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Endocrinology Vol. 141, No. 8 2829-2836
Copyright © 2000 by The Endocrine Society

ARTICLES

Identification of a Hormone-Responsive Promoter Immediately Upstream of Exon 1c in the Human Vitamin D Receptor Gene1

Ian M. Byrne, Louise Flanagan, Martin P. R. Tenniswood and JoEllen Welsh

Department of Biological Sciences (I.M.B., L.F., M.P.R.T., J.W.), University of Notre Dame, Notre Dame, Indiana 46556; and Departments of Zoology (I.M.B.), and Botany (L.F.), University College Dublin, Belfield, Ireland

Address all correspondence and requests for reprints to: Dr. JoEllen Welsh, Department of Biology, University of Notre Dame, Notre Dame, Indiana 46556. E-mail: jwelsh3{at}nd.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To gain insight into the molecular regulation of the human vitamin D3 receptor (hVDR), we have cloned and sequenced the 5' flanking region of exon 1c and examined promoter activity of this region in breast cancer cells. Sequence analysis of the first 1300 bp upstream of exon 1c reveals several characteristics of a class II promoter, including GC-rich regions and the presence of a TATA box at -29 bp. Putative transcription factor binding sites identified in this potential hVDR promoter include AP-2, Sp-1, and glucocorticoid response elements. No consensus vitamin D3 (VDRE) or estrogen (ERE) responsive elements were identified in the promoter sequence. Primer extension analysis performed with a primer specific for exon 1c confirms that transcription initiated in the 5' flanking region of exon 1c occurs in MCF-7 cells. Transient transfection of MCF-7 cells with this putative promoter region cloned into the pRLnull luciferase reporter vector generates significant reporter gene activity that is enhanced by treatment with forskolin, retinoic acid, and 17ß-estradiol. The enhancement of exon 1c promoter activity by 17ß-estradiol is blocked by the selective estrogen response modifier (SERM) tamoxifen and is not observed in estrogen receptor-negative breast cancer cells. In summary, we have cloned and characterized a TATA containing promoter upstream of exon 1c of the hVDR and provide evidence that this region represents a hormonally regulated hVDR promoter.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
1,25DIHYDROXYCHOLECALIFEROL [1,25(OH)2D3] is the hormonal form of vitamin D3 that regulates calcium homeostasis, immune responses, and cell growth. The growth regulatory effects of 1,25(OH)2D3, which include inhibition of proliferation, induction of differentiation, and activation of apoptosis, have been demonstrated in numerous cancer cell lines and tumors, including those derived from breast (1, 2), and are mediated through the vitamin D receptor (VDR), a ligand dependent transcription factor with similarity to other nuclear receptors. Because the genomic actions of 1,25(OH)2D3 are dependent on the presence of the VDR, receptor abundance is an important determinant of cellular sensitivity to 1,25(OH)2D3 (3). VDR abundance is affected by many physiological factors and is likely achieved through a variety of mechanisms, including transcriptional regulation, messenger RNA (mRNA) stability, posttranslational modifications, and ligand induced stabilization of the receptor protein (4). Specific regulators of the VDR include 1,25(OH)2D3 itself and other steroids such as 17ß-estradiol, dexamethasone (Dex), and retinoic acid, all of which have been shown to up-regulate the VDR protein and/or mRNA in vitro (4). Despite these observations, characterization of the relative contributions of transcriptional and posttranscriptional mechanisms in regulation of VDR levels and activity has been difficult due to the scarcity of information available on the VDR promoter region and its regulation.

Elucidation of the transcriptional regulation of the human VDR (hVDR) gene has primarily been hampered by the complexity of its promoter region. The hVDR gene has a similar intron/exon structure to other members of the steroid receptor superfamily with the exception of the untranslated exon 1, which is present in multiple copies and is associated with at least two and probably three differentially used promoters (5, 6). Attempts to demonstrate hormone regulation of these promoter regions, using reporter gene assays in numerous cell lines, have been largely unsuccessful, with one notable exception—the demonstration of a retinoic acid-responsive region in the intronic region located between exon 1c and exon 2 (5); however, no further characterization of this region has been reported.

We show that VDR transcripts containing exon 1c alone are present in MCF-7 human breast cancer cells and are regulated by a promoter immediately upstream of this exon that has the characteristics of a TATA containing promoter. Luciferase reporter constructs containing either 1300 bp or 800 bp of the region immediately upstream of the transcription start site show significant activity following transient transfection in MCF-7 cells. Agents known to up-regulate the VDR, including 17ß-estradiol (E2), forskolin, all trans-retinoic acid (ATRA) and Dex, enhance activity of both promoter constructs in breast cancer cells. 17ß-Estradiol treatment enhances hVDR promoter activity in estrogen receptor positive MCF-7 cells, but not in estrogen receptor-negative SUM 159PT breast cancer cells. These studies provide evidence of a hormonally responsive promoter region upstream of exon 1c in the hVDR gene and support the hypothesis that estrogen, and possibly other hormones, regulates breast cancer cell sensitivity to 1,25(OH)2D3 via transcriptional regulation of the VDR promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
MCF-7 and SUM159PT human breast cancer cell lines were cultured in phenol red free Ham’s F12 media (Life Technologies, Inc., Gaithersburg, MD) containing 10 mM HEPES, 5 µg/ml insulin, 1 mg/ml hydrocortisone and supplemented with 5% FBS (Life Technologies, Inc.) or 5% charcoal-stripped serum (CSS, HyClone Laboratories, Inc., Logan, UT). The estrogen-responsive MCF-7 cells were obtained from ATCC, and the estrogen independent SUM159PT cells were obtained from the University of Michigan Human Breast Cancer Cell/Tissue Bank (Ann Arbor, MI). Both cell lines endogenously express VDR (7, 8).

XL-PCR amplification of the 5' flanking region of exon 1c
The region upstream of exon 1c of the hVDR gene was amplified by PCR using the Human Genome Walker Kit (CLONTECH Laboratories, Inc., Palo Alto, CA). A nested set of primers specific for exon 1c of the hVDR gene was designed (DNAstar Software) and synthesized. The primer located furthest 3' of the start of exon 1c was designated E1C1 (5'-ACTTCCTCGTCCCCCGTCCATTCACC-3'; +55/+80). The nested primer was designated E1C2 (5'-TCGGGTCCCCACGAGAAGACACTCCAG - 3'; +28/+54). Each of the five libraries supplied by the manufacturer was subjected to two rounds of PCR as described in the manufacturer’s protocol with the exception of the substitution of rtTH XL polymerase Retrotherm reverse transcriptase (Perkin-Elmer Corp., Foster City, CA). The first round of PCR included the adapter primer supplied with the kit and the E1C1 primer. The cycling conditions for the primary PCR amplification were as follows: 7 cycles of denaturation at 94 C for 25 sec followed by annealing and elongation at 72 C for 4 min and 32 cycles of 94 C for 25 sec followed by 67 C for 4 min. One microliter of the primary PCR reaction was diluted into 49 µl of ddH2O and used in the secondary PCR reaction. The primers employed in the secondary PCR reaction were a nested adapter primer (supplied) and the primer E1C2. The cycling conditions for the secondary PCR reaction were as follows: 5 cycles of 94 C for 25 sec followed by 72 C for 4 min and 22 cycles of 94 C for 25 sec and 67 C for 4 min, followed by an additional cycle of 64 C for 4 min. Forty microliters of the secondary PCR reactions were analyzed on a 1.2% agarose gel. Amplification products of 1300 bp and 800 bp were cloned into TA cloning vectors and sequenced at least twice on both strands using automated DNA sequencing.

Construction of luciferase reporter plasmids and transient transfections
The 800- and 1300-bp products were subcloned into the promoterless pRL null vector (Promega Corp., Madison, WI) which contains the renilla luciferase reporter gene. Transient transfections with the pRL constructs were performed in MCF-7 and SUM159PT cells plated in six-well plates at a density of 1.5 x 105 cells per well. The cells were incubated at 37 C overnight, then cotransfected with 0.75 µg of the designated pRL construct and 0.25 µg of pGL-3 SV40 (total of 1 µg of DNA per well) in 1 ml of serum free Ham’s F12 media. After incubation at 37 C for 1 h, 1 ml of Ham’s F12 media containing either 5% FBS or 5% CSS supplemented with the appropriate treatment, or an equal volume of ethanol vehicle, was added to each well. After 18 h, cells were lysed and luciferase activity was determined with the Dual Luciferase Assay Kit (Promega Corp.). Transfection efficiency was normalized using the pGL-3 SV40 construct, and data are expressed as relative luciferase units (RLUs). Each experiment was performed in triplicate and replicated between three and six times. The luciferase assay data were statistically analyzed by ANOVA and either Dunnett’s or Tukey’s posthoc tests, as appropriate, with Graph Pad Instat Software (San Diego, CA). Means were considered significantly different if P values of 0.05 or less were obtained.

Isolation of total RNA and primer extension
RNA isolated from MCF-7 cells growing in 5% FBS was used for primer extension to identify transcripts originating from the promoter upstream of exon 1c of the hVDR. Primer extension was performed using 32P-end labeled primer E1C2 (5'-TCGGGTCCCCACGAGAAGACATCCAG-3"; +28/54) and polyA mRNA isolated with Ultraspec RNA isolation reagent (Biotecx Laboratories Inc., Houston, TX). Primer E1C2 (1 fmol) was hybridized to 10 µg of polyA mRNA in a volume of 10 µl ddH2O at 66 C for 25 min using reagents from Epicentre Technologies (Madison, WI). After addition of 1 µl of Retrotherm reverse transcriptase, reactions were incubated at 66 C for 40 min, heated to 95 C for 10 min and products were separated on a denaturing polyacrylamide gel containing 8% acrylamide, 7 M urea, and 1x TBE. 32P-end labeled Ø Hinf 1 DNA markers (Promega Corp.) were electrophoresed on the gel to serve as molecular weight markers. The gel was electrophoresed at 250 V for 2.5 h, vacuum dried, and exposed to x-ray film for 3 days.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
At the time the studies reported here were initiated, there was no evidence that the hVDR gene contained multiple promoters and exon 1 variants. Consequently, we concentrated on the region immediately upstream of the extant exon 1, now referred to as exon 1c (see Fig. 1Go for location of this region). The region immediately upstream of exon 1c of the hVDR gene was amplified using the Human Genome Walker kit, and products of 1300 bp and 800 bp were obtained. These products were cloned into TA cloning vectors and sequenced. The sequence of the 800- and 1300-bp products indicates that the 5' flanking region of exon 1c displays an organization reminiscent of a typical TATA containing promoter (Fig. 1Go). A consensus TATA sequence (GATAAAA) is present 29 bp upstream from the transcription start site. A number of putative regulatory regions, identified using the Transfac online transcription factor database, are present in the 1300-bp region immediately upstream of exon 1c. These include several SP1 and AP-2 sites upstream of the TATA box in addition to consensus sequences corresponding to AP-1, p53, c-myb, and glucocorticoid receptor (GR) response elements. Notably, no sequences corresponding to consensus direct repeat vitamin D3 response elements (VDRE) or estrogen response elements (ERE) are present in this region.



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  		Figure 1. Map of the human vitamin D receptor (hVDR) gene. Top, Intron/exon organization of hVDR, showing exons 2 through 9 (coding region) and untranslated exons 1a through 1f. Putative promoter regions 1a, 1f, and 1c are indicated in the striped boxes. Bottom, The first 1300 bp upstream of exon 1c was sequenced and analyzed with the Transfac online transcription factor database. Potential regulatory regions are denoted in bold with the name(s) of the consensus binding site(s) indicated below.

 
To establish that the promoter region upstream of exon 1c is used in a cellular context, primer extension analysis was used to identify transcripts initiated immediately upstream of the predicted start site of exon 1c. As can be seen in Fig. 2Go, a 79-bp extension product was identified in MCF-7 cells, consistent with a VDR transcript containing only exon 1c. Several larger primer extension products, ranging from approximately 140 bp up to 450 bp, are also present in MCF-7 cells. These transcripts appear to represent splice variants containing exon 1c and several of the alternative exons identified further upstream. Based on reported exon sizes (5, 6), we have tentatively identified these transcripts as splice products containing exons 1f, 1e, and 1c (447 bp) and 1f, 1a, and 1c (365 bp). Another primer extension product containing exons 1a and 1c, was detected upon longer exposure of the film (not shown). While confirmation of these assignments will require cloning and sequencing of these products, the primer extension analysis indicates that the putative promoter upstream of exon 1c of the hVDR gene is active in MCF-7 human breast cancer cells.



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  		Figure 2. Primer extension of the 5'flanking region of exon 1c of the hVDR. Poly A mRNA (10 µg) isolated from MCF-7 cells grown in FBS was incubated with 32P-endlabeled primer E1C2 specific for exon 1c and then with Retrotherm reverse transcriptase as described in Materials and Methods. Reaction products were separated on 8% acrylamide gels and visualized by autoradiography. Lane 1, Complete reaction; lane 2, reaction minus mRNA. MW, {phi}X174 Hinf molecular weight markers. Arrows indicate position of primer extension products with tentative assignment of exon usage for each transcription. *, Position of band detected upon longer exposure of the gel that likely corresponds to a transcript containing exons 1a and 1c.

 
To determine whether the exon 1c promoter region is capable of regulating transcription, the 800- and 1300-bp sequences were cloned into the promoterless renilla luciferase plasmid, pRLnull, and transiently transfected into MCF-7 cells. As shown in Fig. 3aGo, there is significant promoter activity in MCF-7 cells transfected with either the pRL 800 or the pRL 1300 construct, compared with cells transfected with the pRL null control vector. After correction for transfection efficiency, promoter activity for these constructs is elevated up to 80-fold above baseline activity detected with the pRL null vector. Basal activity of this promoter is also significantly enhanced when MCF-7 cells are stimulated with FBS, indicating that the promoter may be regulated by hormones and growth factors present in the serum (data not shown).



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  		Figure 3. Effects of 1,25 (OH)2D3 and 17ß-estradiol on hVDR exon 1c promoter constructs in MCF-7 cells. A, MCF-7 cells were transfected with the pRL800 or pRL1300 renilla luciferase constructs or the pRLnull empty vector and treated with ethanol vehicle (open bars), 100 nM 1,25 (OH)2D3 (hatched bars) or 10 nM 17ß-estradiol (filled bars) in phenol red-free media containing charcoal-stripped serum. RLUs have been corrected for transfection efficiency with cotransfected pGL3 SV40 using the dual luciferase assay and expressed relative to values obtained with pRLnull, which were normalized to 1. Data reflect the mean ± SEM of three wells and are representative of three or more independent trials. *, Significantly different from ethanol control (P < 0.01). B, MCF-7 cells transfected with the pRL800 construct were treated with ethanol vehicle (open bars), 1 nM 17ß-estradiol (filled bars), 1 µM 4-hydroxytamoxifen (hatched bars), or 1 nM 17ß-estradiol plus 1 µM 4-hydroxytamoxifen (gray bars) for 24 h in phenol red-free media containing charcoal-stripped serum. RLUs have been corrected for transfection efficiency with cotransfected pGL3 SV40 using the dual luciferase assay and expressed relative to values obtained with pRLnull, which were normalized to 1. *, Significantly different from ethanol control (P < 0.001); **, significantly different from 17ß-estradiol treated (P < 0.001).

 
To investigate the potential relevance of the promoter region upstream of exon 1c, we examined whether agents known to regulate VDR expression in breast cancer cells could modulate activity of the pRL800 and pRL1300 promoter constructs. It is well accepted that 1,25(OH)2D3 up-regulates the VDR protein; however, the mechanism of this effect is controversial, with evidence supporting both transcriptional regulation and ligand induced stabilization (4). Thus, we examined the possibility that 1,25(OH)2D3 could modulate reporter gene activity in MCF-7 cells, a cell line in which up-regulation of the VDR by 1,25(OH)2D3 has been demonstrated (7). As shown in Fig. 3aGo, no induction of either hVDR promoter construct was observed after treatment of MCF-7 cells with 100 nM 1,25(OH)2D3 for 18 h. Under the same conditions, 100 nM 1,25(OH)2D3 induced reporter gene activity of the rat 24-hydroxylase luciferase promoter (a know vitamin D responsive promoter) more than 15-fold in MCF-7 cells (data not shown). Even with extended treatment time (up to 72 h) under various culture conditions, no induction by 1,25(OH)2D3 of these promoter constructs is demonstrable in MCF-7 cells (data not shown).

In estrogen-responsive breast cancer cells, estrogens and antiestrogens have been shown to alter VDR expression and sensitivity to 1,25(OH)2D3 (4, 9). Although the sequence upstream of exon 1c does not contain any classical consensus ERE binding sites, numerous AP-1 and SP-1 binding sites, which can also mediate estrogen receptor transactivation (10, 11, 12), are present in the sequence. To establish whether up-regulation of the VDR by estrogen in breast cancer cells is correlated with transactivation of the region upstream of exon 1c, we examined reporter gene activity in MCF-7 cells treated with 17ß-estradiol. As demonstrated in Fig. 3aGo, 10 nM 17ß-estradiol significantly (P < 0.01) up-regulated the activity of both the pRL1300 and pRL800 constructs, by 4- and 2-fold, respectively, relative to ethanol vehicle-treated control cells. To determine whether the effects of 17ß-estradiol on the hVDR constructs are mediated directly by the estrogen receptor, the activity of the pRL800 reporter construct was measured in MCF-7 cells treated with 4-hydroxytamoxifen, the biologically active form of the SERM tamoxifen. At the dose of 1 nM, 17ß-estradiol significantly increase the activity of the pRL800 promoter activity (235% relative to ethanol-treated cells: P < 0.001) as shown in Fig. 3bGo. Treatment of MCF-7 cells with 1 µM 4-hydroxytamoxifen completely blocked the stimulation of the pRL800 reporter construct by 17ß-estradiol (P <0.001; 17ß-estradiol vs. 17ß-estradiol plus 4-hydroxytamoxifen). Similar results were obtained in MCF-7 cells transfected with the pRL1300 construct and treated with 17ß-estradiol with and without 4-hydroxytamoxifen (not shown). Collectively, these data suggest that the effect of 17ß-estradiol on transcription initiated in the promoter region immediately upstream of exon 1c is directly mediated via the estrogen receptor.

To determine whether additional agents known to regulate VDR expression modulate reporter gene activity of the exon 1c constructs, similar experiments were conducted in MCF-7 cells treated for 18 h with ATRA, Dex, the phorbol ester TPA, and forskolin. As shown in Fig. 4Go, ATRA (1 nM) and forskolin (1 µM) up-regulate the promoter activity of both the pRL800 and pRL1300 constructs in MCF-7 cells, whereas neither TPA (1 nM) or the synthetic glucocorticoid Dex (10 nM) induce transcription above the basal levels seen in vehicle-treated controls cells. Forskolin induces the promoter activity by approximately 5-fold with both the pRL1300 and pRL800 constructs, suggesting that the sequences responsible for this regulation are localized in the first 800 bp upstream of the promoter. On the other hand, the induction by ATRA is greater when the longer construct is utilized, suggesting that the retinoid-mediated regulation of the promoter lies, at least in part, in the more distal region of the pRL1300 construct.



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  		Figure 4. Effects of retinoic acid, Dex, forskolin, and TPA on hVDR exon 1c promoter activity in MCF-7 cells. MCF-7 cells were transfected with the pRL800 or pRL1300 hVDR constructs or the empty vector pRLnull and treated with ethanol vehicle (EtOH), Dex (10 nM), ATRA (1 nM), forskolin (Forsk, 1 µM), or TPA (1 nM) in phenol red-free media containing charcoal-stripped serum. After 18 h, RLUs were measured by dual luciferase assay and corrected for transfection efficiency using cotransfected pGL3 SV40. RLU values of the pRL constructs are expressed relative to pRLnull, which was normalized to 1, and represent mean ± SEM of three wells. Similar results were obtained in six independent experiments. *, Significantly different from ethanol value, P < 0.01.

 
Because progression of breast cancer is associated with loss of estrogen responsiveness, we examined regulation of the hVDR promoter constructs in an estrogen receptor-negative human breast cancer cell line, SUM159PT (8). This human breast cancer cell line lacks the estrogen receptor and thus is insensitive to estrogens and antiestrogens. Expression of the VDR in SUM159PT cells is lower than that in MCF-7 cells, and consequently, SUM159PT cells are less sensitive to the growth inhibitory effects of 1,2(OH)2D3 than MCF-7 cells (8). Transient transfection experiments under the same conditions as those used for MCF-7 cells demonstrated that the basal level of transcription for both the pRL800 and pRL1300 constructs is significantly lower in SUM159PT cells than in MCF-7 cells, with activities ranging between 5- and 10-fold over those of the pRLnull vector alone (Fig. 5Go). Notably, 10 nM 17ß-estradiol treatment of SUM159PT cells does not induce transcription of the reporter gene from either construct, consistent with the lack of estrogen receptor expression in these cells (8). Similar to MCF-7 cells, treatment with ATRA and forskolin significantly (P < 0.01) enhanced reporter activity in SUM159PT cells. In contrast to MCF-7 cells, Dex significantly enhanced pRL800 activity in SUM159PT cells (P < 0.05), suggesting differential regulation of the hVDR exon 1c reporter constructs in estrogen-dependent vs. estrogen-independent breast cancer cells.



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  		Figure 5. Activity and regulation of hVDR exon 1c promoter in estrogen independent SUM159PT breast cancer cells. SUM159-PT cells were transfected with the pRL800 or pRL1300 hVDR constructs or the empty vector pRLnull and treated with ethanol vehicle (EtOH), Dex (10 nM), ATRA (1 nM), forskolin (Forsk, 1 µM), or TPA (1 nM) in phenol red-free media containing charcoal-stripped serum. After 18 h, RLUs were measured by dual luciferase assay and corrected for transfection efficiency using cotransfected pGL3 SV40. RLU values of the pRL constructs are expressed relative to pRLnull, which was normalized to 1, and represent mean ± SEM of three wells. Similar results were obtained in two independent experiments. *, Significantly different from ethanol value, P < 0.05 (*); P < 0.01 (**).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper, we describe a TATA-containing promoter immediately upstream of exon 1c of the hVDR and demonstrate that this region represents a hormonally regulated hVDR promoter active in breast cancer cells. Exon 1 of the hVDR gene is present in multiple copies (exons 1a through 1f), and the existence of at least three differentially utilized promoters (exons 1a, 1d, and 1f) has been proposed (5, 6). In the studies reported here, primer extension using RNA isolated from untreated MCF-7 cells identifies transcripts containing only exon 1c, as well as additional transcripts initiated from other promoters further upstream that contain exon 1c. These data are consistent with earlier studies that demonstrated numerous transcripts containing exon 1c by 5'RACE (5, 6). Miyamoto et al. (5) identified transcripts originating upstream of exon 1a that contained exon 1c using human kidney RNA as a template. Crofts et al. (6) identified transcripts initiated upstream of exons 1a, 1d, and 1f that contained exon 1c in a panel of 15 cell lines but did not utilize primers that would identify transcripts initiated immediately upstream of exon 1c. Notably, transcripts originating upstream of exon 1f were restricted to kidney, parathyroid, and intestinal cell lines, whereas transcripts originating upstream of exons 1a and 1d were equally expressed in all 15 cell lines examined, including the estrogen-responsive breast cancer cell line T47D (6). Positive identification of the primer extension products generated in MCF-7 cells will be necessary to determine the extent to which transcription is initiated from the various promoter regions, and if promoter usage is altered by hormonal treatments.

Consistent with the primer extension data demonstrating transcription initiation upstream of exon 1c, 5' flanking sequences of exon 1c were active in reporter gene assays in MCF-7 cells. Similar studies have demonstrated that the promoters upstream of exons 1a and 1f can direct reporter gene activity in mammalian cell lines (COS 7, NIH 3T3, HeLa), whereas the 5' flanking region of exon 1d is inactive in reporter gene assays (5, 6). At the present time, there is no evidence to suggest that the promoter regions upstream of exons 1a, 1d, or 1f are hormonally regulated. A major finding of this paper, therefore, is that the promoter immediately upstream of exon 1c is regulated by hormones and other agents in breast cancer cells. In these studies, we report that exon 1c promoter activity is up-regulated by 17ß-estradiol, retinoic acid, and forskolin in MCF-7 estrogen-responsive breast cancer cells, but only the latter two agents enhance promoter activity in SUM159PT cells, which do not express the estrogen receptor (8). All of the agents that up-regulate hVDR promoter activity in the studies presented here have previously been shown to up-regulate VDR expression in numerous cell lines (4). Our data suggest that the mechanism by which these agents up-regulate VDR expression may involve, at least partially, direct regulation of VDR transcription.

In MCF-7 cells, 17ß-estradiol up-regulates hVDR promoter activity through both the pRL800 and pRL1300 constructs, with a peak induction of 6-fold relative to ethanol-treated control cells. The magnitude of this effect is comparable to the induction of other estrogen-responsive reporter genes known to be regulated by the endogenous estrogen receptor in MCF-7 cells (10, 11). The data presented here indicate that the up-regulation of hVDR promoter activity by 17ß-estradiol is mediated by the estrogen receptor because it is blunted by 4-hydroxytamoxifen and is not observed in the estrogen receptor negative SUM159PT breast cancer cells. Although these data indicate that a functional estrogen receptor is necessary for induction of hVDR promoter activity, no consensus ERE (GGTCAnnnTGACC) is present in either the pRL800 or pRL1300 constructs. This suggests that the 17ß-estradiol-estrogen receptor complex mediates its effects via an alternative pathway, such as through interactions with AP-1 or Sp1 transcription factors. The role of AP-1 in induction of estrogen-responsive genes lacking ERE has been well documented (12), and more recently, evidence that 17ß-estradiol mediates effects through interactions with Sp1 transcription factors has emerged (10). The presence of AP-1 and Sp1 sites in the promoter region upstream of exon 1c suggests that 17ß-estradiol might enhance hVDR promoter activity through one or both of these alternative pathways. In particular, six Sp1 sites in the exon 1c promoter are identical to GC/GA-rich sequences recently shown to confer 17ß-estradiol responsiveness to the bcl-2 promoter in MCF-7 cells (10). These sites (GGAGG at -1112, -386, -291, -215 and -55; GGGCTGG at -268) will be the initial focus of further studies to map the estrogen responsive region of the hVDR promoter. Because the GGAGG site located at -1112 is the only Sp1 site not present in the pRL800 construct, it is possible that the increased responsiveness to 17ß-estradiol of the larger construct is mediated through this region.

Our data are consistent with previous reports that 17ß-estradiol up-regulates VDR protein in estrogen-responsive human breast cancer cells (9) and VDR mRNA levels in human osteoblast-like cells (13, 14). Conversely, 4-hydroxytamoxifen down-regulates the VDR protein (7) and down-regulates the promoter activity in MCF-7 cells. In addition, VDR expression tends to be higher in estrogen receptor-positive than in estrogen receptor-negative breast cancer cells (15). Collectively, these data support the concept that estrogen is an important physiological regulator of VDR expression in breast cancer cells which mediates its effects via transcriptional regulation of the promoter region immediately upstream of exon 1c.

Regulation of the hVDR promoter by 17ß-estradiol has numerous clinical implications arising from the potential use of SERMs and vitamin D3 analogs for prevention and/or treatment of breast cancer and osteoporosis (16, 17, 18). The efficacy and toxicity of vitamin D3 analogs is determined, in part, by the level of VDR in target tissues, and our data suggest that estrogen status influences VDR abundance. In this respect, it will be important to determine whether novel SERMs such as raloxifene act as estrogen agonists or antagonists in regulation of hVDR promoter activity in different 1,25(OH)2D3 target cells. Recent data indicate that transcriptional activation by SERMs is cell type specific, promoter dependent, and different for the two estrogen receptor subtypes, ER{alpha} and ERß (11).

Despite data demonstrating ligand-dependent regulation of VDR expression in MCF-7 and other human derived cell lines (7, 19, 20), treatment with 1,25(OH)2D3 has no measurable effect on activity of the promoter immediately upstream of exon 1c. Similarly, the 5' flanking sequence of exon 1a was unresponsive to 1,25(OH)2D3 when tested in reporter gene assays (5). While it remains possible that 1,25(OH)2D3 may induce hVDR transcription via one or more of the newly identified, or as yet unidentified, promoter regions, up-regulation of the VDR protein by 1,25(OH)2D3 may also result from enhanced mRNA stability (19), ligand induced stabilization (21, 22), or reduced proteosomal degradation (23) rather than transcriptional activation.

The up-regulation of the pRL800 and pRL1300 constructs, which contain promoter sequence upstream of exon 1c, by retinoic acid in MCF-7 cells is particularly interesting in light of previous reporter gene assays in ROS17/2.8 rat osteosarcoma cells, which also identified a retinoid-responsive region in the hVDR gene. In those studies, however, retinoic acid regulation was attributed to a region downstream of exon 1c because a construct including the region upstream of exon 1c was not retinoid responsive (5). These discrepancies in retinoic acid regulation of the region surrounding exon 1c may reflect species (rat vs. human) or cell type (osteosarcoma vs. breast cancer) differences, which can only be resolved by further investigations. Despite these discrepancies, it is likely that the well established ability of retinoic acid to up-regulate VDR is mediated, at least in part, at the transcriptional level.

Forskolin, an activator of adenylate cyclase, is a potent enhancer of the promoter immediately upstream of exon 1c in both estrogen receptor positive and negative breast cancer cell lines. There has been a recent report of a forskolin-responsive region upstream of exon 1a in the mouse VDR promoter (24), although it is not clear if a similar forskolin responsive region is present in the 5' flanking region of exon 1a in the hVDR. These data are intriguing because forskolin has been shown to up-regulate VDR mRNA and responsiveness to 1,25(OH)2D3 in human cells (25). While no consensus cAMP response element has been identified in the exon 1c promoter sequence, recent studies have implicated AP-2 elements in mediating cAMP responsiveness of other gene promoters (26, 27). Thus, the effect of forskolin may be mediated through AP-2 sites present in the 1300 bp upstream of exon 1c. Further studies are necessary to examine the role of AP-2, and to determine whether other hormones, such as PTH, which activate adenylate cyclase and enhance VDR expression (4), also modulate this promoter activity.

The presence of consensus glucocorticoid response elements in the promoter immediately upstream of exon 1c suggests that this promoter activity should be responsive to the synthetic glucocorticoid, Dex. However, Dex fails to induce promoter activity in MCF-7 cells, despite previous reports that Dex enhances the growth inhibitory effects of 1,25(OH)2D3 in MCF-7 cells (28). In contrast to MCF-7 cells, Dex up-regulates the hVDR exon 1c reporter constructs in SUM159PT cells. These discrepancies are consistent with literature indicating that effects of glucocorticoids on VDR abundance may be species and tissue specific (4). The limited studies conducted in human-derived cell lines indicate up-regulation, down-regulation, or no change in VDR expression after treatment with Dex (19, 29, 30). Further studies to examine the molecular basis of glucocorticoid regulation of the VDR in estrogen-dependent vs. estrogen-independent breast cancer cells will be necessary to resolve these differences.

Although TPA has been shown to alter cellular responsiveness to 1,25(OH)2D3 and up-regulate VDR protein expression in MCF-7 cells (31), TPA does not up-regulate exon 1c promoter activity. This suggests that TPA modulates VDR expression in MCF-7 cells via posttranscriptional rather than transcriptional mechanisms. Posttranslational effects of TPA are consistent with data indicating that the VDR protein is phosphorylated by PKC at several sites (32).

In summary, we have used the region immediately upstream of exon 1c of the hVDR to demonstrate hVDR promoter activity, which is regulated by 17ß-estradiol, retinoic acid, and forskolin in MCF-7 cells. Because the promoters upstream of exon 1a and 1f do not appear to be hormone responsive, the data presented here suggest that the promoter region upstream of exon 1c is responsible for the hormone-regulated transcription of the hVDR gene, at least in MCF-7 breast cancer cells, and possibly other hormone-responsive tissues and tumors. Examination of this possibility will require comparison of promoter usage in MCF-7 and other cells after hormonal treatment. Furthermore, additional studies with the exon 1c promoter region are necessary to determine the relative importance of transcriptional, posttranscriptional, and translational mechanisms in overall VDR regulation, and the cell type specificity of such mechanisms. These data suggest that estrogen, and possibly other hormones, regulates breast cancer cell sensitivity to 1,25(OH)2D3 via transcriptional regulation of the hVDR promoter.


   Acknowledgments
 
The authors would like to thank Pamela Adams for technical assistance with sequencing, Leo Pharmaceuticals (Ballerup, Denmark) for supplying 1,25(OH)2D3, Dr. J. Omdahl for the vitamin D3-responsive 24-hydroxylase luciferase construct, and Dr. S. Ethier for the SUM159PT cell line.


   Footnotes
 
1 Portions of this work were presented at the Keystone Symposium on Programmed Cell Death, Breckenridge, Colorado, January 1999. This work was supported by NIH NCI Grant No. RO1-CA069700 (to J.W.). Back

Received February 1, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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