The Vitamin D Receptor Represses Transcription of the Pituitary Transcription Factor Pit-1 Gene without Involvement of the Retinoid X Receptor (original) (raw)

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1Department of Physiology, School of Medicine, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain

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1Department 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 Fisiologia, Facultad de Medicina, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.

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22 November 2005

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Samuel Seoane, Roman Perez-Fernandez, The Vitamin D Receptor Represses Transcription of the Pituitary Transcription Factor Pit-1 Gene without Involvement of the Retinoid X Receptor, Molecular Endocrinology, Volume 20, Issue 4, 1 April 2006, Pages 735–748, https://doi.org/10.1210/me.2005-0253
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Abstract

Pituitary transcription factor-1 (Pit-1) plays a key role in cell differentiation during organogenesis of the anterior pituitary, and as a transcriptional activator for the pituitary GH and prolactin genes. However, Pit-1 is also expressed in nonpituitary cell types and tissues. In breast tumors, Pit-1 mRNA and protein levels are increased with respect to normal breast, and in MCF-7 human breast adenocarcinoma cells, Pit-1 increases GH secretion and cell proliferation. We report here that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] administration to MCF-7 cells induces a significant decrease in Pit-1 mRNA and protein levels. By deletion analyses, we mapped a region (located between −147 and −171 bp from the transcription start site of the Pit-1 gene) that is sufficient for the repressive response to 1,25-(OH)2D3. Gel mobility shift and chromatin immunoprecipitation assays confirmed the direct interaction between the vitamin D receptor (VDR) as homodimer (without the retinoid X receptor), and the Pit-1 promoter, supporting the view that Pit-1 is a direct transcriptional target of VDR. Our data also indicate that recruitment of histone deacetylase 1 is involved in this repressive effect. This ligand-dependent Pit-1 gene inhibition by VDR in the absence of the retinoid X receptor seems to indicate a new mechanism of transcriptional repression by 1,25-(OH)2D3.

PITUITARY TRANSCRIPTION factor-1 (Pit-1), a member of the POU domain factor family (Pit-1, Oct-1, and Unc-86), plays a key role in cell differentiation during organogenesis of the anterior pituitary in mammals (1, 2) and as a transcriptional activator for pituitary gene transcription [i.e. transcription of the genes for GH, prolactin (PRL), and Pit-1 itself] (3, 4). Expression of Pit-1 transcripts and protein is closely regulated, and the presence of Pit-1 protein is correlated both temporally and spatially with activation of the GH gene during pituitary development (5). However, Pit-1 transcripts are also found in nonpituitary cell lines and tissues, such as human placenta (6, 7), hemapoietic lymphoid tissues (8), and breast (9, 10), although the role of Pit-1 in these tissues is not yet clear. Higher expression of Pit-1 has been observed in pituitary tumors in relation to normal pituitary and has been related to a possible role of this transcription factor in the pathogenesis of pituitary tumors (11–13). In addition, it has been suggested that Pit-1 expression may be specifically associated with increased cell proliferation (1, 14). In a recent study, it was demonstrated that, in human mammary gland, Pit-1 expression is significantly increased in breast carcinoma with respect to normal breast, and that overexpression of Pit-1 in the human breast adenocarcinoma cell line MCF-7, directly or by means of other mediators, significantly increases cell proliferation, and induces GH expression (10). Endogenous GH and PRL production have been implicated in mammary disorders, including the development and spread of breast cancer (15). However, the mechanisms of regulation of mammary Pit-1 are unknown.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], the most active metabolite of vitamin D, plays a well-known role in mineral (calcium and phosphorus) homeostasis. This hormone is also involved in a host of cell processes, including immune function, cell growth and differentiation, and secretion of hormones (for reviews, see Refs.16–18). Several studies have explored the role of 1,25-(OH)2D3 in cell growth and differentiation in normal and tumoral mammary gland (19, 20). The antiproliferative effects of 1,25-(OH)2D3 in breast have been linked to suppression of growth-stimulatory signals and potentiation of growth-inhibitory signals, leading to changes in cell-cycle regulators as well as to induction of apoptosis (21). Most of the known biological effects of 1,25-(OH)2D3 occur through the direct transcriptional regulation of specific target genes. These regulatory effects of 1,25-(OH)2D3 are mediated through its interaction with a high-affinity nuclear receptor known as the vitamin D receptor (VDR), triggering binding to specific DNA response elements (VDREs) in the promoter regions of target genes. VDR binds DNA predominantly as a heterodimer with a common partner, the retinoid X receptor (RXR), a nuclear receptor for 9-cis retinoic acid (9-cis RA); binding is to response elements typically composed of direct repeats of the hexameric sequence AGGTCA separated by three nucleotides, called DR3 response elements (22–24). However, some response elements for VDR are not of this type, and VDR can also regulate gene expression by binding to VDREs in different configurations (25–29). These non-DR3 response elements seem to be mainly involved in transcription repression. The association of VDR with some VDREs may induce recruitment of corepressor proteins (i.e. nuclear receptor corepressor, silencing mediator of retinoid and thyroid hormone receptor, and Alien) and associated histone deacetylase (HDAC) complexes that suppress transactivation (30–32). In the model of ligand-dependent activation, ligand binding to VDR promotes dissociation of these corepressor complexes and association of coactivator proteins, such as steroid receptor coactivator-1/p160, glucocorticoid receptor-interacting protein-1, and vitamin D receptor (VDR)-interacting proteins (23), inducing transcriptional activation.

A previous report has demonstrated that the mouse Pit-1 gene has a distal vitamin D-responsive enhancer element located at −10 kb from the initiation codon, suggesting a direct effect of vitamin D and its receptor on transcription regulation of this gene (33). In the present study, with the aim of investigating possible regulation of mammary Pit-1 transcription by 1,25-(OH)2D3, MCF-7 human breast adenocarcinoma cells were treated with 1,25-(OH)2D3 and Pit-1 mRNA and protein expression were then evaluated. Our data demonstrate that Pit-1 expression was significantly reduced in 1,25-(OH)2D3-treated cells. Additional experiments showed that 1,25-(OH)2D3 inhibits Pit-1 gene expression at the transcriptional level, through binding of VDR (without RXR) to a region located between −147 and −171 bp upstream of the Pit-1 transcription start site. This region is composed of a novel response element, a direct repeat separated by only 2 bp (DR-2). Repression of Pit-1 transcription by 1,25-(OH)2D3 is reverted by treatment with the histone deacetylase inhibitor, trichostatin A (TSA), suggesting that recruitment of histone deacetylase activity by VDR may partially explain its inhibitory effect on Pit-1 promoter activity. In fact, treatment of MCF-7 cells with 1,25-(OH)2D3 indicates that HDAC1 is bound to the Pit-1 promoter. These findings indicate a novel pathway of 1,25-(OH)2D3-mediated transcription repression, in which the common heterodimeric partner RXR is not directly involved.

RESULTS

Effects of 1,25-(OH)2D3 Administration and pCMVhVDR Overexpression on Pit-1 mRNA and Protein Levels in MCF-7 Cells

Pit-1 mRNA was quantified by Northern blotting. Administration of 100 nm of 1,25-(OH)2D3 alone (Fig. 1A, lane 3), or 100 nm of 1,25-(OH)2D3 plus transfection with pCMVhVDR (Fig. 1A, lane 4), in both cases induced a significant decrease in Pit-1 mRNA levels with respect to control cells [Fig. 1, A (lane 1) and B]. Basically identical results were obtained with RT-PCR (results not shown). Our finding that 1,25-(OH)2D3 plus VDR overexpression did not reduce Pit-1 mRNA levels any more than 1,25-(OH)2D3 alone may be attributable to high endogenous levels of VDR in MCF-7 cells. To test this possibility, we carried out Western blot analyses to assess endogenous VDR levels in MCF-7 and several other cell lines (COS-7, HeLa, and NIH3T3). The results show that VDR protein levels are indeed higher in MCF-7 cells than in the other cell types tested (Fig. 1C).

Fig. 1.

Pit-1 mRNA and Protein Expression Is Down-Regulated by 1,25-(OH)2D3 in MCF-7 Cells A, Northern blots of Pit-1 and 18S mRNA in MCF-7 cells. Upper panel, Pit-1 mRNA. Lane 1, Control (Pit-1 mRNA in nontreated MCF-7 cells). Lane 2, Positive control (MCF-7 cells transfected with 2 μg of pRSVhPit-1). Lane 3, MCF-7 cells treated with 100 nm of 1,25-(OH)2D3 for 48 h. Lane 4, MCF-7 transfected with 2 μg of pCMVhVDR and treated with 100 nm of 1,25-(OH)2D3 for 48 h. Lower panel, 18S mRNA, treatments as for upper panel. B, Relative Pit-1 mRNA expression in MCF-7 cells was calculated from the Pit-1/18S ratio obtained from densitometric readings in four independent experiments (**, P < 0.01; ***, P < 0.001, with respect to control cells). C, Western blots to detect VDR and Sp1 (used as loading control) in MCF-7 and several other cell lines. Lane 1, COS-7 cells. Lane 2, MCF-7 cells. Lane 3, NIH3T3 cells. Lane 4, HeLa cells. D, Western blots to detect Pit-1 and Sp1 (used as loading control) in nuclear extracts of MCF-7 cells. Lane 1, Rat pituitary Pit-1 protein (Santa Cruz Biotechnology; positive control). Lane 2, Untreated MCF-7 cells. Lane 3, MCF-7 cells transfected with 2 μg of pRSVhPit-1 expression vector. Lane 4, MCF-7 cells incubated with 100 nm of 1,25-(OH)2D3 for 48 h. Lane 5, MCF-7 cells transfected with 2 μg of pCMVhVDR expression vector and incubated with 100 nm of 1,25-(OH)2D3 for 48 h. One hundred micrograms of nuclear extract from MCF-7 cells were used for the analysis. The major 31- and 33-kDa immunoreactive bands are indicated by arrows.

Western blots of Pit-1 in nuclear extracts of MCF-7 cells transfected with pCMVhVDR plus 100 nm of 1,25-(OH)2D3, or of cells treated with 100 nm of 1,25-(OH)2D3 alone, are shown in Fig. 1D. Pit-1 was detectable in controls [200 ng of rat pituitary Pit-1 protein, positive control (Santa Cruz Biotechnology, Santa Cruz, CA) lane 1; and untransfected MCF-7 cells, lane 2], as well as in cells transfected with pRSVhPit-1 at 48 h, lane 3. We observed a clear decrease in Pit-1 protein levels, with respect to control cells, in cells treated with 100 nm of 1,25-(OH)2D3 and transfected with pCMVhVDR expression vector, or treated with 100 nm of 1,25-(OH)2D3 alone (lanes 5 and 4, respectively).

Effect of 1,25-(OH)2D3 Administration to MCF-7 Cells on the Transcriptional Activity of the 1.3-kb Pit-1 Promoter and Localization of the Suppressive Vitamin D Response Sequence in the Pit-1 Promoter

To evaluate the effect of vitamin D on the transcriptional activity of the Pit-1 promoter (pGL2B-hPit-11321), 1,25-(OH)2D3 was added to MCF-7 cell culture to a concentration of 100 mm, and we measured luciferase activity levels after incubation for 48 h. This treatment significantly suppressed Pit-1 promoter activity, by up to 25%, as compared with nontreated MCF-7 cells (Fig. 2). These results are consistent with the above-mentioned results of the effect of 1,25-(OH)2D3 on Pit-1 mRNA expression in Northern blotting analysis and RT-PCR, indicating that this 1.3-kb promoter fragment is necessary and sufficient for suppression of the Pit-1 gene transcription by 1,25-(OH)2D3. As a positive control, we used the pGL2-(Spp1)2 construct, which contains two Spp1/mouse osteopontin VDRE sites. 1,25-(OH)2D3 treatment of cells transfected with this construct led to a 4.5-fold increase in reporter activity (Fig. 2).

Fig. 2.

Deletion Analysis Identified a Vitamin D-Responsive Region in the Human Pit-1 Promoter The Pit-1 promoter fragments fused to the pGL2Basic vector (pGL2B), or the pGL2P-(Spp1)2 construct (containing two copies of Spp1/osteopontin VDRE, used as positive control), were transfected into MCF-7 cells using the FuGene reagent, and the cells were then cultured in the presence of 100 nm 1,25-(OH)2D3 or ethanol 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. Asterisks indicate significant differences (**, P < 0.01; ***, P < 0.001) with respect to normalized RLU calculated for ethanol-treated cells. SV40, Simian virus 40.

To locate the Pit-1 gene region involved in inhibition by 1,25-(OH)2D3, two deletion derivatives of the Pit-1 promoter fused to the LUC gene, pGL2B-hPit-1601 and pGL2B-hPit-1116, were used. These constructs were used to transfect MCF-7 cells in the presence or absence of 100 nm 1,25-(OH)2D3. This deletion analysis indicated that the response region is located within the 500-bp fragment −601/−101: as shown in Fig. 2, in cells transfected with pGL2B-hPit-1601 (containing the −601/−101 fragment), 1,25-(OH)2D3 treatment induced a significant reduction in luciferase activity with respect to nontreated cells; by contrast, transfection with pGL2B-hPit-1116 (containing the −101/+15 fragment) had no significant effect on the response to 1,25-(OH)2D3.

Glutathione-_S_-Transferase (GST)-human (h) VDR Binds to the −601/−71-bp Promoter Sequence of Pit-1 But Not to −71/+1 bp

In view of these results indicating that 1,25-(OH)2D3 inhibits Pit-1 transcription through an effect on the −601/−101 region of the Pit-1 promoter sequence, we next investigated whether the VDR binds to this region. Specifically, we used gel mobility shift assays to investigate VDR binding to two different Pit-1 fragments (Pit-1530 and Pit-172; see Fig. 3A) and to the Spp-1 VDRE (used as positive control); it is well known that purified GST-hVDR binds strongly to the VDRE derived from the Spp-1 gene (34), which comprises a direct repeat of the half-site 5′-GGTTCA-3′ with a 3-bp separation. As expected, purified recombinant GST-hVDR bound to the Spp-1 VDRE (Fig. 3B, lanes 2–4). The results also indicate binding of GST-hVDR to the Pit-1530 fragment (Fig. 3B, lanes 6–8), with a similar binding pattern to that for the Spp-1 VDRE. In contrast, GST-hVDR did not bind to the Pit-172 fragment (Fig. 3B, lanes 11–13). To confirm that the protein binding to the Pit-1530 fragment was GST-hVDR, we preincubated the GST-hVDR with an anti-VDR antibody: as shown in Fig. 3, lane 9, this treatment supershifted the Pit-1530 fragment in the gel mobility shift assay.

Fig. 3.

GST-hVDR Binds to the Human Pit-1 Promoter A, Human Pit-1 fragments and oligonucleotides used in the gel mobility shift assays. Pit-1530 and Pit-172 fragments were isolated from the pGL2B-hPit-1601 construct by digestion with _Kpn_I/_Nsp_I (Pit-1530) or _Nsp_I/_Bgl_II (Pit-172). The Pit-124 and Spp1 VDRE oligonucleotides were purchased from Sigma. B, GST-hVDR binds to the Spp1 VDRE (used as positive control) and to the Pit-1530 fragment, but not to the Pit-172 fragment. Each lane contains 20,000 cpm of labeled DNA. Twenty-five nanograms (lanes 2, 6, and 11), 50 ng (lanes 3, 7, and 12), or 100 ng (lanes 4, 8, and 13) of GST-hVDR were added to the different probes. For supershift assay, 100 ng of GST-hVDR was preincubated with 1 μl of anti-VDR antiserum (lane 9).

Localization of the Suppressive Vitamin D Response Sequence in the −171-bp to −147-bp Region of the Pit-1 Promoter

The results shown in Figs. 2 and 3 thus indicate that 1,25-(OH)2D3 inhibits Pit-1 transcription through binding of the VDR to a VDRE within the −601/−71 region of the Pit-1 promoter sequence. This region contains a hexameric direct repeat (ACTTGA, antisense strand) separated by 2 bp, i.e. a candidate DR2-type VDRE. We therefore synthesized a 24-bp oligonucleotide containing this repeat, Pit-124 (−171/−147; see Fig. 3A), which was cloned into the pGL3 promoter vector and used in transfection assays. As shown in Fig. 4, 1,25-(OH)2D3 treatment of cells transfected with pGL3P-hPit-124 led to a statistically significant reduction in luciferase activity, by up to 30%, with respect to the nontreated MCF-7 cells (Fig. 4A). To further test the functionality of the VDRE, this element was mutated in the context of the pGL3P-hPit-124. Specifically, two different mutations were introduced into the Pit-124 sequence: one affecting one base in each hemisite of the VDRE (pGL3P-hPit-124mut-1) and the other affecting two bases in each hemisite (pGL3P-hPit-124mut-2). These constructs were transfected into MCF-7 cells that were then treated with 1,25-(OH)2D3. Figure 4A shows that, whereas transfection of cells with mut-1 did not affect the inhibition of transcription induced by 1,25-(OH)2D3, transfection with mut-2 significantly impaired the inhibitory response.

Fig. 4.

Vitamin D-Mediated Repression of the Pit-1 Promoter Involves a −171/−147-bp (Pit-124) Fragment A, MCF-7 cells were transfected with the empty vector (pGL3Promoter, pGL3P), the pGL3P-hPit-124 construct, the Pit-124 fragment with a mutation of one base in each hemisite (pGL3P-hPit-124mut-1 construct), or the Pit-124 fragment with a mutation of two bases in each hemisite (pGL3P-hPit-124mut-2 construct), and then cultured with or without 100 nm of 1,25-(OH)2D3. After 48 h treatment with 1,25-(OH)2D3, transcription of the wild-type Pit-124 and the Pit-124mut-1 was repressed, whereas Pit-124mut-2 showed no signs of regulation by 1,25-(OH)2D3. Normalized RLU were calculated as for Fig. 2. Asterisks indicate significant differences (***, P < 0.001) with respect to normalized RLU calculated for ethanol-treated cells. Each value is the average of three different experiments. B, Gel mobility shift analysis of the Pit-124 element in the human Pit-1 promoter. A radiolabeled Pit-124 probe was incubated with increasing amounts of GST-hVDR (25, 50, or 100 ng, lanes 2–4, respectively). For supershift assay, 100 ng of GST-hVDR was preincubated with 1 μl of anti-VDR antiserum (lane 5). Increasing concentrations (10×, 100×, and 200×) of the unlabeled Pit-124 oligonucleotide (lanes 10–12, respectively) were used to compete the retardation caused by GST-hVDR in the mobility shift assay (lanes 7–9). Mutation of two bases in each hemisite (Pit-124mut-2) abolished in vitro DNA binding of GST-hVDR (lanes 18–20), whereas mutation of one base in each hemisite (Pit-124mut-1) did not modify the GST-hVDR binding profile (lanes 14–16) with respect to the wild-type Pit-124 VDRE. SV40, Simian virus 40.

Gel mobility shift assay showed that GST-hVDR is able to bind to radiolabeled Pit-124 (Fig. 4B, lanes 2, 3, and 4). After preincubation of the GST-hVDR with anti-VDR antibody, the band observed in the mobility shift assay was supershifted, confirming that the ligand was VDR (Fig. 4B, lane 5). The complexes were also competed out by a 100-fold molar excess of unlabeled 24-bp Pit-1 probe (Fig. 4B, lane 11). As shown in Fig. 4B, VDR binding was unaffected when Pit-124mut-1 was used in the assays (lanes 14–16), but completely abolished when Pit-124mut-2 (with mutation of two bases in each hemisite) was used (lanes 18–20).

To provide further support for the hypothesis that VDR regulates endogenous Pit-1 transcription, a chromatin immunoprecipitation (ChIP) assay was carried out to confirm in vivo interaction of VDR on the human Pit-1 promoter (Fig. 5A). As shown in Fig. 5B, the ChIP PCR product was detected with the VDR antibody in 1,25-(OH)2D3 MCF-7 cells using proximal primers (A-B), but not using distal primers (C-D). This suggests that endogenous VDR specifically binds to the regulatory region (−171/−147) of the Pit-1 promoter. This is consistent with the in vitro results of our gel retardation assays.

Fig. 5.

1,25-(OH)2D3 Treatment of MCF-7 Cells Induces VDR Binding to the Pit-1 Promoter A, Diagram of the human Pit-1 gene promoter showing the location of the primers used in the ChIP assay. B, Soluble chomatin prepared from MCF-7 cells treated (+) or not treated (−) with 1,25-(OH)2D3 was immunoprecipitated with an anti-VDR antibody or control IgG. The immunoprecipitated DNA was amplified by PCR using primers (A-B) that amplified a 200-bp region of the Pit-1 promoter (−219/−18) that includes the down-regulatory VDR sequence (−171/−147), or using primers (C-D) that amplified a different 280-bp region of Pit-1 promoter (−476/−197) not containing this sequence.

VDR Binds to the Pit-1 Promoter in the Absence of RXR

To evaluate whether the vitamin D response element in the Pit-1 promoter is involved in transcription repression in the absence of RXR, gel shift assays using GST-hRXR were carried out. Figure 6A shows that RXR alone did not bind either the Pit-124 fragment (lane 8) or the Pit-1530 fragment (lane 15), nor did it potentiate the binding of VDR (lanes 3–7 and lane 16), suggesting that RXR is not involved in VDR-mediated transcription repression. In contrast, using Pit-124mut-1 as probe (i.e. one base change in the third position, A to G, in each hemisite of the VDRE), binding of both GST-hRXR alone (Fig. 6A, lane 11) and GST-hRXR plus GST-hVDR (Fig. 6A, lane 12) was observed. Furthermore, using an MCF-7 nuclear extract (1 μg) and labeled Pit-124, we observed a band that was supershifted with the anti-VDR antibody (Fig. 6, lane 3), but not with the anti-RXR antibody (Fig. 6, lane 4).

Fig. 6.

GST-hVDR Binds to the Pit-1 VDRE as a Homodimer, without RXR A, GST-hVDR and GST-hRXR heterodimer binds to Pit-124 mut-1, but only GST-hVDR as homodimer binds to Pit-124 and Pit-1530. In each series, 50 ng (lanes 2–7) or 100 ng (lanes 10, 12, 14, and 16) of GST-hVDR were incubated alone or together with 100 ng (lanes 11, 12, 15, and 16) or increasing concentrations of GST-hRXR (25, 50, 100, 150, and 200 ng, lanes 3–8, respectively), and then mixed with the Pit-124, Pit-124 mut-1, or Pit-1530 probe. Lanes 1, 9, and 13 contain the probe alone. B, Nuclear extract (1 μg) obtained from MCF-7 cells was incubated with 1 μl of anti-VDR antibody (lane 3) or 1 μl of anti-RXR antibody (lane 4) and 15 min later mixed with the Pit-124 probe. C, The transcription repression effect of 1,25-(OH)2D3 is not modified by 9-cis RA. MCF-7 cells were transfected with pGL2Basic, pGL2B-hPit-1601, pGL3Promoter, or pGL3P-hPit-124, and were then treated with 100 nm 1,25-(OH)2D3 and/or 1 μm 9-cis RA. Relative luciferase activity was calculated as the ratio of luciferase activity in vitamin D-treated cells to that in the corresponding control cells. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; and ***, P < 0.001) with respect to relative luciferase activity calculated for cells transfected with pGL2B or pGL3P.

In addition, MCF-7 cells transfected with pGL3P-hPit-124 and treated with 9-cis RA alone (1 μm) did not down-regulate transcription of Pit-1 with respect to control cells (Fig. 6C). The transcription repression response induced by 1,25-(OH)2D3 plus 9-cis RA (1 μm) did not differ significantly from that induced by 1,25-(OH)2D3 alone (67.5 ± 3.7% vs. 66.1 ± 0.6%) (Fig. 6C). Using a larger fragment of the Pit-1 promoter (pGL2B-hPit-1601), the transcriptional response of transfected MCF-7 cells treated with 9-cis RA (1 μm) was not significantly modified with respect to that obtained with pGL3P-hPit-124 (97.1 ± 14.5% vs. 95.4 ± 0.5%). Likewise, cells transfected with the pGL2B-hPit-1601 construct and treated with 1,25-(OH)2D3 plus 9-cis RA (100 nm and 1 μm, respectively) did not show a significantly modified transcription repression response to 1,25-(OH)2D3 (69.2 ± 9.5% and 73.8 ± 8.5%, respectively).

Effects of Histone Acetylation on 1,25-(OH)2D3-Mediated Transcription Repression

To assess whether or not histone deacetylase activity is involved in the VDR-mediated repression of the Pit-1 gene, we investigated whether TSA, a potent inhibitor of histone deacetylase, affects 1,25-(OH)2D3-induced promoter repression. The effects of 1,25-(OH)2D3 were simultaneously assayed in MCF-7 cell cultures with or without 100 nm TSA. As expected, in the absence of TSA, 1,25-(OH)2D3 addition inhibited Pit-1 activity (Fig. 7A). Treatment with TSA (100 nm) alone potentiated Pit-1 promoter activity, with a 5-fold increase in luciferase expression. However, 1,25-(OH)2D3 addition to TSA-treated cultures did not significantly reduce this TSA-induced promoter activity. Thus, our results suggest that recruitment of histone deacetylase activity by VDR may be partially responsible for its inhibitory effect on Pit-1 promoter activity.

Fig. 7.

Histone Deacetylase Activity Is Involved in 1,25-(OH)2D3-Induced Pit-1 Gene Repression A, Treatment of MCF-7 cells with a histone deacetylase inhibitor, TSA, inhibits the repressive effect of 1,25-(OH)2D3 on Pit-1 transcription. MCF-7 cells were transfected with pGL2B-hPit-11321 and 12 h later incubated for 48 h with 100 nm 1,25-(OH)2D3 and/or 100 nm TSA. Luciferase activity was measured in relative luciferase units (RLU) and normalized by reference to the estimated β-galactosidase activity in each reaction mixture. B, Soluble chromatin prepared from MCF-7 cells treated (+) or not treated (−) with 1,25-(OH)2D3 was immunoprecipitated with anti-HDAC1 or anti-HDAC2 antibodies or control IgG. The immunoprecipitated DNA was amplified by PCR using primers A-B (as in legend of Fig. 5). n.s., Not significant.

To further investigate the involvement of HDAC proteins in 1,25-(OH)2D3-mediated Pit-1 gene repression, we next carried out ChIP assays using specific antibodies for HDAC1 and HDAC2. These proteins belong to class I of the HDAC family: the proteins of this class have been shown to be associated with certain corepressors, and thus to repress gene expression. As shown in Fig. 7B, treatment of MCF-7 cells with 1,25-(OH)2D3 induced specific recruitment of HDAC1, but not HDAC2, by the Pit-1 promoter. Thus, our findings indicate that repression of the Pit-1 gene by the 1,25-(OH)2D3-VDR complex is mediated by HDAC1 chromatin deacetylation.

DISCUSSION

Since 1988, when the pituitary transcription factor Pit-1 was identified (35, 36) and confirmed as a transcriptional activator of the PRL and GH genes (37, 38), numerous studies have demonstrated a key role of Pit-1 in pituitary function. In addition, interactions between Pit-1 and nuclear receptors have been demonstrated in the pituitary and extrapituitary regulation of gene expression. For example, Pit-1 acts synergically with thyroid hormone and retinoic acid receptors (39, 40) to increase GH gene expression. In addition, simultaneous binding of VDR/RXR and Pit-1 to the PRL promoter facilitates promoter occupancy and is thus involved in transcriptional activation of the PRL gene (41). Conversely, it has been also reported that thyroid hormones decrease Pit-1 expression in pituitary GH4C1 cells by interference with other regulators of transcription (42), and that VDR may act as a repressor of Pit-1 signaling in several cell lines, via a direct protein-protein interaction between VDR and Pit-1 (43). Although Pit-1 is mainly produced in the pituitary gland, Pit-1 expression has also been demonstrated in extrapituitary cell types and tissues, including human breast (9, 10). In human breast, both PRL and GH are expressed, suggesting that Pit-1 may be involved in mammary GH and PRL regulation, as in the pituitary. A recent study from our group (10) has shown that Pit-1 increases both mRNA and protein levels of GH in mammary tissues, supporting this hypothesis at least for GH.

However, although transcription of the Pit-1 gene is subject to multiple mechanisms of control, indicated by the complex regulatory motifs within the Pit-1 enhancer (12), there have been few reports of direct transcriptional regulation of Pit-1 by nuclear receptors. One of these regulatory motifs has been described for 1,25-(OH)2D3 by Rhodes et al. (33) in the promoter of the mouse Pit-1 gene. This site, which confers inducibility by 1,25-(OH)2D3, is comprised of a direct repeat of the sequence AGTTCA separated by 4 bp, located about 10 kb 5′ of the Pit-1 transcription initiation site. According to Rhodes et al., other investigators, using an oligonucleotide containing the DR4-type element located in the mouse Pit-1 gene, have also demonstrated that heterodimeric RXR and VDR complexes induce a stimulatory response, at least as effective as that induced by DR3-type VDREs. Moreover, it has been suggested that 5′-flanking sequences of the downstream and upstream motif of DR4-type response elements should be considered integral parts of the response element (44, 45). In view of these data, and given the known interactions between Pit-1 and VDR, the present study aimed to investigate whether treatment with 1,25-(OH)2D3 regulates Pit-1 gene transcription in an extrapituitary cell line (MCF-7, human breast adenocarcinoma cells).

First, we treated MCF-7 cells with 1,25-(OH)2D3, and evaluated Pit-1 expression. Surprisingly, and in contrast to results obtained by Rhodes et al. (33), we found that treatment with 1,25-(OH)2D3 induced a significant decrease in Pit-1 mRNA and protein levels, suggesting that this hormone inhibits Pit-1 gene transcription. To evaluate whether the down-regulatory effect of 1,25-(OH)2D3 on Pit-1 mRNA and protein levels acts at the transcriptional level, we did experiments using successive promoter truncations of the Pit-1 gene. The results obtained demonstrate that a region located between −601 and −71 bp from the transcription start site is sufficient to enable repression by 1,25-(OH)2D3. We therefore explored the possibility that this transcriptional repression of the Pit-1 gene may involve binding of the VDR to some VDRE in the sequence. We found a vitamin D-responsive sequence located between −147 and −171 bp from the transcription start site, comprising an imperfect DR2 motif (antisense strand, 5′-ACTTCA GT AGTTCA-3′) that resembles the canonical VDRE responsible for vitamin D-induced up-regulation of genes (the hexameric AGGTCA motif arranged as direct repeats with 3-bp separation, DR3). When the putative Pit-1 VDRE was subcloned into the pGL3-promoter vector and treated with 1,25-(OH)2D3, a significant inhibition of luciferase activity was observed, suggesting that vitamin D represses Pit-1 transcription through a specific element located in the Pit-1 promoter. In addition, mutation of this element in the Pit-1 promoter abolished the inhibitory response to vitamin D, demonstrating that this element is functional. Furthermore, we demonstrated with VDR ChIP assay that VDR binds in vivo to the endogenous Pit-1 promoter (containing the VDRE site). Gel mobility shift assays indicated that VDR, i.e. without RXR, binds to this sequence as homodimer. Thus, the contrasting results obtained in the present study with respect to the previous studies mentioned above may be attributable a) to the fact that we used an in vivo approach, i.e. whole MCF-7 cells (which express Pit-1) were treated with 1,25-(OH)2D3 to evaluate Pit-1 expression, and b) to the fact that we used a proximal Pit-1 promoter (−1380 bp from the transcription start site) to locate the response region. The above-mentioned studies used only distal sequences (0.5 kb containing the DR4 element) located approximately −10 kb from the transcription start site, not the complete Pit-1 gene. Thus, it seems that transcription of Pit-1 in MCF-7 cells is inhibited by 1,25-(OH)2D3, and that this effect is mediated by a negative proximal VDRE. However, we cannot rule out the possibility that under some circumstances 1,25-(OH)2D3 may enhance Pit-1 transcription through binding of VDR/RXR heterodimers to isolated sequences in the distal region of the Pit-1 promoter.

VDR is a ligand-dependent transcription factor that down-regulates the expression of genes either by binding to negative VDREs or by antagonizing the action of certain transcription factors (for review see Ref.23). Moreover, depending on the nature of the response element, it may determine the type of VDR complex assembled on it. For example, as mentioned above, RXR-VDR heterodimers bind effectively to all natural DR3 motifs (AGGTCA), whereas VDR monomers or homodimers bind preferentially to response elements containing PuGTTCA half-sites; thus, it has been suggested that the T/A base pair at the third position of each half-site, opposite a G/C pair, is of primary importance for homodimeric hVDR recognition (34). This seems to be the case for the sequence found in the Pit-1 promoter. Specifically, in the absence of RXR, purified GST-hVDR protein incubated with the labeled DR2 oligonucleotide (including the Pit-1 VDRE) gave two bands in gel mobility shift assay, suggesting that GST-hVDR bound to the DR2 sequence as homodimer. In contrast, when GST-hRXR protein was added in the gel shift assay (i.e. RXR alone, or GST-hRXR plus GST-hVDR) neither binding of RXR to Pit-1 VDRE nor increased affinity of the VDR for the Pit-1 VDRE were observed. Our data obtained using MCF-7 nuclear extract and specific antibodies to VDR and RXR support these results. In this connection, using PCR-mediated random site selection, it has been demonstrated that the binding of RXR as a homodimer (1/81) or RXR-VDR as a heterodimer (1/152) to DR2-type motifs is very infrequent (46). DR2-type sequences have been reported to be responsive to binding by RXR-retinoic acid receptor (RAR) heterodimers (47–51). In DR2-type sequences, Polly et al. (52) found that VDR-mediated repression of retinoid signaling in the human TNFαR1 gene is induced by VDR sequestering either RXR or RAR from the RXR-RAR complex. However, to our knowledge, there have been no reports of DR2-type sequences for direct VDR-mediated negative transcription. In addition, in the present study, neither treatment of MCF-7 cells with 9-cis RA alone nor with 9-cis RA plus 1,25-(OH)2D3 modified the transcriptional response of cells transfected with Pit-1 promoter linked to the LUC reporter gene, by comparison with the response of transfected cells with 1,25-(OH)2D3 alone. Thus, it seems that at least in MCF-7 cells vitamin D down-regulates Pit-1 gene transcription directly via the VDR, not through interference with or in collaboration with other nuclear receptors like RXR.

In the present study, we also found that TSA, a potent histone deacetylase inhibitor, induces a significant increase in Pit-1 gene transcription that is not reduced by 1,25-(OH)2D3. This suggests that recruitment of histone deacetylase activity by VDR may partially explain its inhibitory effect on Pit-1 promoter activity. In fact, when MCF-7 cells were treated with 1,25-(OH)2D3 we observed, using ChIP assays, that HDAC1 but not HDAC2 is bound to the Pit-1 promoter. HDAC1 only displays activity within a complex of proteins [for example, SIN3 homolog A (Sin3A)], which are necessary to modulate its deacetylase activity and to enable it to bind DNA (53, 54). It has been demonstrated (55) that the association of HDAC corepressor complex components (i.e. nuclear receptor corepressor, HDAC2, and Sin3A) results in 1,25-(OH)2D3-induced transrepression of the 1α-hydroxylase gene. Thus, we can speculate that the 1,25-(OH)2D3-dependent repression of Pit-1 gene transcription may be associated with recruitment of the HDAC1/Sin3A complex by VDR bound to the VDRE of the Pit-1 promoter.

Repression of transcription of Pit-1 by vitamin D in human breast adenocarcinoma cells may have important physiological and/or pathophysiological consequences. It has been demonstrated that Pit-1 antisense oligonucleotides not only block GH and PRL transcription, but also inhibit [3H] thymidine incorporation by somatotroph and lactotroph cell lines, suggesting that Pit-1 may regulate DNA replication and cell proliferation (1). At the extrapituitary level, Pit-1 expression is specifically associated with cell proliferation in the human myeloid leukemic cell line HL-60 (14), supporting the idea that one of the functions of nonpituitary Pit-1 may be the control of cell proliferation. Pit-1 is expressed in human breast where it regulates endogenous GH secretion (10). GH and PRL have been implicated in the pathogenesis of breast cancer (for reviews see Refs.15 and 56–58); conversely, vitamin D and its analogs have antiproliferative and proapoptotic effects in a number of cell types, including breast cancer cells (for review see Ref.19). Thus, we might speculate that inhibition of Pit-1 by vitamin D may reduce the increase in proliferation induced by this transcription factor. Although this hypothesis cannot be confirmed on the basis of the present experimental data, our results indicate that in a human extrapituitary cell line, MCF-7, 1,25-(OH)2D3 exerted its suppressive effect on Pit-1 gene transcription through a direct effect on the Pit-1 promoter, via binding of VDR (without involvement of RXR) to an imperfect DR2-type VDRE located between positions −147 and −171 (antisense strand, 5′-ACTTCA GA AGTTCA-3′) with respect to the transcription start site.

MATERIALS AND METHODS

Cell Culture and Reagents

MCF-7 human breast adenocarcinoma cells were obtained from the European Collection of Cell Cultures (Salisbury, UK). Stock culture was grown in 90-mm Petri dishes in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mml-glutamine in a 95:5 air-CO2 atmosphere at 37 C. Twelve to 24 h before transfection, cells were cultured in DMEM containing 10% charcoal-stripped fetal calf serum. A total of 75 × 103 cells per well were seeded in six-well dishes and allowed to attach overnight. Crystalline 1,25-(OH)2D3 was a kind gift from Cristina Badia (Roche-Pharma, Madrid, Spain). 9-cis RA and TSA were obtained from Sigma-Aldrich (St. Louis, MO).

Oligonucleotides, Plasmids, and Transfections

The promoter region of the human Pit-1 gene (−1321/+1; pGL2B-hPit-11321) and PCR-generated 5′ deletion sequences (−601/+1, pGL2B-hPit-1601; and −102/+15, pGL2B-hPit-1116) subcloned into pGL2Basic vector were obtained from M. Delhase (59). VDR expression vector (pCMVhVDR), the pGL2P-(Spp1)2 construct containing two copies of the mouse Spp1 gene VDRE (positive control), pGEX-3X-VDR, and pGEX-2T-RXR were gifts from L. P. Freedman (Merck Research Laboratories, West Point, PA) and R. Evans (The Salk Institute for Biological Studies, La Jolla, CA). The Pit-1 expression vector pRSVhPit-1 was obtained from J. L. Castrillo (Centro de Biologia Molecular “Severo Ochoa,” Madrid, Spain).

A double-stranded oligonucleotide containing a 24-bp sequence corresponding to the −171/−147 region of the upstream response sequence of the human Pit-1 promoter was synthesized with a _Kpn_I or _Bgl_II site (italicized below) at each end. The top strand (5′-_C_GGTCTGAACTACTGAAGTTGTC_A_-3′) and the bottom strand (3′-_CATGG_CCAGACTTGATGACTTCAACAG_TCTAG_-5′) were annealed and subcloned into the _Kpn_I and _Bgl_II site of the pGL3Promoter vector (named pGL3P-hPit-124). Binding sites containing direct repeats separated by 2-bp (DR2) are underlined. Similarly, two types of double-stranded mutated oligonucleotides of the above-mentioned 24-bp sequence were also inserted into the same vector to obtain the mutated upstream response sequence constructs pGL3P-hPit-124mut-1, and pGL3P-hPit-124mut-2. The top strands of pGL3P-hPit-124mut-1 and pGL3P-hPit-124mut-2 are as follows, with mutations indicated by boldface: pGL3P-hPit-124mut-1, 5′-CGGTCTGAGCTACTGAGGTTGTCA-3′; pGL3P-hPit-124mut-2, 5′-CGGTCTTTACTACTTTAGTTGTCA-3′. The nucleotide sequences of all the cloned inserts were confirmed by sequencing.

Cells were incubated for 48 h with 100 nm TSA and/or with 100 nm of crystalline 1,25-(OH)2D3 diluted in ethanol and/or with 1 μm 9-cis RA in hormone-depleted medium. Transfections were carried out in wells containing 0.3 μl of FuGene (Roche Molecular Biochemicals, Indianapolis, IN) and 300 ng of total DNA, 200 ng of the corresponding hPit-1 promoter fused to the luciferase vector, and 100 ng of Rous sarcoma virus galactosidase (pRSV-gal). The cells were harvested in buffer (5× lysis buffer; Promega, Madison, WI), and luciferase activity was then measured. β-Galactosidase activity was measured at 420 nm using _o_-nitrophenyl-β-d-galactopyranoside as substrate.

To evaluate Pit-1 mRNA and protein expression, MCF-7 cells were 1) treated with 100 nm of crystalline 1,25-(OH)2D3; 2) treated with 100 nm of crystalline 1,25-(OH)2D3 and transfected with 2 μg of pCMVhVDR construct; or 3) transfected with 2 μg of pRSVhPit-1 construct (used as positive control). The cells were then incubated for 48 h. Total RNA isolation, Northern blotting, and Western blotting were then performed as described below.

Northern Blot Analysis

Total RNA was isolated from cultured cells with TRIzol reagent (Invitrogen, Carlsbad, CA). For Northern blot analysis, 30 μg of each RNA sample were separated on 1% agarose formaldehyde gels, transferred to a nylon membrane (Hybond-N+), and hybridized with a 0.9-kb _Hin_dIII/_Not_I fragment isolated from the pRSVhPit-1 construct labeled by random priming. Hybridizations were done at 42 C with 50% formamide, and the more stringent wash was done at 65 C with 2× saline sodium citrate (SSC) [containing 0.1% sodium dodecyl sulfate (SDS)], and 0.1× SSC for 30 min at room temperature. The filters were dried and exposed to Hyperfilm (Amersham Biosciences, Barcelona, Spain) with an intensifying screen at −80 C. A rat 18S ribosomal RNA oligonucleotide probe (Amersham Biosciences) was used to assess the amount and integrity of total RNA loaded in each gel. Final results were obtained by densitometric scanning of the x-ray films with a Bio-Rad (Hercules, CA) video densitometer coupled to a computer running the Analysis 1 D program.

Western Blot Analysis

Western blotting of Pit-1 was performed using nuclear extract [obtained as described by Andrews and Faller (60)] from MCF-7 cells. Briefly, 100 μg of nuclear extract was subjected to 12% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane, which was blocked and washed. The blot was immunolabeled with a rabbit polyclonal anti-Pit-1 antiserum (Santa Cruz Biotechnology) and rabbit polyclonal anti-Sp1 antiserum (Santa Cruz Biotechnology), as nuclear loading control, then incubated with alkaline-phosphatase-conjugated goat antirabbit IgG (1:5000). Bound antibodies were detected using the disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate (CSPD) alkaline phosphatase substrate (Tropix; PE Biosystem, Bedford, MA), and visualized by placing the blot in contact with standard X-ray film according to the manufacturer’s instructions.

Western blotting for detection of VDR and Sp1 in the MCF-7, COS-7 (African green monkey kidney), HeLa (human epithelial carcinoma), and NIH3T3 (mouse embryonic fibroblast) cell lines was performed as follows. Cells were lysed at 4 C in 1 ml of lysis buffer [50 mm HEPES (pH 7.5), 150 mm NaCl, 5 mm EGTA, 1.5 mm MgCl2, 1% SDS, 10% glycerol, 1% Triton X-100, 10 mm sodium orthovanadate, 4 mm phenylmethylsulfonyl fluoride (PMSF), and 50 μg/ml aprotinin] and sonicated. The cell lysate was then centrifuged at 17,000 × g for 15 min at 4 C. The resulting supernatant was collected, and protein concentration determined by the Bradford method. A lysate supernatant containing 100 μg of total protein from MCF-7, COS-7, HeLa, and NIH3T3 cells was then resuspended in 2× SDS-sample buffer [50 mm Tris-HCl (pH 6.8), 50% glycerol, 2% SDS, 2% β-mercaptoethanol, and bromophenol blue], and boiled for 5 min. The samples were subjected to 10% SDS-PAGE. Proteins were transferred for 2 h at 4 C to nitrocellulose membranes, which were blocked with 0.1 g of casein in PBS containing 0.1% Tween 20 (PBST) for 2 h at room temperature. The membranes were then immunolabeled for 2 h at room temperature with the monoclonal anti-VDR antiserum (1:500) (Chemicon International, Temecula, CA). After five washes for 5 min each in PBST, the membranes were incubated with alkaline-phosphatase-conjugated goat antirat IgG (1:5000) in the presence of CSPD as substrate for 1 h at room temperature. After washing again in PBST (5 × 5 min), immunolabeling was detected by placing the blot on standard x-ray film according to the manufacturer’s instructions (Tropix). To confirm that equivalent amounts of total protein were added to each well, membranes were stripped by incubation in 0.2 m glycin, pH 2.5, containing 0.1% SDS and 1% Tween 20 at room temperature for 1 h, and then re-probed with an anti-Sp1 rabbit polyclonal antiserum (1:500; Santa Cruz Biotechnology).

Overexpression and Purification of GST-hVDR and GST-hRXR Fusion Proteins and MCF-7 Nuclear Extract

The GST-hVDR and GST-hRXR fusion proteins were overexpressed as described previously (61). Briefly, 500 ml of bacterial culture expressing the recombinant GST fusion protein were grown at 37 C to an OD600 of 0.3, at which time the temperature was reduced to 20 C. Cells were induced by the addition of 0.1 mm isopropyl-β-d-thiogalactopyranoside at OD600 0.6. After 3.5 h, bacteria were collected by centrifugation and resuspended in 5 ml of lysis buffer (PBS containing 0.5 mm PMSF, 0.5 mg/ml leupeptin, and 1 mm dithiothreitol), sonicated, and centrifuged. Soluble extracts were incubated with glutathione-Sepharose matrix (Amersham Biosciences) for 30 min at 4 C before washing three times in lysis buffer. The amount of protein immobilized on beads was estimated by SDS-PAGE with Coomassie blue staining, by comparison with a titration of BSA.

Nuclear extract from MCF-7 cells, used for gel retardation assays, was obtained as described by Andrews and Faller (60).

Gel Mobility Shift Assays

Gel mobility shift assays were performed as previously described (34, 61). The DNA probes (Pit-1530, Pit-172) were isolated (see Fig. 3A) by digesting the plasmid pGL2B-hPit-1601 with _Kpn_I and _Nsp_I (Pit-1530) or _Nsp_I and _Bgl_II (Pit-172), then gel-purifying the fragments and end-labeling with γ-[32P] ATP using T4 polynucleotide kinase. The Spp-1/osteopontin vitamin D response element (5′-GATCCACAAGGTTCACGAGGTTCACGTCTG-3′) and the Pit-124 oligonucleotide (5′-AGGTCTGAACTACTGAAGTTGTCA-3′) (purchased from Amersham Biosciences) were also end-labeled with γ-[32P] ATP using T4 polynucleotide kinase, then purified through a MicroSpin G-25 column. For each gel mobility shift assay, 20,000 cpm of probe (10 fmol) was mixed with increasing amounts of bacterially expressed GST-hVDR and/or GST-hRXR, and/or 1 μg of MCF-7 nuclear extract, for 45 min at room temperature in a buffer consisting of 20 nm Tris (pH 7.9), 1 mm EDTA (pH 7.9), 10% glycerol, 0.05% Nonidet P-40, 50 mm KCl, 1 mm dithiothreitol, and 1 μg poly(deoxyinosine:deoxycytosine). In the supershift experiments, 1 μl of anti-VDR antibody (Chemicon International) or 1 μl of anti-RXR antibody (Santa Cruz Biotechnology) was added to the protein (GST-hVDR or GST-hRXR or nuclear extract), and this mixture was then incubated for 15 min before addition of 20,000 cpm of probe followed by incubation for a further 30 min. Increasing concentrations (10×, 100×, and 200×) of the unlabeled Pit-124 oligonucleotide were used to compete the retardation caused by GST-hVDR in mobility shift assay. Half of the reaction mixture (10 μl) was electrophoresed at 15 V/cm at 4 C on 5%–10% polyacrylamide gels. After electrophoresis, gels were dried and exposed to x-ray film.

ChIP Assays

ChIP assays were performed using the Upstate protocol (Upstate, Charlottesville, VA). MCF-7 cells were cultured in DMEM containing 10% charcoal-stripped fetal calf serum and treated for 2 h in the absence or presence of 100 nm 1,25-(OH)2D3. Cells were then fixed with 1% formaldehyde for 10 min at 37 C, washed, and collected in ice-cold PBS containing protease inhibitors (1 mm PMSF, 1 μg/ml aprotinin, and 1 μg/ml pepstatin A). Cell pellets were resuspended in lysis buffer [1% SDS, 10 mm EDTA, 50 mm Tris-HCl (pH 8.1), with protease inhibitors as described above] with each 200 μl sample containing 106 cells. Samples were incubated for 10 min on ice and sonicated at 15 W with 5-sec bursts for fourteen rounds. The supernatant was diluted 10-fold with ChIP dilution buffer [0.01% SDS, Triton X-100, 1.2 mm EDTA, 16.7 mm Tris-HCl (pH 8.1), 150 mm NaCl] containing protease inhibitors after removing cell debris. Twenty-microliter aliquots of the diluted supernatant (the total chromatin fraction as INPUT) were incubated for 4 h at 65 C to reverse cross-links. To reduce nonspecific background, the diluted soluble chromatin fractions were precleared with salmon sperm DNA/protein A agarose 50% slurry for 30 min at 4 C. Four microliters of anti-VDR antibody (Chemicon), or 5 μl of anti-HDAC1 antibody (Upstate), or 5 μl of anti-HDAC2 antibody (Upstate), or control human IgG (Sigma) were added and incubated overnight at 4 C. The immune complexes were then incubated with 60 μl of salmon sperm DNA/protein A agarose-50% slurry for 1 h at 4 C, and the supernatant was discarded. The beads were washed with the following buffers: low-salt wash buffer [SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl (pH 8.1), 150 mm NaCl], high-salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl (pH 8.1), 500 mm NaCl], LiCl wash buffer [0.25 m LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mm EDTA, 10 mm Tris-HCl (pH 8.1)], and TE buffer [10 mm Tris-HCl, 1 mm EDTA (pH 8.0)]. The histone complexes were eluted in freshly prepared elution buffer (1% SDS, 0.1 m NaHCO3) and the histone-DNA cross-links 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 32 cycles in total. The four pairs of Pit-1 primers were: the distal promoter pair (C-D) [C (forward, −476/ −454 bp) 5′-TCGTTTTTAAAAGCTGTGCATT-3′; D (reverse, −197/−177 bp) 5′-TTGAAAACACGTCCCTCAAA-3′; PCR product 280 bp long] and the proximal promoter pair (A-B) [A (forward, −219/−198 bp) 5′-TTTTTGAGGGACGTGTTTTCA-3′, B (reverse, −40/−18 bp) 5′-AGTCTTTTCCCAGGAGTATTG C-3′, PCR product 200 bp long] (Fig. 6A).

Statistical Analysis

Each experiment was performed at least three times. All values are expressed as mean ± 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 M. Delhase (Laboratory of Gene Regulation and Signal Transduction, La Jolla, CA), L. P. Freedman (Merck Research Laboratories, West Point, PA), R. Evans (The Salk Institute for Biological Studies, La Jolla, CA), and J. L. Castrillo (Centro de Biologia Molecular “Severo Ochoa,” Madrid, Spain) for plasmids. We also thank J. A. Costoya (Department of Physiology, Santiago de Compostela, Spain) for critical review and comments on this manuscript. Crystalline 1,25-(OH)2D3 was generously provided by Cristina Badia (Roche Pharma, Spain).

This work was supported by grants from the Xunta de Galicia (PGIDIT03PXIB20802PR) and the Fondo de Investigaciones Sanitarias, Ministerio de Sanidad (PI040518), Spain.

Abbreviations

• ChIP
  Chromatin immunoprecipitation;
• 9-_cis_RA
• DR
• GST
  glutathione-_S_-transferase;
• h
• HDAC
• PBST
  PBS containing 0.1% Tween 20;
• Pit-1
  pituitary transcription factor-1;
• PMSF
  phenylmethylsulfonyl fluoride;
• POU
  Pit-1, Oct-1, and Unc-86;
• PRL
• RXR
• SDS
• TSA
• VDR
• VDRE
  vitamin D response element.

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