn.edu/tess)], we found that the VDR promoter contains a Pit-1 consensus binding site (35), positioned between –254 and –246 bp from the transcription start site of the hVDR gene. Thus, to delineate the location of Pit-1 binding to the VDR promoter, we performed a site-directed mutagenesis on this putative Pit-1 response element (obtaining pGL3B-hVDR623mut) (Fig. 2AGo) and then cotransfected this construct with the pRSV-hPit-1 expression vector. As shown in Fig. 2BGo, the transcriptional increase obtained using the wild-type pGL3B-hVDR623 construct was completely abolished when the mutated VDR promoter element was cotransfected with the pRSV-hPit-1 vector, indicating that this element was functional.

Gel Retardation and ChIP Assays Indicate that Pit-1 Binds to the VDR Promoter in a Region between –254 and –246 bp from the Transcription Start Site
To delineate the site of Pit-1 binding to the VDR promoter, we used a gel retardation assay. As positive control, we used a labeled fragment of the hGH promoter, containing the distal Pit-1 response element (GH28). As shown in Fig. 3AGo, this probe was shifted when MCF-7 nuclear extract was added (lane 2). To confirm that the protein binding to the GH28 fragment was Pit-1, we preincubated the nuclear extract with an anti-Pit-1 antiserum. This treatment supershifted the GH28 fragment in the gel retardation assay (lane 3). Using a labeled oligonucleotide containing the Pit-1 consensus site in the VDR promoter (from –266 to –238 bp, VDR28) (Fig. 2AGo) and MCF-7 nuclear extract (1 µg), we observed a band that was supershifted with the anti-Pit-1 antibody (Fig. 3Go, lanes 5 and 6, respectively). In contrast, when we used a labeled oligonucleotide containing the mutated Pit-1 consensus binding site (VDR28mut) in the VDR promoter region, binding was completely abolished when MCF-7 nuclear extract was added.


Figure 3
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  		Fig. 3. Pit-1 Binds to the hVDR Promoter in a Region Located between –254 and –246 bp Upstream of the Start Transcription Site of the VDR Gene

A, Gel mobility shift analysis show Pit-1 binding to an oligonucleotide containing a VDR promoter fragment (VDR28, –266 to –238 bp from VDR transcription start site) (Fig. 2AGo). A radiolabeled VDR28 probe was incubated with 1 µg MCF-7 nuclear extract (lane 5). For supershift assay, 1 µg MCF-7 nuclear extract was preincubated with 1 µl anti-Pit-1 antiserum (lane 6). Mutation of 5 bases in the VDR28 element (VDR28mut) abolished in vitro Pit-1 binding. A radiolabeled GH fragment (GH28) containing the distal Pit-1 response element in the GH promoter, used as a positive control, was shifted (lane 2) or supershifted (lane 3) using MCF-7 nuclear extract or MCF-7 nuclear extract plus 1 µl anti-Pit-1 antibody, respectively. B, Diagram of the hVDR gene promoter showing the location of primers used in the ChIP assay. The black square shows the location of the Pit-1 response element in the VDR promoter. C, Soluble chromatin prepared from MCF-7 cells transfected with pRSV-hPit-1 was immunoprecipitated with an anti-Pit-1 antibody or control IgG. The immunoprecipitated DNA was amplified by PCR using primers D, which amplified a 482-bp region of the VDR promoter including the up-regulatory Pit-1 sequence (–254/–246) or primers A–C, which amplified different regions of the VDR promoter not containing this sequence. As positive control, we used specific GH promoter primers that amplified a 201-bp sequence in the GH promoter containing the distal Pit-1 response element. Lane 1, Molecular weight marker; lanes 2–5, inputs from PCR products using primers corresponding to regions A, B, C, and D, respectively; lane 6, IgG-immunoprecipi-tated DNA amplified using primers D; lanes 7–10, PCR products from Pit-1-immunoprecipitated DNA amplified using primers A, B, C, and D, respectively; lanes 11 and 12, IgG or Pit-1-immunoprecipitated DNA (respectively) amplified using GH promoter primers. The results are typical of three similar experiments.

 
To seek additional support for the hypothesis that Pit-1 induces endogenous VDR transcription, a ChIP assay was performed to confirm in vivo interaction of Pit-1 with the hVDR promoter (Fig. 3Go, B and C). As shown in Fig. 3CGo, the ChIP PCR product was detected with the Pit-1 antibody in pRSV-hPit-1-transfected MCF-7 cells using proximal D primers (lane 10) but not using medial (B, lane 8; and C, lane 9) or distal (A, lane 7) primers. This suggests that endogenous Pit-1 specifically binds to the regulatory region (–266/–238) of the VDR promoter. As a positive control, we used the hGH promoter containing both the proximal and distal Pit-1 response elements. Figure 3Go, lane 12, shows a 201-bp band corresponding to the ChIP PCR product, detected with the anti-Pit-1 antibody using specific GH promoter primers.

Increased VDR Transcription Induced by Pit-1 Is Mediated by Recruitment of CBP, Ac-H4, and RNA Pol II
To study the mechanism by which Pit-1 induces increased VDR transcription, we performed ChIP assays using specific antibodies for CBP, Ac-H4, and RNA Pol II. As shown in Fig. 4Go, using a CBP antibody and proximal D primers, the ChIP PCR product of pRSV-hPit-1-transfected MCF-7 cells (lane 4) was increased with respect to the product from untransfected MCF-7 cells (lane 3). Figure 4Go also shows that, using proximal D primers, the ChIP PCR product was detected with the Ac-H4 antibody (lane 4) or increased with the RNA Pol II antibody (lane 4) in pRSV-hPit-1-transfected MCF-7 cells. Binding of Pit-1 to the VDR promoter, using proximal D primers, was also observed in both control (untransfected) cells and pRV-hPit-1-transfected cells (Fig. 4Go, top, lanes 3 and 4, respectively). This suggests that binding of Pit-1 to its response element in the regulatory region (–266/–238 bp) of the VDR promoter induces recruitment of endogenous CBP, Ac-H4, and RNA Pol II.


Figure 4
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  		Fig. 4. The Increase in VDR Transcription Induced by Pit-1 Is Mediated by Recruitment of the CBP, Ac-H4, and RNA Pol II

Soluble chromatin prepared from MCF-7 cells transfected (+) (lane 4) or not transfected (–) (lane 3) with the pRSV-hPit-1 vector was immunoprecipitated with anti-Pit-1, anti-CBP, anti-Ac-H4, or anti-RNA Pol II antibodies or control IgG (lane 2). The immunoprecipitated DNA was amplified by PCR (38 cycles for Pit-1 and 34 cycles for CBP, Ac-H4, and RNA Pol II) using primers D (see legend of Fig. 3Go). The results are typical of several separate experiments.

 
Pit-1 Synergizes with VDR to Increase Transcription of Vitamin D-Regulated Genes
To evaluate the effect of the Pit-1-dependent VDR expression increase on vitamin D-regulated genes, we performed transfection experiments in COS-7 cells (which show low levels of VDR expression). As a positive target control, we used the pGL2-(Spp-1)2 construct, which contains two Spp-1/mouse osteopontin vitamin D response element (VDRE) sites. As shown in Fig. 5Go, 1, 25-(OH)2D3 treatment of cells transfected with this construct and cotransfected with pCMV-hVDR led to a significant increase in VDR reporter activity. Indeed, when COS-7 cells were also cotransfected with pCMV-hVDR and pRSV-hPit-1 and treated with 1,25-(OH)2D3, a significant increase in reported activity was observed with respect to both cells not treated with 1,25-(OH)2D3 and cells not transfected with pRSV-hPit-1. This seems to indicate that Pit-1 synergistically cooperates with 1,25-(OH)2D3/VDR to induce enhanced Spp-1 transcription.


Figure 5
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  		Fig. 5. Pit-1 Expression Synergizes with 1,25-(OH)2D3-VDR to Increase Transcription of the Mouse Osteopontin (Spp-1) Gene

MCF-7 cells were transfected with the pGL2-(Spp-1)2 construct and with pRSV-hPit-1 (or control plasmid, pRc/RSV) and/or with pCMV-hVDR construct (or control vector, pRc/CMV) and then treated with ethanol or 100 nM 1,25-(OH)2D3 for 48 h in hormone-depleted medium. Normalized relative luciferase units (RLU) were calculated as the ratio of luciferase activity in vitamin D-treated cells to that in the corresponding control (ethanol-treated) cells. **, P < 0.01 and ***, P < 0.001 with respect to normalized relative luciferase units calculated for ethanol-treated cells or 1,25-(OH)2D3-treated and pCMV-hVDR-transfected cells.

 
VDR Protein Is Also Recruited by the Pit-1/CBP/Ac-H4/RNA Pol II Complex in the VDR Promoter
Given that it has been demonstrated that Pit-1 interacts with several nuclear receptors, including VDR (33), our next objective was to evaluate whether VDR may itself be involved in Pit-1-induced VDR transcription. A total protein extract from MCF-7 cells was immunoprecipitated with a polyclonal anti-Pit-1 antibody (Fig. 6AGo, lane 2) or control IgG (Fig. 6AGo, lane 1) and then blotted with a monoclonal anti-VDR antibody. Figure 6AGo shows that a band corresponding to immunoreactive VDR was present in the anti-Pit-1-immunoprecipitated MCF-7 cell extract. To confirm that VDR is recruited in vivo by Pit-1 to transactivate VDR gene transcription, a ChIP assay was performed. As shown in Fig. 6BGo, the ChIP PCR product, using proximal D primers, was detected with the anti-VDR antibody in pCMV-hPit-1-transfected MCF-7 cells (lane 4) but not in untransfected MCF-7 cells (lane 3) or in control IgG-treated cells (lane 2). This suggests that recruitment of endogenous VDR by Pit-1 may be partially responsible for its stimulatory effect on VDR promoter activity.


Figure 6
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  		Fig. 6. Pit-1 Also Recruits VDR Protein to Bind the VDR Promoter

A, MCF-7 cells were immunoprecipitated overnight with anti-Pit-1 antibody (lane 2) or with IgG (as specificity control, lane 1) and then blotted with an anti-VDR antibody. ns, Not specific. B, Soluble chromatin was obtained from nontransfected MCF-7 cells and immunoprecipitated with anti-VDR antibody (lane 3) or from pRSV-hPit-1-transfected MCF-7 cells and immunoprecipitated with anti-VDR antibody (lane 4) or control IgG (lane 2). The immunoprecipitated DNA was amplified by PCR using primers D (see legend of Fig. 3Go, B and C).

 
VDR Protein Levels Are Critical for Regulating Pit-1-Dependent VDR Transcription
The presence of VDR in the complex of proteins binding the VDR promoter and regulating VDR transcription suggests that VDR may be necessary for the transcriptional regulation of its own expression. To evaluate this hypothesis, experiments were performed with several human cell lines showing different VDR expression levels. Western blotting of EA.hy926 (a human endothelial cell line) and COS-7 (African green monkey kidney cells) indicated low levels of VDR expression, in contrast with HeLa (human cervix adenocarcinoma) and MCF-7 cells, which showed high levels of VDR expression. These cell lines were then transfected with pRSV-hPit-1, and VDR mRNA and protein were evaluated by RT-PCR and Western blotting. As shown in Fig. 7BGo, no significant increase in VDR mRNA level was observed in EA.hy926 or COS-7 cells, whereas VDR mRNA level was significantly increased in MCF-7 and HeLa cells. Western blotting of VDR in the COS-7 and EA.hy926 cell lines is shown in Fig. 7CGo. No significant increase in VDR protein level was observed when pRSV-hPit-1 expression vector was transfected into either COS-7 or EA.hy926 cells (lane 3), whether with respect to the control (untransfected cells; lane 1) or pRc/RSV-transfected cells (lane 2). However, overexpression of Pit-1 vector in MCF-7 and HeLa cells (Fig. 7DGo) induced a significant increase in VDR protein levels (lane 3) with respect to the control cells (lane 1, untransfected cells; lane 2, pRc/RSV-transfected cells).


Figure 7
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  		Fig. 7. VDR mRNA and Protein Expression Induced by Pit-1 Is Dependent on the Levels of VDR in the Cell

A, Western blots to detect VDR and ß-actin (loading control) expression in human endothelial (EA.hy926), African green monkey kidney (COS-7), human cervix adenocarcinoma (HeLa), and MCF-7 cells. B, Pit-1, VDR, and 18S PCR products obtained from pRSV-hPit-1-transfected (+) or untransfected (–) COS-7, EA.hy926, MCF-7, and HeLa cells. C, Western blots of Pit-1, VDR, and ß-actin (loading control) in COS-7 and EA.hy926 cells. Lane 1, Control (untreated cells); lane 2, pRc/RSV-transfected cells; lane 3, pRSV-hPit-1-transfected cells. D, Western blots of Pit-1, VDR, and ß-actin (loading control) in MCF-7 and HeLa cells. Lanes 1–3 as in C.

 
Using reporter gene assays, we also evaluated the effect of Pit-1 overexpression in EA.hy926, COS-7, and HeLa cells (Fig. 8Go). Cotransfection of pRSV-hPit-1 and pGL3B-hVDR623 into EA.hy926 (Fig. 8AGo.1) or COS-7 (Fig. 8AGo.2) cells did not modify luciferase reporter activity with respect to cells not transfected with pRSV-hPit-1. In contrast, cotransfection of pCMV-hVDR into these cell lines significantly increased reporter activity. However, no changes in reporter activity were observed when the pGL3B-hVDR623mut construct was cotransfected with pRSV-hPit-1 and pCMV-hVDR into COS-7 cells (data not shown). In HeLa cells (Fig. 8AGo.3), overexpression of Pit-1 alone (that is, without pCMV-hVDR) was sufficient to induce a significant increase in VDR reporter activity. In addition, in COS-7 cells, increasing the concentration of pCMV-hVDR transfected into cells cotransfected with pRSV-hPit-1 and pGL3B-hVDR623 led to an increased reporter response (Fig. 8BGo), suggesting that, depending on VDR protein levels, the transcriptional response of the VDR gene to Pit-1 may be rescued.


Figure 8
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  		Fig. 8. VDR Transcriptional Reporter Activity Induced by Pit-1 Is Dependent on the Levels of VDR in the Cell

A.1–A.3, A total of 250 ng of the pGL3B-hVDR623 construct (containing the VDR promoter) was transfected alone or cotransfected with 500 ng pRSV-hPit-1 and/or 500 ng of pCMV-hVDR into EA.hy926, COS-7, or HeLa cells using FuGene reagent. Luciferase activity was normalized to ß-galactosidase activity. B, COS-7 cells were transfected with 250 ng of the pGL3B-hVDR623 reporter vector and/or co-transfected with 500 ng pRSV-hPit-1 and increasing concentrations (125–1000 ng) of pCMV-hVDR. Normalized relative luciferase units (RLU) were calculated as A.1. n.s., Not significant. ***, P < 0.001 with respect to pGL3B-hVDR623-transfected cells.

 
To evaluate in vivo VDR expression requirements for Pit-1-dependent VDR transcription, ChIP analyses were performed. As shown in Fig. 9Go, transfection of COS-7 cells with pRSV-hPit-1 did not induce Pit-1 binding to the VDR promoter (lane 4), but Pit-1 binding was observed when pCMV-hVDR was cotransfected with pRSV-hPit-1. Similar ChIP experiments were performed using EA.hy926 and HeLa cells. The pattern of binding of Pit-1 to the VDR promoter in EA.hy926 cells (with very low levels of VDR expression) is similar to that observed in COS-7 cells, with VDR protein expression being necessary for binding of Pit-1 to the VDR promoter. In contrast, in the HeLa cell line (with higher levels of VDR expression than in COS-7 and EA.hy926 cells), the VDR transcriptional response was similar to that in MCF-7 cells, in which Pit-1 binds the VDR promoter in vivo without any need for transfection of a VDR expression vector.


Figure 9
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  		Fig. 9. Pit-1 Binding to the VDR Promoter Is Dependent on the Levels of VDR Protein

Soluble chromatin prepared from HeLa, COS-7, and EA.hy926 cells not transfected (–) (lane 3) or transfected (+) with pRSV-hPit-1 (lane 4) or cotransfected (++) with pRSV-hPit-1 and pCMV-hVDR (lane 5) were immunoprecipitated with an anti-Pit-1 antibody or control IgG (lane 2). The immunoprecipitated DNA was amplified by PCR (38 cycles) using primers D (see diagram of Fig. 3BGo) that amplified a 482-bp region of the VDR promoter, which includes the up-regulatory Pit-1 sequence (–254/–246). All results are typical of several separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Interactions between Pit-1 and nuclear receptors have been observed previously. For example, Pit-1 and estrogen receptor are required for estrogen responsiveness of the rat prolactin (PRL) gene (36); thyroid hormone receptor and retinoic acid receptors (RAR and RXR) cooperate with Pit-1 to activate the rat GH gene (37, 38), and VDR and Pit-1 cooperate to activate PRL transcription (32). In view of this, and given that direct repression of Pit-1 transcription by VDR has been demonstrated (34), in the present study we evaluated the possibility of regulation of VDR transcription by Pit-1. To this end, cells of the MCF-7 line were transfected with a Pit-1 expression vector (pRSV-hPit-1), and VDR mRNA and protein levels were then evaluated using real-time RT-PCR and Northern blotting or by Western blotting, respectively. Our results clearly indicate that overexpression of Pit-1 induces a significant increase in VDR expression. In contrast, VDR protein expression was almost suppressed when endogenous Pit-1 protein was blocked using a Pit-1 siRNA. Using a reporter gene containing the VDR promoter (pGL3B-hVDR623), it was also observed that the up-regulation of VDR gene expression by Pit-1 was at the transcriptional level. By mapping the VDR promoter, a Pit-1 consensus sequence was found, located between –246 and –254 bp from the VDR transcription start site, and responsible for VDR transcriptional activation. Selective mutation of 5 bp in this sequence completely abolished the Pit-1-induced VDR transcription. In vitro gel retardation assays demonstrated that nuclear Pit-1 bound to a VDR fragment containing the Pit-1 response element but not to a VDR fragment containing the mutated Pit-1 site. In addition, using ChIP analysis, it was observed that Pit-1 binds to the VDR promoter in vivo. It has been suggested that Pit-1 activity is determined by a regulatory balance between N-CoR (nuclear receptor corepressor)/SMRT (silencing mediator of retinoid and thyroid hormone receptor), mouse Sin3 homolog A/B, and histone deacetylase corepressor complexes and CBP coactivator complexes (39) and that the POU domain of Pit-1 is crucial for recruitment of CBP (40). In accordance with these data, our findings also show that binding of Pit-1 to the VDR promoter recruits CBP, Ac-H4, and RNA Pol II. CBP recruitment by Pit-1 may lead to acetylation of histones and chromatin remodeling (as indicated by acetylation of histone H4) and thus facilitate entry of RNA Pol II, necessary for increased transcription (41).

The present results, together with the previously demonstrated Pit-1 transcriptional repression by VDR (34), suggest a negative regulatory feedback between VDR and Pit-1, in which Pit-1 stimulates VDR transcription and, conversely, VDR inhibits Pit-1 transcription. Negative feedback regulation between nuclear receptors (and their ligands) and transcription factors has been reported previously. For example, 1,25-(OH)2D3 inhibits transcription of ß-catenin, whereas ß-catenin can potentiate VDR transcription activity (23, 40). The regulatory relationship between VDR and Pit-1 could be involved in the physiological regulation of breast development. Notably, overexpression of Pit-1 in MCF-7 cells increases cell proliferation and raises both GH (29) and PRL (Ben, I., S. Seoane, and R. Perez-Fernandez, unpublished observation) levels. In addition, Pit-1 mRNA expression is significantly increased in breast carcinoma compared with normal breast (29). Both hormones GH and PRL have been implicated in the pathogenesis of breast cancer (43, 44, 45, 46). In contrast, 1,25-(OH)2D3 and its analogs have antiproliferative and proapoptotic effects (47, 48), and clinical trials have been performed with 1,25-(OH)2D3 analogs against some types of cancer (49, 50). Thus, we might speculate that the balance between the transcription factor Pit-1 (which stimulates cell proliferation, either directly or indirectly) and 1,25-(OH)2D3/VDR (which inhibits proliferation and induces differentiation) could be deregulated in some circumstances, with Pit-1 prevailing, leading to cell transformation.

To evaluate whether the Pit-1-dependent increase in VDR expression may in turn induce increased expression of 1,25-(OH)2D3-responsive genes, COS-7 cells (which express low VDR levels) were transfected with a reporter plasmid linked to the promoter of the mouse osteopontin/Spp-1 gene (a direct target of VDR) (51), containing two copies of a VDRE (52). Our results indicate that Pit-1 synergizes with 1,25-(OH)2D3/VDR to increase the transcriptional response of the Spp-1 gene. Synergisms between VDR and other transcription factors have been reported previously. Specifically, Kommagani et al. (21) demonstrated that overexpression of p63 in SaoS2 cells sensitizes the cells to 1,25-(OH)2D3 treatment by inducing VDR and therefore that p63 has a synergistic effect with 1,25-(OH)2D3/VDR on osteopontin expression. In the present study, however, when only the Pit-1 expression vector (i.e. without the VDR expression vector) was transfected into COS-7 cells and these were then treated with 1,25-(OH)2D3, no significant increase in Spp-1 transcriptional activity was observed, indicating that endogenous VDR protein is necessary for the synergistic 1,25-(OH)2D3/VDR-Pit-1-mediated Spp-1 gene transcription. However, this may also indicate a VDR-Pit-1 protein-protein interaction in the regulation of a 1,25-(OH)2D3 target gene. We therefore evaluated possible VDR-Pit-1 protein-protein interactions in the regulation of the VDR promoter by Pit-1. Total protein extracts from MCF-7 cells were first immunoprecipitated using an anti-Pit-1 antibody and then blotted using an anti-VDR antibody. VDR immunoreactivity was detected in the Pit-1-immunoprecipitated cell extract. To confirm in vivo that VDR binds to the Pit-1 protein, a ChIP analysis was performed. The results again show VDR binding to the VDR promoter in Pit-1-transfected MCF-7 cells but not in untransfected cells. 1,25-(OH)2D3 up-regulates VDR expression, probably to maintain constant levels of unoccupied receptor and amplify the effect of 1,25-(OH)2D3 (53, 54). Recently, Zella et al. (24) has described regulatory regions downstream of the VDR gene transcriptional start site, located within two introns, that confer 1,25-(OH)2D3 regulation to a minimal VDR gene promoter. It is generally accepted that VDR expression level is a key factor in the response to 1,25-(OH)2D3 in specific target cells, because responsiveness to 1,25-(OH)2D3 may differ depending on VDR level (55, 56). In the present experiments, however, Pit-1-mediated VDR gene transcription does not seem to have been mediated by 1,25-(OH)2D3/VDR, because 1,25-(OH)2D3 was not administered to the MCF-7 cells. In addition, the 1,25-(OH)2D3/VDR-responsive regulatory regions in the VDR promoter found by Zella et al. (24) are similar to classical VDREs, comprising two hexanucleotide half-sites separated by 3 bp (55), and are not present in the VDR fragment to which Pit-1/VDR is bound.

To explore in greater depth why VDR is present in this complex with Pit-1 and to evaluate whether VDR protein is necessary for the up-regulation of VDR transcription by Pit-1, we studied this regulation in several other cell lines, including EA.hy926 and COS-7 cells (with low levels of VDR gene expression compared with the high levels in MCF-7 cells) and HeLa cells (with moderate levels of VDR gene expression compared with MCF-7 cells). The results obtained suggest that the increased VDR gene transcription induced by Pit-1 is dependent on VDR protein levels. Thus, in both COS-7 and EA.hy926 cells, with low endogenous VDR levels, Pit-1 does not induce an increase in VDR transcription, either at mRNA or protein level, or in reporter assays. However, the Pit-1-induced increase in VDR transcription is rescued when VDR is overexpressed in COS-7 and EA.hy926 cells. These findings are supported by the in vivo results obtained using ChIP analysis, in which Pit-1 binding to the VDR promoter was observed in COS-7 and EA.hy296 cells only after cotransfection with both VDR and Pit-1 (in contrast to MCF-7 and HeLa cells, in which Pit-1 binding to the VDR promoter was observed after transfection with Pit-1 only). The present results differ from those obtained by other authors (22) using COS-7 cells: specifically, these authors found a significant increase in VDR reporter activity when only the ZEB transcription factor was transfected (i.e. without VDR cotransfection). This discrepancy may be attributable to the fact that these authors used a mouse VDR promoter that shows low sequence homology with the hVDR promoter (13); alternatively, the transformed COS-7 cell line they used may have had higher VDR expression levels.

Our findings seem to indicate that, depending on VDR expression levels, cells may respond to Pit-1 either with increased VDR expression (in cell types in which VDR protein levels are at a sufficient level to allow cooperation with Pit-1) or with no VDR expression response (in cell types in which VDR protein is absent or present only at a low level). Although in the present study the VDR transcriptional response to other regulators was not evaluated, we can speculate that the Pit-1/VDR relationship observed here may represent a general mechanism of VDR transcription regulation. Thus, in a 1,25-(OH)2D3 target cell in which VDR is necessary to mediate the 1,25-(OH)2D3 action, Pit-1 (and/or other transcriptional regulators?) may induce VDR transcription, because the cell has enough VDR protein for the VDR to cooperate with Pit-1 (and/or other transcriptional regulators?) and thus induce VDR transcription. In contrast, in other cell types not targeted by 1,25-(OH)2D3 and thus probably with low levels of VDR, the cell will not respond to Pit-1 (or to other transcriptional regulators?) despite the fact that the VDR gene (and its promoter) is present, because not enough VDR is expressed for cooperation with Pit-1. It remains unclear why some cells become responsive to Pit-1 or other transcription factors (thus inducing VDR expression, which leads in turn to additional VDR cooperation with these factors), whereas other cells are unresponsive. Other specific factors present in 1,25-(OH)2D3 target cells may perhaps be responsible for these differences in mechanisms of transcriptional response.

In conclusion, the present study has demonstrated that the transcription factor Pit-1 up-regulates VDR gene expression at the transcriptional level by binding to a region located between –254 and –246 bp from the VDR transcription start site. However, VDR transcriptional regulation by Pit-1 is dependent on the presence of VDR protein in a cell-type-specific manner: specifically, VDR up-regulation by Pit-1 appears to depend on the levels of VDR already present in the cell.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and Reagents
MCF-7 (human breast adenocarcinoma) and COS-7 (African green monkey kidney) cells were obtained from the European Collection of Cell Cultures (Salisbury, UK). HeLa (human cervix adenocarcinoma) cells were obtained from American Type Culture Collection (via LGC Promochem, Barcelona, Spain). The human endothelial cell line EA.hy926 was a kind gift from Dr. Oglesbee and Dr. Edgell. Stock culture was grown in 90-mm Petri dishes in DMEM supplemented with 10% fetal bovine serum [or 10% charcoal-stripped fetal calf serum when COS-7 cells were treated with 1,25-(OH)2D3], 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine in a 95:5 air/CO2 atmosphere at 37 C. 1,25-(OH)2D3 (Sigma, St. Louis, MO) was used at 100 nM.

Oligonucleotides, Plasmids, Site-Directed Mutagenesis, and Transfections
The hVDR expression vector pCMV-hVDR was a kind gift from L. P. Freedman. The hPit-1 expression vector, pRSV-hPit-1, was obtained from J. L. Castrillo. The promoter region of the hVDR gene (–601/+23) subcloned into the pGL3-basic vector (pGL3B-hVDR623) was obtained from A. Muñoz (20). The pGL3B-hVDR623 vector was mutated at one Pit-1 consensus binding site, positioned between –254 and –246 bp from the transcription start site of the hVDR gene. Site-directed mutagenesis was performed with the QuikChange kit (Stratagene, La Jolla, CA) under the conditions recommended by the manufacturer. The mutagenized oligonucleotide primer was as follows (mutagenized bases on the sense strand identified by lowercase letters): 5'-GCGGGTATCCGCACCTAgAtcttTCGACAACTCTGTCCC-3'. The newly constructed mutant plasmid (designated pGL3B-hVDR623mut) was verified by DNA sequence.

Transfections were performed in wells containing 0.6 µl of FuGene (Roche Molecular Biochemicals, Indianapolis, IN) and 350 ng total DNA [250 ng pGL3B-hVDR623, pGL3B-hVDR623mut, or pGL3-Basic vectors and 100 ng of Rous sarcoma virus galactosidase (pRSV-gal)]. The cells were cotransfected with 500 ng Pit-1 (pRSV-hPit-1) and/or VDR (pCMV-hVDR) expression vectors and/or pRc/RSV and pRc/CMV empty vectors (control cells). The cells were harvested in buffer (5x lysis buffer; Promega, Madison, WI), and luciferase activity was then measured. ß-Galactosidase activity was measured at 420 nm using o-nitrophenyl-ß-D-galactopyranoside as substrate.

To evaluate VDR mRNA and protein expression, MCF-7, COS-7, HeLa, and EA.hy926 cells were cultured in 90-mm Petri dishes and transfected with 1) 4 µg pRSV-hPit-1 construct or 2) 4 µg pRc/RSV (control) or pCMV-hVDR (positive control in the Northern blot) constructs. The cells were then incubated for 48 h. Total RNA isolation, RT-PCR, real-time PCR, Northern blotting, and Western blotting were then performed as described in what follows.

RNA Isolation and RT-PCR Analysis
Isolation of total RNA from the MCF-7, COS-7, HeLa, and EA.hy926 cell lines was done with the TRIzol reagent (Invitrogen, Barcelona, Spain) according to the instructions of the manufacturer. cDNA synthesis was performed as described previously (29). Samples were denatured at 94 C for 1 min, annealed at 58 C (Pit-1 and 18S) or 60 C (VDR) for 1 min, and extended at 72 C for 1 min, for a total of 30 cycles, with an extension step of 10 min at 72 C in the final cycle.

Primer sequences for VDR PCR amplification were as follows: primer A (5'-GACTTTGACCGGAACGTGCC-3') was a 20-mer corresponding to nucleotides 43–62 in exon 2 of VDR cDNA, and primer B (5'-CATCATGCCGATGTCCACAC-3') was an antisense 20-mer corresponding to nucleotides 251–279 in exon 3 of VDR cDNA. The PCR product obtained was 227 bp long. Primer sequences for Pit-1 PCR amplification were as follows: primer A, 5'-GTGTCTACCAGTCTCCAACC-3', a 20-mer corresponding to nucleotides 570–589 in exon 1 of Pit-1 cDNA; and primer B, 5'-ACTTTTCCGCCTGAGTTCCT-3', an antisense 20-mer corresponding to nucleotides 269–288 in exon 3 of Pit-1 cDNA. The PCR product obtained was 247 bp long. Human 18S was used as internal reference. Primer sequences were as follows: forward primer, 5'-GTAACCCGTTGAACCCCATT-3'; and reverse primer, 5'-CCATCCAATCGGTAGTAGCG-3'. The product obtained was 131 bp long.

Analysis of VDR Gene Expression by Real-Time PCR
Total RNA isolation from cultured cells, and cDNA synthesis, were performed as described above. VDR and 18S mRNA levels were quantified using real-time PCR in a fluorescent temperature cycler (LightCycler; Roche Molecular Biochemicals, Mannheim, Germany) as per the instructions of the manufacturer. The 20-µl amplification mixture contained 2 µl of RT reaction products plus MgCl2 at 4 mM, each primer at 0.5 µM, and 2 µl LightCycler DNA Master SYBR Green I mix (Roche Molecular Biochemicals). After initial denaturation at 94 C for 30 sec, reactions were cycled 40 times as follows: denaturation at 95 C for 2 sec, annealing at 60 C for 10 sec, and extension at 72 C for 15 sec. The amount of PCR products formed in each cycle was evaluated on the basis of SYBR Green fluorescence. At the end of each run, melting curve profiles were produced (cooling the sample to 68 C and heating slowly to 95 C, with continuous measurement of fluorescence) to confirm amplification of specific transcripts (data not shown). Cycle-to-cycle fluorescence emission readings were monitored and quantified using the second derivative maximum method of the LightCycler software package (Roche Molecular Biochemicals). VDR mRNA levels were normalized with respect to the 18S level in each sample.

Northern Blot Analysis
Total RNA was isolated from cultured cells with TRIzol reagent (Invitrogen). For Northern blot analysis, 30 µg of each RNA sample was separated on 1% agarose formaldehyde gels, transferred to a nylon membrane (Hybond-N+; GE Healthcare, Little Chalfont, UK), and hybridized with a 0.4-kb BstXI fragment isolated from the pCMV-hVDR construct labeled by random priming. Hybridizations were done at 42 C with 50% formamide, with an initial stringent wash at 65 C with 2x sodium saline citrate containing 0.1% SDS, and a second wash with 0.1x sodium saline citrate for 30 min at room temperature. The filters were dried and autoradiographed on Hyperfilm (GE Healthcare) with an intensifying screen at –80 C. A rat 18S ribosomal RNA oligonucleotide probe (GE Healthcare) was used to assess the amount and integrity of the total RNA loaded in each gel.

Immunoprecipitation and Western Blot Analysis
For immunoprecipitation assay, 1 µg primary antibody (Pit-1, rabbit polyclonal from Santa Cruz Biotechnology, Santa Cruz, CA) or control human IgG (Sigma) was added to the MCF-7 total protein extract in 500 µl immunoprecipitation buffer [50 mM Tris HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100] and incubated overnight with rotation at 4 C. Twenty-five microliters of GammaBind G Sepharose (GE Healthcare) were then added to the extract-antibody mixture, and it was further incubated with rotation at 4 C for 1 h. After centrifugation at 1500 x g at 4 C for 5 min, the supernatant was discarded and the coimmunoprecipitated VDR was detected by Western blotting with an anti-VDR monoclonal antibody (1:500; Chemicon, Temecula, CA).

Western blotting of Pit-1 from MCF-7 cells or VDR from MCF-7, COS-7, HeLa, or EA.hy926 cells was done as described previously (34). Briefly, 60 µg of total protein was subjected to 10% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane, which was blocked and washed. The blot was immunolabeled with a polyclonal anti-Pit-1 antiserum (Santa Cruz Biotechnology) or monoclonal anti-VDR antiserum (Chemicon) and with a monoclonal anti-ß-actin antiserum (Santa Cruz Biotechnology) and then incubated with alkaline-phosphatase-conjugated goat antirat VDR or antirabbit Pit-1 IgG (1:5000) and goat antimouse ß-actin IgG. Bound antibodies were detected using the enhanced chemiluminescence Western blotting analysis system (GE Healthcare), and visualized by placing the blot in contact with standard x-ray film according to the instructions of the manufacturer.

Pit-1 siRNA
The Ambion Silencer siRNA Construction kit (Ambion, Austin, TX) was used for in vitro synthesis of three sense and antisense RNAs and then double-stranded RNAs by annealing. The primer sequences comprised a 29-bp primer containing 8 bases complementary to the T7 promoter and 21 complementary to the target gene. Primer sequences were as follows: gene sequence of RNA interference (RNAi) site-1, 5'-AACCCCTTGTCTTTACAAGTTCCTGTCTC-3' (antisense) and 5'-AAAACTTGTAAAGACAAGGGGCCTGTCTC-3' (sense); gene sequence of RNAi site-2, 5'-AATTAAGTTAGGATACACCCACCTGTCTC-3' (antisense) and 5'-AATGGGTGTATCCTAACTTAACCTGTCTC-3' (sense); gene sequence of RNAi site-3, 5'-AATTGAATCTCGAGAAAGAAGCCTGTCTC-3' (antisense) and 5' AACTTCTTTCTCGAGATTCAACCTGTCTC-3' (sense); gene sequence of missense control (universal scrambled siRNA; Ambion), 5'-AAGCTTCATAAGGCGCATAGC-3'.

MCF-7 cells were seeded into 90-mm plates (106 cells per plate) and transfected using JetSI transfection reagent (Polyplus Transfections, Illkirch, France) for 48 h. A total of 20 nM Pit-1 siRNA or nontargeting siRNA (control) was used for transfection. After transfection, protein was isolated as described above.

Gel Mobility Shift Assays
Two double-stranded oligonucleotides, both containing 28-bp sequences corresponding to the –266/–238-bp region of the upstream response sequence of the hVDR promoter, containing the wild-type or the mutated form of the Pit-1 consensus binding site, were obtained by synthesis (wild type, VDR28, top strand, 5'-GTATCCGCACCTATAATCATCGACAACT-3'; mutated, VDR28mut, mutations indicated by lowercase letters, top strand, 5'-GTATCCGCACCTAgAtcttTCGACAACT-3'). A double-stranded 28-bp oligonucleotide containing the distal Pit-1 response element in the hGH promoter (from –131 to –104 bp with respect to the transcription start site of the hGH gene, top strand, 5'-AGGAGCTTCTAAATTATCCATTAGCACA-3') was used as control. In all cases, top strand and bottom strand were annealed and end labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase and then purified through a MicroSpin G-25 column. Gel mobility shift assays were performed as described previously (34). Briefly, 20,000 cpm of probe (10 fmol) was mixed with 1 µg MCF-7 nuclear extract [obtained as described by Andrews and Faller (56)] for 45 min at room temperature in buffer. In the supershift experiment, 1 µl anti-Pit-1 antibody (Santa Cruz Biotechnology) was added to the nuclear extract, and this mixture was first incubated for 15 min before addition of 20,000 cpm of probe and then incubated for an additional 30 min. Half of the reaction mixture (10 µl) was electrophoresed at 15 V/cm at 4 C on 10% polyacrylamide gel. After electrophoresis, the gel was dried and visualized by autoradiography.

ChIP Assays
ChIP assays were performed using the protocol of Upstate (Upstate, Charlottesville, VA) as described previously (34). Diluted soluble chromatin fractions were immunoprecipitated with 1 µg polyclonal anti-Pit-1 antibody (Santa Cruz Biotechnologies), polyclonal anti-CBP, anti-acetyl H4, monoclonal anti-RNA Pol II (Upstate), monoclonal anti-VDR (Chemicon), or control human IgG (Sigma). The histone-DNA crosslinks were reversed by 4 h incubation at 65 C. The DNA from these samples was extracted through phenol/chloroform and ethanol precipitated with 20 µg of glycogen. The DNA extracted was then dissolved in 30 µl of H2O. PCR was used to analyze the DNA fragments from ChIP assays. Five microliters of assayed DNA sample and 5 µl of input/start material were used in each 50-µl reaction. The PCR was run for 60 sec at 95, 60, and 72 C within each cycle, for 34 or 38 cycles in total.

The four pairs of VDR primers were as follows: (A) forward (–1522/–1504 bp from transcription initial site), 5'-CACGATGCTTTGGGCAAG-3' and reverse (–1157/–1137 bp), 5'-GTGCTAGAGCCCAGCAAATC-3', PCR product 385 bp long; (B) forward (–1157/–1137 bp), 5'-GATTTGCTGGGCTCTAGCAC-3' and reverse (–771/–751 bp), 5'-AGACCTGGAATTGTGGATGG-3', PCR product 405 bp long; (C) forward (–771/–751 bp), 5'-CCATCCACAATTCCAGGTCT-3' and reverse (–391/–372 bp), 5'-CTGACCAGGCCAGGACTTC-3', PCR product 395 bp long; (D) forward (–463/–446 bp), 5'-GGGATTTCCCATTCGTG-3' and reverse (–1/+18 bp), 5'-CGCCTTTTGACAAGCAGAG-3', PCR product 482 bp long. As positive control, the hGH promoter was used. This promoter contains two Pit-1 binding sites. The forward primer (–228/–206 bp from transcription initial site) was 5'-TGGCTGACACTCTGTGCA-3', and the reverse primer was (–47/–27 bp) 5'-TATACCCTGGCCCCTTCTCT-3'; the PCR product was 201 bp long.

Statistical Analysis
Each experiment was performed at least three times. Values are expressed as means ± SD. Means were compared by unpaired t tests or one-way ANOVA with the Tukey-Kramer multiple comparison test for post hoc comparisons. Statistical significance is taken to be indicated by P < 0.05.


   ACKNOWLEDGMENTS
 
We thank L. P. Freedman (Merck Research Laboratories, West Point, PA), J. L. Castrillo (Centro de Biología Molecular "Severo Ochoa," Madrid, Spain), and A. Munoz (Instituto de Investigaciones Biomédicas "Alberto Sols," Consejo Superior de Investigaciones Científicas-Universidad Autónoma, Madrid, Spain) for plasmids, and S. Oglesbee and C. J. Edgell (University of North Carolina Lineberger Comprehensive Cancer Center, Chapel Hill, NC) for the EA.hy926 cell line.


   FOOTNOTES
 
This work was supported by Fondo de Investigaciones Sanitarias, Ministerio de Sanidad Grant PI040518 and Xunta de Galicia Grant PGIDIT05PXIC20805PN (Spain).

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 24, 2007

Abbreviations: Ac-H4, Acetylated histone H4; CBP, CREB (cAMP response element binding protein) binding protein; ChIP, chromatin immunoprecipitation; h, human; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; Pit-1, pituitary transcription factor-1; Poly II, polymerase II; PRL, prolactin; RNAi, RNA interference; siRNA, small interference RNA; VDR, vitamin D receptor; VDRE, vitamin D response element.

Received for publication December 27, 2006. Accepted for publication April 20, 2007.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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Nuclear Receptors:   VDR
Coregulators:   CBP
Ligands:   Calcitriol



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C. J. Fabian
If Vitamin D Prevents Breast Cancer, How Does It Do It, and How Much Does It Take?
ASCO Educational Book, January 1, 2009; 2009(1): 71 - 74.
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