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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
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1). 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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, 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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, 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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, 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. 3a, 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. 3b. 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. 4, 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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). 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
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.
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Acknowledgments |
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Footnotes |
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Received February 1, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3 are mediated
exclusively through the genomic signaling pathway. Endocrinology 137:1554–1561[Abstract]
and ß is cell type- and
promoter-dependent. J Biol Chem 274:32008–32014 and ER ß at AP1 sites. Science 277:1508–1510This article has been cited by other articles:
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  F. E. Nashold, K. M. Spach, J. A. Spanier, and C. E. Hayes Estrogen Controls Vitamin D3-Mediated Resistance to Experimental Autoimmune Encephalomyelitis by Controlling Vitamin D3 Metabolism and Receptor Expression J. Immunol., September 15, 2009; 183(6): 3672 - 3681. [Abstract] [Full Text] [PDF]
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 S. Safe and K. Kim Non-classical genomic estrogen receptor (ER)/specificity protein and ER/activating protein-1 signaling pathways J. Mol. Endocrinol., November 1, 2008; 41(5): 263 - 275. [Abstract] [Full Text] [PDF]
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S. Khan, F. Wu, S. Liu, Q. Wu, and S. Safe Role of specificity protein transcription factors in estrogen-induced gene expression in MCF-7 breast cancer cells J. Mol. Endocrinol., October 1, 2007; 39(4): 289 - 304. [Abstract] [Full Text] [PDF]
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 S. Seoane, I. Ben, V. Centeno, and R. Perez-Fernandez Cellular Expression Levels of the Vitamin D Receptor Are Critical to Its Transcriptional Regulation by the Pituitary Transcription Factor Pit-1 Mol. Endocrinol., July 1, 2007; 21(7): 1513 - 1525. [Abstract] [Full Text] [PDF]
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L. A. Zella, S. Kim, N. K. Shevde, and J. W. Pike Enhancers Located within Two Introns of the Vitamin D Receptor Gene Mediate Transcriptional Autoregulation by 1,25-Dihydroxyvitamin D3 Mol. Endocrinol., June 1, 2006; 20(6): 1231 - 1247. [Abstract] [Full Text] [PDF]
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L. A. Gilad, T. Bresler, J. Gnainsky, P. Smirnoff, and B. Schwartz Regulation of vitamin D receptor expression via estrogen-induced activation of the ERK 1/2 signaling pathway in colon and breast cancer cells J. Endocrinol., June 1, 2005; 185(3): 577 - 592. [Abstract] [Full Text] [PDF]
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L. Schneider, C. El-Yazidi, A. Dace, M. Maraninchi, R. Planells, A. Margotat, and J. Torresani Expression of the 1,25-(OH)2 vitamin D3 receptor gene during the differentiation of mouse Ob17 preadipocytes and cross talk with the thyroid hormone receptor signalling pathway J. Mol. Endocrinol., February 1, 2005; 34(1): 221 - 235. [Abstract] [Full Text] [PDF]
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 K. D. Healy, J. B. Zella, J. M. Prahl, and H. F. DeLuca Regulation of the murine renal vitamin D receptor by 1,25-dihydroxyvitamin D3 and calcium PNAS, August 19, 2003; 100(17): 9733 - 9737. [Abstract] [Full Text] [PDF]
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 S. Palomba, F. G. Numis, G. Mossetti, D. Rendina, P. Vuotto, T. Russo, F. Zullo, C. Nappi, and V. Nunziata Raloxifene administration in post-menopausal women with osteoporosis: effect of different BsmI vitamin D receptor genotypes Hum. Reprod., January 1, 2003; 18(1): 192 - 198. [Abstract] [Full Text] [PDF]
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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.
