Novel N-Terminal Variant of Human VDR
K. L. Sunn,
T.-A. Cock,
L. A. Crofts1,
J. A. Eisman and
E. M. Gardiner
Bone and Mineral Research Program, Garvan Institute of Medical
Research, Sydney, New South Wales, 2010 Australia
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ABSTRACT
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The importance of N-terminal regions of nuclear hormone receptors
in transcriptional regulation is increasingly recognized. As variant
VDR gene transcripts indicated possible N-terminally extended
receptors, we investigated their natural occurrence, transactivation
capacity, and subcellular localization. A novel 54-kDa VDRB1 protein,
in addition to the previously recognized 48-kDa VDRA form, was detected
in human kidney tissue as well as in osteoblastic (MG63), intestinal
(Int-407, DLD-1, and COLO 206F), and kidney epithelial (786) human cell
lines by Western blots using isoform-specific and nonselective anti-VDR
antibodies. VDRB1 was present at approximately one-third the level of
VDRA. Isoform-specific VDRB1 expression constructs produced lower
ligand-dependent transactivation than VDRA when transiently transfected
with a vitamin D-responsive promoter into cell lines with low
endogenous VDR. Intracellular localization patterns of the green
fluorescent protein-tagged VDR isoforms differed. VDRB1 appeared
as discrete intranuclear foci in the absence of 1,25-dihydroxyvitamin
D3, whereas VDRA produced diffuse nuclear fluorescence.
After 1,25-dihydroxyvitamin D3 treatment, both VDR
isoforms exhibited similar diffuse nuclear signal. In the absence of
1,25-dihydroxyvitamin D3, the VDRB1 foci partially
colocalized with SC-35 speckles and a subset of promyelocytic leukemia
nuclear bodies. These data provide the first evidence of VDRB1,
a novel N-terminally variant human VDR that is expressed at a level
comparable to VDRA in human tissue and cell lines. It is characterized
by reduced transactivation activity and a ligand-responsive
speckled intranuclear localization. The intranuclear
compartmentalization and altered functional activity of VDRB1 may
mediate a specialized physiological role for this receptor
isoform.
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INTRODUCTION
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THE BIOLOGICALLY ACTIVE form of vitamin D,
1,251,25-dihydroxyvitamin D3
[1,25-(OH)2D3], acts
through the VDR, a member of the nuclear hormone receptor (NHR)
superfamily, which includes steroid, thyroid, retinoid, and orphan
receptors. Upon ligand binding, VDR forms homodimers or VDR:RXR
heterodimers that bind to specific vitamin D-responsive elements,
recruit additional coactivators, and interact with the general
transcription apparatus to initiate or inhibit gene transcription
(1). Vitamin D regulates calcium homeostasis as well as
specialized functions in cell proliferation and differentiation
(2). Multiple species of ERs, TRs, RARs, and RXRs have
been reported to be derived from separate genes. By contrast, variant
PRs and PPARs have been reported to be generated through differential
promoter usage and/or alternative splicing (3, 4, 5, 6, 7, 8). The
functional diversity contributed by these variants is increasingly
understood. For example, the human PR isoforms exert striking
differences in promoter specificity, suggesting that the two forms have
a different potential to synergize with one another and/or other
factors involved in modulation of transcription (4). The
PR isoforms are also unique in that only B receptors can activate
transcription in the presence of antiprogestins (9, 10);
furthermore, A-receptors can dominantly inhibit B receptors (8, 11) as well as other members of the steroid receptor superfamily
(12, 13). Overall, the existence of these isoforms
suggests that variant receptors may modulate different
physiological responses.
These alternate receptors vary in the length of their N-terminal A/B
domains, the region of greatest diversity in the NHR (1),
from 23 to more than 600 amino acids (aa) (14, 15).
Although vitamin D exhibits functional diversity, the VDR differs from
the other NHRs with its very short A/B domain (23 aa) and limited
structural variability (1, 2, 14). Two VDR isoforms
differing in the N terminus by 14 aa and generated by alternative
translation initiation have been described in avian species
(16). Until recently, there has been limited evidence for
human VDR (hVDR) isoforms. A 3-aa N-terminal extension due to a common
start codon polymorphism (17) has been reported to cause
elevated transactivation activity in some studies (18). A
dominant-negative VDR generated by intron 8 retention in the rat
(19) has uncertain physiological significance. Overall,
however, there has been little evidence of functionally different
isoforms of the hVDR.
Recently, we reported alternatively spliced upstream exons in the hVDR
gene locus with the potential to encode variant proteins termed VDRB1
and VDRB2, with 50- or 23-aa N-terminal extensions, respectively
(14). There was one variant transcript potentially
encoding each variant protein isoform, compared with six transcripts
encoding the standard A form of VDR in all tissues and cell lines
examined, as well as four additional VDRA transcripts initiating at a
distal promoter that was active in the major vitamin D target tissues
(14). In the present study we report that the novel VDRB1
protein coexists with the previously identified VDRA, and show that it
has altered ability to transactivate a vitamin D-responsive
promoter-reporter construct. Based on studies with chimeric green
fluorescent protein (GFP)-VDR proteins, we also provide evidence that
VDRB1 localizes to unique intranuclear foci, unlike the uniform
distribution of VDRA. The ligand response of the VDRB1 foci differs
from those reported for other NHRs (20, 21, 22, 23, 24). The VDRB1
foci associate with a subset of SC-35 splicing factor domains, but not
with PML or p80 coilin.
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RESULTS
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Immunodetection of hVDR Isoforms
Initial Western blots did not show the presence of additional
band(s) in the predicted size range. Thus, large quantities of
whole-cell lysate were analyzed. Using this approach, two
immunoreactive proteins corresponding in size to transfected VDRA and
VDRB1 were detected in empty vector transfected MG63 whole-cell lysates
by the commercial anti-VDR antibody 9A7 (Fig. 2A
). These identities
were confirmed using isoform-specific antisera with reactivities
previously demonstrated by probing Western blots of extracts from COS-1
cells expressing individual VDR isoforms (Fig. 1
). Both the VDRB-common and the
VDRB1-specific antisera detected only the upper band in the MG63 lysate
but bound appropriately to VDRB1 and VDRB2 in transfected cell lysates
(Fig. 2
, B and C). VDRA and VDRB1 were
also present in the intestinal cell lines Int-407, COLO 206F, and
DLD-1, as well as the 786 kidney cell line (Fig. 3A
). Furthermore, both VDRA and VDRB1
were detected in human kidney (Fig. 3B
). Quantification of band
intensities suggested that VDRB1 is present at approximately one-third
the level of VDRA in MG63 cells, with comparable intensities for both
isoforms in kidney tissue and the other cell lines. A band of the
predicted size and immunoreactivity of the VDRB2 protein was not
detected in any sample.

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Figure 2. VDRB1 in Human Osteosarcoma MG63 Cells
Whole-cell lysates from MG-63 cells either mock transfected (400 µg
protein) or transfected (7 µg protein) with individual VDR
isoform-specific constructs to provide size markers. The membrane was
probed with the anti-VDR antibody 9A7 (A), VDRB-common (B), and
VDRB1-specific antiserum (C).
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Figure 1. Exon 1d Transcripts of the hVDR
The 427-aa VDR A protein corresponds to the published cDNA sequence
(75 ). VDRB1 and VDRB2 proteins are 50 or 23 aa larger,
respectively, at the N terminus (14 ). A, Amino acid
sequence of N-terminal variant A/B domains encoded by novel hVDR
transcripts with regions used for peptide production
underlined (B). Immunoblotting of transiently
transfected cell lysates expressing each individual isoform confirmed
that each antiserum detected only the appropriate proteins. Neither
VDRB-common nor VDRB1-specific antiserum detected VDRA which lacks the
unique B region, as evident in the lanes for VDRA transfected cell
lysate and baculovirus-expressed recombinant hVDR (rec hVDR) C, CMV,
empty vector transfected cell lysate.
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Figure 3. Detection of 54-kDa VDRB1 in Human Cell Lines and
Tissue
Whole-cell lysates (400 µg protein) from Int-407, COLO 206F, DLD-1,
and 786 cells were run on a 12% SDS-PAGE, transferred to PVDF
membrane, and probed with the anti-VDR antibody 9A7 (A), or
VDRB1-specific antiserum (B). Whole-cell protein lysates from human
kidney (300 µg protein) were compared with individual VDR
isoform-transfected protein lysates (7 µg protein) and probed with
the anti-VDR antibody 9A7 (C) VDRB1-specific antiserum (D).
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VDR Isoform Transactivation
Hormone-stimulated transactivation by the novel receptor
VDRB1 was lower than that of VDRA on the rat 24-hydroxylase reporter
construct in two cell lines with low endogenous VDR. In COS-1 monkey
kidney cells, hormone-stimulated transactivation by the N-terminally
extended VDRB1 was 60% of the level of VDRA (Fig. 4A
), despite comparable protein
expression levels (Fig. 4C
). Transactivation was similarly less
efficient (
60% of the VDRA level) in the P19 mouse embryonal
carcinoma cell line (Fig. 4B
). There was no consistent effect of either
VDR isoform on basal transcriptional levels in either cell line,
indicating that there was neither ligand-independent transcriptional
activation nor repression by either isoform.

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Figure 4. Transactivation of Rat 24-Hydroxylase Promoter by
hVDR Isoforms
Transcriptional activation of the rat 24-hydroxylase promoter reporter
construct was determined after cotransfection with VDRA or VDRB1
expression construct in COS-1 (A) or P19 cells (B).
1,25-(OH)2D3 (1 nM)-stimulated
transactivation by VDRB1 was approximately 60% of the level of VDRA
in both cell lines, despite comparable protein expression levels
(C).
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Ligand-Regulated Subcellular Localization of hVDR-Enhanced Green
Fluorescent Protein (EGFP) Fusion Proteins
Short stretches of basic amino acids (RNKKR and RPHRR) in
the N terminus of VDRB1 resemble a typical nuclear localization
consensus sequence (14, 25, 26, 27); therefore, intracellular
localization was examined by transient expression of VDR-EGFP fusion
proteins in COS-1 cells. Protein bands of appropriate sizes (75 and 81
kDa for VDRA- and VDRB1-EGFP fusion proteins, respectively) were
detected by both anti-GFP and 9A7 anti-VDR antibodies (Fig. 5
, A and B). These fusion proteins were
transcriptionally active, with the VDRB1 exhibiting less
1,25-(OH)2D3-stimulated
transactivation than the VDRA fusion protein, as seen with the
unmodified VDR isoforms (Fig. 5C
).

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Figure 5. Full-Length Expression of Transcriptionally Active
hVDR-EGFP Isoforms
Western blot (5 µg total protein per lane) extracted 48 h after
transfection shows detection of full-length VDR-EGFP fusion proteins
probed with anti-VDR antibody, with no significant truncation products
detected by either anti-VDR (A) or anti-GFP (B) antibodies. Both VDR
fusion proteins effectively transactivated the rat 24-hydroxylase
promoter reporter construct in response to 10 nM
1,25-(OH)2D3 (C).
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VDRB1-EGFP appeared as distinct nuclear foci with little signal
in the cytoplasm (Fig. 6
, A and C). All
VDRB1-EGFP expressing cells with a strong signal exhibited the distinct
punctate distribution. A small proportion of cells (15%) exhibited a
homogeneous faint signal comparable to EGFP alone. Bright homogeneous
nuclear signal was seen in all VDRA-EGFP expressing cells (Fig. 6
, B
and D). Both proteins were excluded from nucleoli. Varying VDR plasmid
concentration (0.022 µg) or altering time before fixation (2472 h
posttransfection) had no effect on the punctate distribution of either
VDR isoform. After exposure to
1,25-(OH)2D3 (18 h), the
VDRB1-EGFP nuclear pattern had become diffuse with more cytoplasmic
signal apparent. Nuclear VDRA-EGFP signal was not significantly altered
by ligand treatment, although the level and distribution of cytoplasmic
signal were more variable (Fig. 6
, B and D, lower
panel).

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Figure 6. Distinct VDRB1 Intranuclear Localization in COS-1
Cells Does Not Colocalize with Basal Transcription Factors TBP and RNA
PolIIo
VDRB1 isoform fusion protein localized to discrete nuclear foci (A and
C, upper panel), whereas VDRA appeared as a diffuse
nuclear signal (B and D, upper panel). Both isoform
types exhibited diffuse nuclear distribution after exposure of cells to
10 nM 1,25-(OH)2D3 (AD,
lower panels ). Cytoplasmic signal was greater for VDRA
transfected cells, but was not markedly altered by ligand treatment.
The EGFP empty vector control produced faint diffuse cytoplasmic and
nuclear signal in both the presence and absence of ligand (not shown).
COS-1 cells were transfected with GFP-tagged hVDR isoforms
(green), then immunostained (red) with
antibody against the transcription factor TBP (A and B) or H5 mAb
against RNA PolIIo (C and D). Merged images (yellow)
indicate the extent of overlap between the VDR isoforms and other
nuclear proteins. Immunocytochemistry was performed 16 h after
treatment with 10 nM 1,25-(OH)2D3.
Bar = 20 µM.
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Immunofluorescence of VDR Isoforms with Nuclear Speckled Proteins,
Sites of Transcription, and RNA Processing
The VDRB1-GFP foci were compared with the speckles of active RNA
Polymerase II (RNA PolIIo) (28, 29, 30, 31), SC-35, the non-small
ribonucleoprotein spliceosome assembly factor (32, 33),
p80 coilin, which localizes to sites of RNA processing (31, 34, 35, 36) and PODs (PML oncogenic domains), which are discrete
interchromosomal accumulations of several proteins including PML and
Sp100 (37, 38, 39). The localization patterns of VDR
and TATA Binding Protein (TBP) and RNA PolIIo were examined using the
GFP-tagged VDR isoforms and immunostaining for endogenous TBP and RNA
PolIIo. There was incomplete nuclear overlap between TBP and both VDR
isoforms, as indicated by the nonuniform staining in the merged images
(Fig. 6
, A and B). Although treatment with
1,25-(OH)2D3 dispersed the
characteristic VDRB1 speckles, thereby increasing the extent of overlap
with TBP, the general background of mottled TBP nuclear staining was
not altered with
1,25-(OH)2D3 treatment.
Hence, the degree of colocalization was due merely to the
generally homogenous nuclear distribution of both proteins rather than
specific association. Coincidental overlap with either VDR isoform and
RNA PolIIo was also due to the even distribution of active RNA Pollo
throughout the nucleoplasm, irrespective of
1,25-(OH)2D3 status (Fig. 6
, C and D). The degree of colocalization was increased upon
1,25-(OH)2D3 treatment,
with both VDR isoforms exhibiting a uniform distribution overlying that
of active RNA PolIIo, although the enhancement of apparent overlap upon
treatment was more striking for VDRB1.
There was a clear correlation between the GFP-tagged VDRB1 and SC-35
indirect immunofluorescence signals (Fig. 7A
). Each of the VDRB1 foci was
immediately adjacent to but not absolutely colocalized with an SC-35
speckle, but not every SC-35 speckle was associated with a VDRB1 focus.
In contrast, VDRB1 and PML nuclear body signals were only
coincidentally colocalized, as the VDRB1 foci only partially associated
with the PML domains; their distribution was randomized in comparison
to the PODs (Fig. 7B
.). There was no detectable colocalization between
VDRB1 and the punctate pattern of protein p80 coilin (Fig. 7C
).
Detection of physical associations is precluded by limitations of the
resolution of indirect immunofluorescence and is beyond the scope of
this study. Apparent overlap of VDRA and SC-35 speckles, PML domains,
or coiled bodies was due to the uniform distribution of the VDR and
coincidental (data not shown).

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Figure 7. Subcellular Localization of VDRB1-EGFP Chimeras
Compared with SC-35, PML Nuclear Bodies, and p80 Coilin
COS-1 cells were transfected with GFP-tagged hVDRB1
(green), and then immunostained (red)
with antibody against the spliceosome component SC-35 (A), PML nuclear
bodies (B), or p80 Coilin (C). Merged images (yellow)
indicate the extent of overlap between the VDRB1 and other nuclear
focal proteins. Expanded view of merged region (400% enlargement) is
indicated by the white dotted square and image on
far right of each figure. Bar = 20
µM.
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DISCUSSION
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This study provides evidence for a previously unknown variant of
the hVDR that corresponds in size and immunoreactivity to the predicted
54-kDa VDRB1 protein (14). VDRB1 was present at
approximately a 1:3 ratio with the well studied 48-kDa VDRA protein in
human kidney and in the five cell lines examined. These findings
suggest a role for posttranscriptional regulation in determining
variant VDR protein levels, as the number of VDRA-encoding transcripts
(ten) exceeds the single transcript encoding VDRB1. A role for
posttranscriptional regulation of VDR isoform expression is further
supported by the apparent absence of the predicted VDRB2 isoform,
despite the previously reported evidence for VDRB2-encoding transcripts
(14). It is possible that VDRB2 may exist at a level lower
than was detected by the current analyses. Our preliminary studies
indicate that this receptor would produce a level of transactivation
intermediate between VDRA and VDRB1 (our unpublished
observations).
The N-terminal domains of other NHRs modify transcriptional activity by
interacting with basal transcription machinery (40, 41, 42),
serving as phosphorylation substrates (43, 44), or
functioning as activation function-1 domains
(45, 46, 47). In agreement with this pattern,
transactivation by VDRB1 was clearly lower than by VDRA on the rat
24-hydroxylase promoter (T.-A. Cock, manuscript submitted). This
difference was seen in both COS-1 and P19 cells, indicating that the
effect of the N-terminal extension on transactivation function is not
cell line specific.
The VDRB1-EGFP fusion protein accumulated in nuclear speckles that were
not seen with VDRA-EGFP. Ligand treatment dispersed VDRB1 to a
homogenous distribution similar to that of VDRA-EGFP. The ligand
sensitivity of the speckles suggests functional significance, as
proposed for other NHRs that are also distributed in dynamic clusters
or foci (20, 48). Importantly, however, the VDRB1 speckle
distribution and ligand response pattern differed from that reported
for other receptors such as GR and MR, which accumulate in discrete
nuclear clusters only after ligand treatment (20, 48, 49, 50, 51, 52).
The distribution of the VDRA-EGFP fusion protein throughout the nucleus
and cytoplasm in the absence of ligand is similar to that of an
analogous fusion protein reported elsewhere (53). In that
study, fluorescence became entirely nuclear and homogeneous within 30
min of 1,25-(OH)2D3
treatment, and all cytoplasmic signal was restored within 30 min of
hormone removal. These findings are consistent with cell fractionation
and immunocytology studies, which indicated that VDR was both nuclear
and cytoplasmic (20, 48, 49, 54). More recently, standard
VDR (i.e. VDRA) has been shown to form ligand-induced
intranuclear foci upon heterodimerization with RXR, consistent with a
role for heterodimer binding to DNA target sites in the formation of
these foci (55). The foci seen upon heterodimerization
with RXR, however, do not resemble the VDRB1 foci, and endogenous RXR
did not colocalize with GFP-VDRB1 in the present study (data not
shown).
NHR nuclear localization and distribution in discrete nuclear domains
appear to be important aspects of functional coordination
(56). TRß1 is present in both the nucleus and the
cytoplasm in the absence of ligand but translocates to the nucleus and
is uniformly distributed in the presence of T3
(57). In contrast, MR is predominantly nuclear in the
absence of ligand but concentrates to prominent clusters within the
nucleus when bound to aldosterone (22). Similarly,
unliganded GR resides predominantly in the cytoplasm, with hormone
activation leading to translocation to the nucleus and gene activation
(50). Transcriptional activation of GR by ligand is also
correlated with focal nuclear accumulation similar to MR
(20). In colocalization studies of VDRB1 and GR, we have
also seen nuclear clustering of GR within 15 min of dexamethasone
treatment, but this punctate distribution did not colocalize with the
VDRB1 speckles, which themselves were not affected by dexamethasone
treatment (data not shown).
Nuclear accumulation may be attributable to chromatin or hormone
response element targeting (23), but more complex
intranuclear interactions are also possible (52, 58).
Proficient transcription and RNA processing may occur at the periphery
of speckles (29, 32, 59, 60) or nuclear foci may be
storage sites of splicing and transcription factors distal from the
sites of bulk transcription (61). The VDRB1 speckles could
therefore relate to regulation of specific genes, as has been suggested
for clustered GR molecules, or may represent sites of receptor
storage.
Mammalian nuclei contain subnuclear foci of various transcription
factors, hnRNP proteins, heterochromatin proteins, and even elements of
the cleavage and polyadenylation machinery (38). The
various morphologically distinct substructures, or nuclear bodies,
include sphere organelles, interchromatin granule clusters, coiled
bodies, and the PML nuclear bodies or PODs (61, 62, 63). The
emerging view is that many of these subdomains are associated with
specific genetic loci and that interactions with these various domains
and loci are dynamic and can change in response to cellular signals
(38).
A number of nuclear proteins have been associated with a speckled
localization, including SC-35, PML, ND10, and Sp100 (37, 38, 64). The VDRB1 foci were associated with the periphery of the
SC-35 splicing factor speckles, a pattern of overlap similar to that
previously described for transcriptionally active genes
(65). PODs or PML nuclear bodies have been implicated in
several intracellular processes including cell-cycle regulation, viral
infection, growth inhibition, apoptosis, and transcriptional regulation
(39, 66, 67, 68). The lack of significant association of the
VDRB1 foci with the PML nuclear bodies is indicative that in the
absence of ligand and further modifications, VDRB1 does not localize
within the POD (38, 66, 67, 68, 69, 70, 71). Similarly, the VDRB1 foci
were not associated with coiled bodies in which small
ribonucleoproteins, nucleolar proteins, and cell-cycle control
proteins, as well as several basal transcription factors, are highly
enriched (38). Similarly, TBP and PolIIo were shown in
coincidental overlap with both VDR isoforms after ligand treatment due
to their similar uniform distributions within the nucleus.
This study presents the first direct evidence that the VDRB1, with its
extended A/B domain, is produced in vivo, albeit at lower
levels than the previously described VDRA, and is transcriptionally
active. This novel isoform is localized in dynamic intranuclear
speckles. This localization, which is ligand-sensitive, does not
coincide with other known nuclear "speckled" proteins, but appears
to be juxtaposed to the focal domains containing the SC-35 protein. The
data imply that VDRA and nuclear speckle-localized VDRB1 may coexist
and that
1,25-(OH)2D3-induced
release of VDRB1 from speckles may modulate the cellular response
to ligand. Differential transcriptional activity of the VDR
isoforms could preferentially modulate subsets of target genes in
vitamin D-responsive pathways.
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MATERIALS AND METHODS
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Production and Characterization of Isoform-Specific Antipeptide
Antibodies
Polyclonal B-common and B1-specific antisera were generated by
intraperitoneal injection of rabbits with 300 µg of KLH-coupled
synthetic peptides in Freunds complete adjuvant. The peptides were
NH2-MEWRNKKRSDWLSMVLRTAGC-COOH encoded by exon 1d hVDR,
NH2-SVRPHRRAPLGSTYLPPAPSC-COOH encoded by exon 1c (Fig. 1A
). Animals
were boosted and bled at 4-wk intervals with a terminal bleed after 7
months (Chiron Corp. Technologies, Melbourne, Australia).
Antibody titers were determined by ELISA and specificities confirmed by
Western blots of lysates prepared from COS-1 cells transfected with
individual isoform-specific expression constructs (see below). Lysate
protein (5 µg) was electrophoresed on a 10% SDS-PAGE and
immunoblotted on polyvinylidene difluoride membrane
(Amersham Pharmacia Biotech, Buckinghamshire, UK). The
membrane was sequentially probed with the test antisera. The 9A7
antibody detected all VDR forms, whereas the B-common antiserum
detected both B form VDRs but not VDRA; and the B1-specific antiserum
detected only VDRB1 (Fig. 1C
).
Isoform-Specific Constructs
hVDR A, B1 and B2 cDNAs were cloned from SAOS-2
osteosarcoma RNA by RT-PCR. Forward primer for VDRA was
5'-GAGTCAAGCTTTCAGGGATGGAGGCAATTGCGG-3' and for VDRB1 and VDRB2 was
5'-GAGTCAAGCTTCTTGGCATGGAGTGGAGGAATAAG-3'. The common reverse primer
from exon 9 was 5'-GACTCGGGCCCCTAGTCAGGAGATCTCATTGCCAAAC-3'. To
ensure translation of only a single variant from a specific ATG codon
in each expression construct, noninitiating methionine codons in
exons 1d and 1c were mutated to isoleucine, using forward PCR primers
(M23I 5'-TGGCTGTCGATTGTGCTCAGAAC-3' and M60I
5'-TCAGGGATTGAGGCAATTGCGGCC-3') and the common
reverse primer.
For expression studies, modified cDNAs were cloned into pRC-CMV
(Stratagene, La Jolla, CA). For subcellular localization, the cDNAs
were subcloned into pEGFP-N1 vector (CLONTECH Laboratories, Inc., Palo
Alto, CA). All plasmids were verified by DNA sequencing (Perkin-Elmer
Corp., Norwalk, CT).
Cell Extract Preparation and Immunoblotting
Human osteosarcoma MG63, embryonal intestinal Int-407,
colon carcinoma DLD-1 and COLO 206F, and renal adenocarcinoma 786 cell
lines, all known to express transcripts encoding the putative VDR
isoforms (14), were grown in DMEM with 10% FBS. For
isoform size standards, MG63 cells were transfected in 15-cm dishes
with 5 µg pRC-CMV-VDR isoform constructs using FUGENE6 reagent
(Roche Molecular Biochemicals, Mannheim GmbH,
Germany). Whole-cell extracts were prepared from untransfected
cultures, and 48 h after transfection, cells were harvested,
washed in 1xPBS, and resuspended in boiling SDS-lysis buffer (10%
SDS, 0.15 M Tris-HCL, 1% ß-mercaptoethanol) with 10%
DNase/RNase, and vortexed for 30 sec (72). The lysates
were then boiled for 30 sec and immersed in liquid nitrogen. Samples
were lyophilized on a Speedvac drier and resuspended in rehydration
buffer [8 M urea, 2% dithiothreitol (DTT), 2%
3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1- propane
sulfonate (CHAPS)]. Total protein was separated by 12% SDS-PAGE,
transferred to PVDF membrane, and blocked overnight at 4 C in
Tris-buffered saline with Triton-X100 containing 10% skim milk powder,
0.5% BSA before incubation in isoform-specific antiserum (1 µg/ml)
or with 0.4 µg/ml 9A7 rat anti-VDR monoclonal (Affinity BioReagents, Inc., Golden, CO) as primary antibody for 2 h
at room temperature, followed by incubation with donkey antirabbit
horseradish peroxidase (HRP) or goat antirat-HRP secondary antibody
(Zymed Laboratories, Inc., South San Francisco, CA) for
1.5 h at room temperature. Immunoreactive proteins were identified
by enhanced chemiluminescence (Amersham Pharmacia Biotech). Band intensities were estimated by densitometry of a
lightly exposed film containing a serial dilution (400100 µg
protein) of MG63 cell lysate (Molecular Dynamics, Inc.,
Sunnyvale, CA; Amersham Pharmacia Biotech).
Total Protein Preparation from Human Tissue
Frozen tissue was ground to a fine powder using a ceramic
mortar and pestle kept cool under liquid nitrogen immersion.
Approximately 500 µl of dry powder were transferred to a clean
Eppendorf tube and washed three times in low-salt washing
buffer (3 mM KCl; 1.5 mM
KH2PO4; 68 mM
NaCl; 9 mM
NaH2PO4) to remove excess
blood and cellular debris. The cell pellet was washed three times in
low-salt washing buffer and resuspended in boiling SDS-lysis buffer
(10% SDS, 0.15 M Tris-HCl, 1% ß-mercaptoethanol) with
10% DNase/RNase solution, and vortexed for 30 sec. The lysates were
then boiled for 30 sec and immersed in liquid nitrogen
(72). Samples were lyophilized on a Speedvac drier and
resuspended in rehydration buffer (8 M urea, 2% DTT, 2%
CHAPS). The lysate was cleared of cell debris by centrifugation at
13,000 rpm for 10 min at room temperature. The supernatant was then
transferred to a new tube. To the total protein sample, an equal volume
of ammonium sulfate was added and mixed for 3060 min at 4 C. The
sample was centrifuged at 3,000 x g for 15 min at 4 C.
The supernatant was removed and discarded, with the pellet washed five
times in low-salt washing buffer. The protein pellet was resuspended in
rehydration buffer (8 M urea, 2% DTT, 2% CHAPS)
and quantified.
Transactivation Studies
COS-1 and P19 cells, both with low endogenous VDR, were
maintained in DMEM with 10% FCS. For comparison of transactivation by
VDRA and VDRB1 cells were seeded in 24-well plates at a density of
2 x 104 cells per well for cytomegalovirus
(CMV)-VDR, or 106 cells per well for GFP-VDR
studies, in DMEM with 2% charcoal-stripped FBS. Cells were transfected
24 h later in serum-free medium using FUGENE6 with 540 ng
DNA/well. Transfected DNA consisted of 20 ng pRC/CMV-VDR isoform
expression plasmid, 250 ng of rat 24-hydroxylase promoter-luciferase
reporter construct (24(OH)ase-Luc), 230 ng empty pRC/CMV vector, and 40
ng pRous sarcoma virus-ß-galactosidase reporter contruct as a
transfection efficiency control.
The 24-hydroxylase luciferase reporter construct contains 1,475 kb
(-1,401 to +74) of promoter sequence from the rat 25-hydroxyvitamin
D-24-hydroxylase gene (73) (a generous gift from Dr. B.
May, Adelaide, South Australia). Assays of EGFP-chimeric constructs
included 100 ng pVDR-EGFP, 2 µg 24(OH)ase-Luc, 230 ng empty
pEGFP vector, and 0.5 µg pRous sarcoma virus-ß-galactosidase
reporter construct. After 5 h cells were treated with 1 or
10 nM
1,25-(OH)2D3 or vehicle
(isopropanol), as indicated, in DMEM with 2% charcoal-stripped FBS,
incubated at 37 C for 18 h, and then lysed in cell lysis buffer.
Reporter activity was determined by the Luciferase Reporter System
assay (Promega Corp.). Results are expressed as mean
± SEM from at least three separate transfections, each in
triplicate, and normalized to ß-galactosidase level (Tropix, PE Applied Biosystems, Bedford, MA).
Fluorescence Microscopy and Immunocytochemistry
For fluorescence microscopy, 105 COS-1
cells per well in two-well Labtek-II chamberwell slides (NUNC,
Naperville, IL) were transfected with 2 µg of pVDRA-EGFP or
pVDRB1-EGFP or pEGFP. After 18 h, cells were fixed with 2%
paraformaldehyde/PBS for 30 min, washed three times with PBS, and
coverslipped. For each construct, 180 cells were examined.
Colocalization studies (74) used mouse monoclonal
antibodies H5 against hyperphosphorylated RNA PolIIo (28)
(Babco, Richmond, CA), the rabbit anti-transcription factor IID
(TBP) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA),
the spliceosome assembly factor SC-35 (Sigma, St. Louis,
MO), PML nuclear bodies (5E10- GeneTex, ), or p80 Coilin rabbit
antisera (kind gift from Prof. A. Lamond) as primary antibody and Alexa
Fluor 594 antimouse, and 594 antirabbit secondary antibodies
(Molecular Probes, Inc., Eugene, OR). Nuclear
integrity was confirmed for all dual-labeling experiments by
4,6-diamidino-2-phenylindole staining (Roche Molecular Biochemicals). Cells were photographed on a DMR microscope
(Leica Corp. Microsystems, Nussloch, Germany) using
standard fluorescein isothiocyanate/Texas Red filter sets and 100x
objective with oil immersion.
 |
ACKNOWLEDGMENTS
|
---|
We thank Prof. Angus Lamond for the p80 coilin antiserum, Drs.
G. Corthals and V. Wasinger for their assistance with the protein
extractions, and J. Flanagan and other members of the Bone and Mineral
Program for helpful discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Edith Gardiner, Bone and Mineral Research Program, Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, Sydney, NSW 2010, Australia, E-mail:
e.gardiner{at}garvan.unsw.edu.au
This work was supported by the National Health and Medical Research
Council of Australia, and an Australian Postgraduate Award (APA).
1 Current Address: Cell Biology Program Memorial Sloan-Kettering Cancer
Center, New York, New York 10021. 
Abbreviations: aa, amino acid; CHAPS,
3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate;
CMV, cytomegalovirus; DTT, dithiothreitol; EGFP, enhanced green
fluorescent protein; hVDR, human VDR; 24(OH)ase-Luc, 24-hydroxylase
promoter-luciferase reporter construct; PML, promyelocytic leukemia;
POD, PML oncogenic domain; PVDF, polyvinylidene difluoride; RNA PolIIo,
RNA polymerase II; TBP, TATA-binding protein.
Received for publication December 12, 2000.
Accepted for publication May 24, 2001.
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