A Negative Vitamin D Response DNA Element in the Human
Parathyroid Hormone-related Peptide Gene Binds to Vitamin D Receptor
Along with Ku Antigen to Mediate Negative Gene Regulation by Vitamin
D*
Toshihide
Nishishita
,
Tomoki
Okazaki
§,
Toshio
Ishikawa
,
Tetsuya
Igarashi
,
Keishi
Hata¶,
Etsuro
Ogata
, and
Toshiro
Fujita
From the
Endocrine Genetics and Hypertension Unit,
4th Department of Internal Medicine, University of Tokyo School of
Medicine, Bunkyo-ku, Tokyo 112, ¶ Mitsubishi Kagaku Bio-clinical
Laboratory Inc., Itabashi-ku, Tokyo 174, and
Cancer
Institute Hospital, Toshima-ku, Tokyo 173, Japan
 |
ABSTRACT |
We found that the human parathyroid
hormone-related peptide (hPTHrP) gene contained a DNA element
(nVDREhPTHrP) homologous to a negative vitamin D
response element in the human parathyroid hormone gene. It bound to
vitamin D receptor (VDR) but not retinoic acid X
receptor (RXR
)
in the human T cell line MT2 cells. VDR binding to this element was
confirmed by the Southwestern assay combined with immunodepletion using
anti-VDR monoclonal antibody, and this binding activity was repressed
by 1,25-dihydroxyvitamin D3. Such a repression was reversed
by acid phosphatase treatment, suggesting that 1,25-dihydroxyvitamin
D3 phosphorylates VDR to weaken its binding activity to
nVDREhPTHrP. In electrophoretic mobility shift assay,
we found anti-Ku antigen antibody specifically supershifted the MT2
nuclear proteinnVDREhPTHrP complex. The
nVDREhPTHrP-bearing reporter plasmid produced vitamin
D-dependent inhibition of the reporter activity in MT2
cells, which was markedly masked by the introduction of the Ku antigen
expression vector in the antisense orientation. On the other hand, such
a procedure did not perturb the vitamin D response element-mediated
gene stimulation by vitamin D. These results indicate that
nVDREhPTHrP interacts with Ku antigen in addition to VDR to
mediate gene suppression by vitamin D.
 |
INTRODUCTION |
The mechanism by which steroid/thyroid nuclear hormone receptors
activate gene transcription has been extensively studied (1-8). Among
them there have been so many lines of solid evidence showing that
vitamin D receptor (VDR),1
thyroid hormone receptor, and retinoic acid receptor employ a common
machinery to exert their ligand-dependent specific effects; all of them utilize a retinoic acid X receptor (RXR) in common as a
partner of a heterodimer (2, 4). However, this mechanism seems to be
confined only to gene stimulation but not to gene repression. This
situation led us to address one of the key points in hormonal biology,
negative feedback mechanism. The levels of almost all the hormones
synthesized at a specific organ are under rigid control to keep their
levels within a very narrow range. One of the representative mechanisms
is the so-called end product inhibition. In the cases of the nuclear
hormone receptors, negative transcriptional regulation of several
pituitary trophic peptide hormone genes by glucocorticoid or sex
steroid hormones is a good example. Although individual nuclear hormone
receptors might well be involved in such regulation, detailed unified
molecular mechanisms such as the manner of dimerization are largely
unknown, with a few exceptions (9-14). Likewise, the mechanism of
negative gene regulation by VDR and vitamin D is only partially
understood (15-21). In this case, an active form of vitamin D,
1,25-dihydroxyvitamin D3, can be considered an end product
of parathyroid hormone (PTH) action, and this metabolite, in turn,
inhibits the synthesis of PTH mRNA to keep the blood calcium level
constant. Demay et al. (16) first reported that a negative
vitamin D response element (nVDRE) exists in the upstream region of the
human PTH gene to mediate such gene repression. This element contains a
homologous sequence to only one of the two hexameric DNA sequences that
form the core sequence of the consensus DNA sequence (VDRE) for
positive gene regulation by vitamin D (2, 4, 22-25). In this process, it was shown that VDR, but not RXR, was involved, and the presence of
another unknown partner protein(s) of VDR was proposed (17). However,
there have been no reports confirming that the nVDRE is conserved
among the genes whose expression is negatively regulated by vitamin D. We have found that the human PTH-related peptide (PTHrP) gene, which is
assumed to be derived from an ancestoral gene in common with the PTH
gene (26), contains a DNA sequence very homologous to the nVDRE of
hPTH. Inoue et al. (27) reported that expression of the
hPTHrP gene was inhibited by 1,25-dihydroxyvitamin D3 at
the transcriptional level in human adult T cell lymphoma/leukemia virus-infected T cells, MT2 cells. With these cells, the therapeutic potential of vitamin D to decrease the blood calcium level due to the
inhibition of PTHrP synthesis was discussed. Unlike parathyroid cells,
from which it is hard to establish cultured cell lines, MT2 cells are
transfectable cultured cell lines. Here, we examined the molecular
mechanism of negative regulation of the PTHrP gene by vitamin D in MT2
cells.
 |
MATERIALS AND METHODS |
Homology Search by Computer Analysis--
The DNA sequences
homologous to the nVDRE of human PTH (16) were searched in the EMBL
gene bank, including the human PTHrP gene, and some of them are shown
in Fig. 1. We also compared the nVDRE with other DNA elements reported
to be responsible for negative as well as positive gene regulation by
vitamin D (16-24).
Synthetic Oligonucleotides and Plasmid Constructions--
All
the oligonucleotides used in this report were made by a DNA synthesizer
(Biosearch 8700). They were synthesized as follows: nVDREhPTHrP,
5'-(GATCC)TGCTATAGATTCATATTTGGTTTATA(T)-3';
3'-(G)ACGATATCTAAGTATAAACCAAATAT(AGATC)-5'. VDREmop,
5'-(GATCC)ACAAGTTCACGAGGTTCACGTCT(T)-3';
3'-(G)TGTTCAAGTGCTCCAAGTGCAGA(AGATC)-5'. Bases in the parentheses are
BamHI and XbaI cohesive ends to facilitate subsequent ligations to a BamHI-XbaI larger
fragment of PUTKAT1 (28). The methods to prepare double-stranded DNAs
and to construct the Tk promoter-based CAT reporter plasmids were also
described (28). The plasmids encoding p70 subunit of Ku antigen in the antisense orientation were described (29).
Transfection and CAT Assay--
MT2 cells were grown in RPMI
1640 medium supplemented with 10% fetal bovine serum unless otherwise
mentioned. The CAT plasmids or the expression vector encoding p70
subunits of Ku antigen in the antisense orientation (29) were
introduced into MT2 cells by the DEAE-dextran method (30). After
transfection, cells were equally split into several dishes to avoid
differences in transfectional efficiency among dishes. Final amounts of
the transfected plasmids per dish were 5 µg. Twelve h later, the
medium was changed to RPMI 1640 containing 1% fetal bovine serum with
several different concentrations of vitamin D metabolites (purchased
from Dupher Co.) or vehicle (100% ethanol) alone. In each case,
40 h after transfection, cells were harvested, and CAT assay was
performed. Where indicated, average CAT activity was calculated after
three separate transfections using 14C scintillation
counting, and typical results are shown in the figures with a given CAT
activity as 100 in each case. Details are shown in each figure.
Preparation of Nuclear Extracts and Gel Shift Assay--
Nuclear
extracts were prepared by the method of Schreiber et al.
(31) from MT2 cells 40 h after maintaining the cells in the media
as indicated. The synthetic oligonucleotides were end-labeled with
[
-32P]ATP by T4 polynucleotide kinase.
104 cpm of the probe (109 cpm/µg) was
incubated with 10 µg of the nuclear protein along with 1 µg of
poly(dI-dC) in each reaction for 30 min at room temperature. Final KCl
concentration was adjusted to 80 mM by the binding buffer containing no KCl (10, 31). The total reaction volume was 25 µl.
DNA-protein complexes were resolved on 4% nondenaturing acrylamide
gels, dried, and visualized by autoradiography as described (12-14).
Where indicated, 0-50-fold molar excesses of nonradiolabeled oligonucleotides were used as competitors. When anti-human VDR rat
monoclonal antibody, 9A7 (BIOMOL Research Laboratory Inc.), or
anti-human RXR
rabbit polyclonal antibody, D-20 (Santa Cruz Biotech.
Inc.) was used, each of 1/20 and/or 1/4 dilution and the respective
control IgGs (1 µl) supplied by manufacturers were included in the
reaction for 15 min before the addition of the radiolabeled probes. We
also used 1 µl of anti-Ku antigen human antiserum, which specifically
recognizes the p70 subunit of Ku antigen (29) and control serum in the
same way in electrophoretic mobility shift assay (EMSA). To explore the
effects of vitamin D metabolites on the protein-DNA interaction, cells
were treated in the same manner as described in transfection method.
Where indicated, 0.1 unit of potato acid phosphatase (Sigma) was
directly included in the reaction mixture at 37 C° for the indicated
period. Protein concentrations were determined by the Bio-Rad assay
kit.
Southwestern Analyses, Immunodepletion, and
Immunoblotting--
Thirty µg of the nuclear proteins obtained as
described above was mixed with an equal volume of 2× denaturing buffer
(5% N-lauroylsarcosine, 5 mM Tris-Cl (pH 6.8),
25% glycerol, 0.05% pyronin Y, and 200 mM
dithiothreitol). After a 15-min incubation at room temperature, samples
were loaded onto an 8% (or 10%) SDS-polyacrylamide gel. Further
details were described elsewhere (28), except that we used a
radiolabeled oligo(nVDREhPTHrP or VDREmop) in
this study. Where indicated, we used MT2 nuclear proteins in which VDR
was immunodepleted with the 9A7 shown above, according to the method reported previously (28). Immunoblotting with 1 µg/ml of the 9A7 or
1:1000 diluted anti-Ku antigen human antiserum also shown above was
performed by the enhanced chemiluminescence method as reported
(28).
 |
RESULTS |
Putative nVDRE Sequences in PTH and PTHrP Genes from Several
Species--
In the upstream region of the human PTHrP gene, we found
a DNA sequence homologous to a nVDRE in the hPTH gene (16). In the
anti-stranded sequence of this DNA in the hPTHrP gene, 11 out of 13 bases or 10 out of 11 bases were identical to the nVDRE of hPTH (Fig.
1). Both contained one copy of the DNA
motif AG(G/A)TTCA. This motif mimics a core sequence (AG(G/A)TCA) for
the binding of the specific set of the nuclear hormone receptor
including VDR (2). Interestingly, one VDRE found in the mouse
osteopontin gene (VDREmop; Ref. 22) consists of a direct
repeat of the same motif with two (or three) spacers. Furthermore, 4 bases, CTAT, flanking this motif were common between the two. These
findings suggest that both might mediate negative gene regulation by
1,25-dihydroxyvitamin D3. Falzon (18) recently reported
that the rat PTHrP gene possessed two distinct DNA elements (nVDREs),
both of which were responsible for negative gene regulation by vitamin
D. We compared our nVDRE of hPTHrP with Falzon's nVDREs in the rat
PTHrP gene (18) and speculated that one of them might be equivalent to
our nVDRE because of the similar location and overall sequence
similarity (see "Discussion"). Furthermore, we also found a DNA
sequence in the chicken TGF
2 gene very homologous to nVDREs of both
hPTH and hPTHrP. Intriguingly, expression of the chicken TGF
2 gene
was shown to be inhibited by vitamin D in mesangial cells (20). On the
other hand, as shown in Fig. 1, the sequences of other recently
proposed nVDREs such as those in the chicken PTH (21) gene and the rat
PTHrP gene (19) or positive VDRE in the human osteocalcin gene (23) are
considerably different from these three nVDREs.

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Fig. 1.
DNA sequences homologous to a putative
negative vitamin D response element (nVDRE) in the human PTHrP gene
(nVDREhPTHrP). The numbers shown above each
sequence represent the locations from the respective transcription
start sites. The bases similar to the consensus direct repeat,
AG(G/A)TTCA (2), are underlined. The identical bases between
nVDREhPTHrP and a given sequence are shown as bold
letters. The sources of each putative nVDRE are as follows:
nVDREhPTH (16), nVDRErPTHrP-1 and
nVDRErPTHrP-2 (18), nVDRErPTHrP-3 (19),
nVDREcPTH (21), and the nVDRE-like sequence of chicken
TGF 2 (20). One of the positive VDREs, VDREmop (22), was
also included.
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Interaction between Nuclear Protein(s) in MT2 Cells and
nVDREhPTHrP--
We synthesized oligonucleotides
corresponding to nVDREhPTHrP and VDREmop and
examined the interactions between each of these oligonucleotides and
nuclear protein(s) from MT2 cells by EMSA. As shown in Fig.
2, both of the oligonucleotides formed
two protein-DNA complexes. Since each of the lower bands did not
consistently appear, we focused on the upper bands in this study.
Although the migrating positions of both complexes seemed similar in
EMSA, the MT2 protein-nVDREhPTHrP complex was competed out
only by a 10-50-fold molar excess of the nonradiolabeled
nVDREhPTHrP. The same amount of VDREmop was
ineffective as a competitor. Conversely, only a 10-50-fold molar
excess of VDREmop, but not that of nVDREhPTHrP, was able to abolish the formation of MT2 protein-VDREmop
complex. Nevertheless, a 200-fold molar excess of the reciprocal
competitors in each EMSA similarly abolished the binding between MT2
nuclear proteins and each probe (not shown and see "Discussion").
These results indicate that the composition of the nuclear proteins is
different, at least in part, between the two protein-DNA complexes.

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Fig. 2.
EMSA using MT2 nuclear proteins and
nVDREhPTHrP (left panel) and
VDREmop (right panel) as radiolabeled
probes. On each panel, a 10-fold (lanes 2 and 4) and a 50-fold (lanes 3 and
5) molar excess of nonradiolabeled VDREmop
(lanes 2 and 3) or nVDREhPTHrP
(lanes 4 and 5) were included. The
arrows indicate the specific protein DNA complex.
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We next explored the possibility that VDR and RXR
, both of which
were shown to be crucially involved in VDREmop-mediated gene regulation by vitamin D in a wide variety of cells (2 and 4) other
than MT2 cells, were present in the two MT2 nuclear protein-DNA complexes shown here. As shown in Fig. 3,
both antibodies supershifted or attenuated the binding between
VDREmop and MT2 nuclear proteins. On the other hand, the
formation of the complex between nVDREhPTHrP and MT2
nuclear proteins was hardly affected by the addition of anti-RXR
antibody. Although the 1/4-diluted anti-VDR antibody weakened this
complex, it did not supershift the complex (lane 3) nor did
its 1/20 dilution affected the formation of the complex (lane
2), which was in contrast to the case of VDREmop and
MT2 nuclear proteins (lanes 4-6). Together, these results
suggest that nVDREhPTHrP-MT2 protein complex contained VDR
but not RXR
, and VDR might be included in this complex in a manner
different from the authentic VDR-RXR
heterodimer formation. Indeed,
MT2 cells are known to contain large amounts of VDR (27).

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Fig. 3.
VDR, but not RXR , exists in the
complex. The effects of anti-human VDR rat monoclonal antibody,
9A7 (A, lanes 2 and 5, 1 to 20 dilution; lanes 3 and 6, 1 to 4 dilution), or
anti-human RXR rabbit polyclonal antibody, D-20 (B,
lanes 8 and 10, 1 to 4 dilution) on the complex
between MT2 nuclear proteins and either nVDREhPTHrP
(lanes 1-3 and 7-8) or VDREmop
(lanes 4-6 and 9-10) in EMSA. In lanes
1 and 4, 1 µl of rat IgG2, and in lanes 7 and 9, 1 µl of preimmune rabbit IgGs were included. The
arrows indicate the specific protein-DNA complex.
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nVDREhPTHrP Is Necessary for Negative Gene Regulation
by Vitamin D in MT2 Cells--
To examine whether
nVDREhPTHrP is necessary for negative gene regulation by
vitamin D in MT2 cells (Fig. 4), we
transfected the cells either with the parental TkCAT (28) or with the
same CAT plasmid bearing nVDREhPTHrP in the sense
orientation. As shown in Fig. 4, 10
8 M
1,25-dihydroxyvitamin D3 selectively inhibited CAT activity driven by nVDREhPTHrP-TkCAT but not TkCAT. Furthermore,
less active vitamin D metabolites were ineffective.

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Fig. 4.
CAT assay in MT2 cells.
nVDREhPTHrP-TkCAT (A) or the parental TkCAT
(B) was introduced into MT2 cells. Cells were equally split
into five conditions after transfection. Forty h after the addition of
vehicle (veh) alone (lane 1), 10 9
M (lane 2) or 10 8 M
(lane 3) 1,25-dihydroxyvitamin D3 (1.25 D3, or 10 8 M
24,25-dihydroxyvitamin D3 (24.25 D3
(lane 4) or 10 8 M
25-dihydroxyvitamin D3 (25 D3
(lane 5), cells were harvested, and a CAT assay was
performed. Typical results were shown. The average CAT activity after
three different transfections is indicated below each condition. CAT
activity driven by TkCAT in MT2 cells treated with vehicle alone
(B, lane 1) was arbitrarily represented as 100.
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Vitamin D Attenuates the Binding between nVDREhPTHrP
and VDR--
Our next question was what is the effect of vitamin D on
the binding between nVDREhPTHrP and MT2 proteins. As shown
in EMSA (Fig. 5A),
1,25-dihydroxyvitamin D3 weakened the binding between nVDREhPTHrP and MT2 nuclear proteins, although it
strengthened the binding between VDREmop and these proteins
in a dose-dependent manner. In the Southwestern assay,
using a radiolabeled nVDREhPTHrP as a probe (Fig.
5B), similar dose-dependent effects of
1,25-dihydroxyvitamin D3 were observed on the band
corresponding to about 50 kDa in size but not on the other bands. We
then employed MT2 nuclear proteins in which VDR was immunodepleted with
the 9A7 shown above in the Southwestern assay. As shown in Fig.
5C, immunodepletion treatment clearly and selectively
abolished the binding of this 50-kDa band but not higher molecular mass
bands, strongly suggesting this 50-kDa band corresponded to the VDR
itself. On the other hand, the radiolabeled VDREmop yielded
only one band around 96 kDa in size, which was unaffected by the
immunodepletion. We assume these higher molecular weight proteins are
nonspecific DNA binding proteins. Immunoblotting with 9A7 revealed that
treatments with 1,25-dihydroxyvitamin D3 did not affect the
amounts of VDR protein (Fig. 5D). Together, these results
suggest that 1,25-dihydroxyvitamin D3 treatment weakens the
binding between nVDREhPTHrP and VDR in MT2 cells,
thereby exerting its inhibitory effect on the expression of
nVDREhPTHrP-bearing gene(s) in these cells.

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Fig. 5.
Dose-dependent effects of
1,25-dihydroxyvitamin D3 on VDR or the formation of
nVDREhPTHrP-MT2 nuclear protein complex. In
A, EMSA using radiolabeled VDREmop (lanes
1-4) or nVDREhPTHrP (lanes 5-8) and
nuclear proteins obtained from MT2 cells grown in the presence of
10 9 M (lanes 2 and 6),
10 8 M (lanes 3 and 7),
or 10 7 M 1,25-dihydroxyvitamin D3
(1.25(OH)2D3) (lanes 4 and 8) and vehicle alone (v, lanes 1 and
5) is shown. M, molarity. In B, a
Southwestern assay using the same set of MT2 protein and radiolabeled
nVDREhPTHrP is shown. The arrow indicates a
presumptive VDR monomer, which is strongly suggested in C.
In C, VDR was immunodepleted with the 9A7 (each,
right) or rat IgG2 (each, left) from nuclear
proteins obtained from untreated MT2 cells and Southwestern assay with
the radiolabeled nVDREhPTHrP (left panel) or
VDREmop (right panel) was performed. In
D, immunoblotting assay with the 9A7 and MT2 nuclear
proteins treated similarly to A and B are shown.
Immunoblotting of proteins corresponding to those in lane 1 with rat IgG2 alone is shown in lane 5. The arrow
indicates the position of human VDR. The higher molecular weight
proteins might be nonspecific proteins. In B, C,
and D, molecular weight markers are shown on the left
side.
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Ku Antigen, Along with VDR, Mediates Negative Regulation of the
Gene(s) Containing nVDREhPTHrP by Vitamin D--
In order
to find a clue for a probable partner of VDR in the
nVDREhPTHrP-MT2 protein complex, we reexamined the sequence
of nVDREhPTHrP and noticed that it contained an octamer
ATTTGCAT-like sequence following the core motif. This sequence,
ATTTGGTT, is reminiscent of one of the negative calcium-responsive
elements, oligo(A). We previously showed that the footprint in the
oligo(A) HeLa nuclear protein was localized exactly in this position
(15) and demonstrated that the nuclear protein specifically binding to
oligo(A) contained Ku antigen (29). Various roles of Ku antigen such as
double strand break repair or DNA recombination by virtue of its
ability to bind DNA ends nonspecifically have been reported (32-34).
However, a recent report demonstrating that Ku antigen recognizes a
specific internal DNA element in the mouse mammary tumor virus long
terminal repeat to inhibit glucocorticoid receptor-mediated transcriptional stimulation (35) prompted us to examine whether Ku
antigen is involved in nVDREhPTHrP-mediated gene inhibition by vitamin D. As shown in Fig. 6,
inclusion of anti-Ku antigen antibody (29) in EMSA significantly
reduced the nVDREhPTHrP-MT2 protein complex but not the
VDREmop-MT2 protein complex. Of note, this treatment seemed
to strengthen the binding activity of the faster migrating band when we
used nVDREhPTHrP as a probe. However, as noted earlier,
because the migrating position of the faster migrating band overlaps
that of the occasionally observed lower band (Fig. 2), we did not
pursue this issue in this manuscript.

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Fig. 6.
Ku antigen exists in the
nVDREhPTHrP-MT2 nuclear protein complex but not in the
VDREmop-MT2 protein complex. EMSA using MT2 nuclear
proteins and nVDREhPTHrP (lanes 1-2) or
VDREmop (lanes 3-4) is shown. One microliter of
preimmune human serum (lanes 1 and 3) or anti-Ku
antigen antibody (lanes 2 and 4, see Ref. 29) was
added in each lane. The solid arrows indicate the
specific DNA protein complexes (Fig. 2). The upper dashed
arrow in lane 2 indicates the supershifted band.
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To show the effect of Ku antigen on nVDREhPTHrP-mediated
gene repression by vitamin D in vivo, we transiently
introduced an expression vector encoding the p70 subunit of Ku antigen
in its antisense orientation (29) along with the TkCAT-based reporter plasmids bearing either nVDREhPTHrP or VDREmop.
Immunoblotting with anti-Ku antigen antiserum, which specifically
recognizes the p70 subunit of Ku antigen (Fig.
7A), revealed that this
treatment inhibited the synthesis of the p70 subunit of Ku antigen by
about 50%. As shown in Fig. 7B, although
1,25-dihydroxyvitamin D3 had similar repressive effects on
nVDREhPTHrP-CAT activity in MT2 cells transfected with an
empty vector, it lost the inhibitory action in MT2 cells when the p70
subunit of Ku antigen was expressed in the antisense orientation. On
the other hand, 1,25-dihydroxyvitamin D3 exerted similar
stimulatory effects on the CAT activity by VDREmop-TkCAT
with or without expression of Ku antigen.

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Fig. 7.
Involvement of Ku antigen in
nVDREhPTHrP-mediated CAT activity by vitamin D. A, immunoblotting of nuclear proteins obtained from the MT2
cells transiently transfected with an expression vector encoding the
p70 subunit in the antisense orientation (right; see Ref.
29) or a pRc-cytomegalovirus empty vector (left, E) with the
anti-Ku antigen antiserum, which specifically recognizes the p70
subunit (indicated by an arrow). Molecular mass markers are
shown on the left side. B,
nVDREhPTHrP-TkCAT (left panel) and
VDREmop-TkCAT (right panel) were introduced into
MT2 cells transiently transfected with a cytomegalovirus
promoter-driven pRc/CMVneo empty vector (Invitrogen) (lanes
1 and 2) or with the same vector encoding the p70
subunit of Ku antigen in its antisense orientation (lanes 3 and 4, see A and Ref. 29). After transfection,
cells were treated with vehicle alone (lanes 1 and
3) or 10 7 M
1,25-dihydroxyvitamin D3
(1.25(OH)2D3) (lanes 2 and 4). Typical results are shown. The average CAT activity
after three different transfections is indicated below each condition.
To focus on the effect of the antisense-oriented Ku antigen on the CAT
activity by vitamin D, CAT activity of each of the first
lanes was arbitrarily represented as 100.
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Phosphatase Treatment of VDR Restores Its Binding to
nVDREhPTHrP Once Attenuated by Vitamin
D--
Recently, Ku antigen was shown to have an inhibitory effect on
the glucocorticoid receptor-mediated transcriptional stimulation of the
mouse mouse mammary tumor virus long terminal repeat gene (35). It was
subsequently shown that the catalytic subunit of Ku antigen
(DNA-dependent protein kinase), along with both regulatory subunits of Ku antigen (p70 and p86), phosphorylates glucocorticoid receptor after binding to its specific binding DNA element called negative response element (36). Although the authors did not refer to
the direct role of this type of phosphorylation on gene repression
(36), their report prompted us to speculate that presumable
phosphorylation of VDR by the Ku antigen, which would have been
triggered by the treatment with 1,25-dihydroxyvitamin D3,
might weaken its activity to bind to nVDREhPTHrP, leading to vitamin D-mediated gene repression. To examine this possibility, we
treated nuclear proteins obtained from vehicle- or
1,25-dihydroxyvitamin D3-administered MT2 cells with potato
acid phosphatase for different times and examined their activity to
bind nVDREhPTHrP by the Southwestern assay as shown in Fig.
8. As expected, the binding of the VDR to
nVDREhPTHrP, once attenuated by the treatment with
10
7 µM 1,25-dihydroxyvitamin D3
(see Fig. 5), but not with vehicle alone, was up-regulated by the
phosphatase treatment. On the other hand, the binding of the
nonspecific bands above the VDR was not altered by the treatment. These
results suggest, albeit indirectly, that phosphorylation of VDR by a
certain kinase (e.g. DNA-dependent protein
kinase) might decrease its activity to bind to
nVDREhPTHrP.

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Fig. 8.
Phosphatase treatment of the nuclear proteins
from 1,25-dihydroxyvitamin
D3-preadministered MT2 cells up-regulates the binding of VDR to nVDRE>hPTHrP.
MT2 nuclear proteins obtained from vehicle alone (V) (first
and second lanes) or 1,25-dihydroxyvitamin D3
(1.25(OH)2D3) (10 7
M)-administered MT2 cells (third and fourth
lanes) were treated with 0.1 unit of potato acid phosphatase
(PAP) for 0 min (second and fourth lanes) or 30 min (first and third lanes) individually at 37 °C
followed by SDS-polyacrylamide gel electrophoresis and Southwestern
assay. The radiolabeled nVDREhPTHrP was used as a probe. An
arrow indicated the position of VDR (Fig. 5C).
Unlike VDR, whose binding activity was up-regulated by phosphatase
treatment, several upper nonspecific bands were not affected by the
treatment (compare the third and fourth lanes. On the other
hand, the intensity of VDR in the first and second lanes did
not differ. Molecular mass markers are shown on the left side.
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DISCUSSION |
We have shown that the human PTHrP gene contains a DNA element
(nVDREhPTHrP) very homologous to the negative vitamin D
response element found in the human PTH gene (16). Although it is in reverse orientation compared with the nVDREhPTH, we
demonstrated that nVDREhPTHrP functioned similarly to
nVDREhPTH in terms of transcriptional repression by vitamin
D. Sequence comparison between nVDREhPTHrP and
nVDREhPTH revealed 11 identical bases out of 13 nucleotides. Furthermore, it contains a heptameric AG(G/A)TTCA, mimicking a hexameric AG(G/A)TCA motif proposed to be a core element (2) for the binding of the nuclear receptors for thyroid hormone, vitamin D (VDR), or retinoic acid (retinoic acid receptor and RXR).
However, unlike VDRE, consisting of two such motifs separated by a
three-base spacer (Refs. 2 and 22; see VDREmop),
nVDREhPTHrP and nVDREhPTH possess only one
motif. This observation led us to speculate that the content of nuclear
proteins binding to the nVDREs was different from the usual VDR-RXR
heterodimer, as is found in the authentic VDRE (see below). On the
other hand, by computer search, we found the nVDRE motif in the
1100-bp upstream region of the mouse TGF
2 gene (Fig. 1). 12 out of
13 bases were identical between the two. The only difference lay in the
heptameric region. In the chick TGF
2 gene, AGATTCA in the hPTHrP
gene was changed to AGGTTCA, which was, in turn, identical to the
heptamer in the hPTH gene. Very interestingly, expression of this gene in the mesangial cells was reported to be inhibited by
1,25-dihydroxyvitamin D3 (20). This finding further
supports that nVDRE might play a general role in vitamin D-mediated
gene inhibition.
A couple of recent reports demonstrated that the rat PTHrP gene
contains several DNA sequences responsible for negative gene regulation
by vitamin D. Falzon (18) reported two nVDREs in the upstream region of
the rat PTHrP gene. He showed that both of them were necessary for
vitamin D-mediated gene inhibition. One of them located at the
726-bp
upstream region possesses one heptameric sequence, AGGTTCT. This
sequence is oriented similar to nVDREhPTHrP and in reverse
when compared with nVDREhPTH. The similarity between
nVDREhPTHrP and nVDREhPTH was found in the sequences adjacent to the heptamer. The five nucleotides, GTGCT, lying
7 to
3 bases preceding the core heptamer were identical between the
two nVDREs found in the human and rat PTHrP genes; such identity was
not observed in nVDREhPTH. Interestingly, this AGGTTCA was
repeated with a two-base spacer in the VDREmop (22). Like
our nVDREhPTHrP (Figs. 3 and 5C), the nVDRE in
the human PTH gene was reported to bind to VDR as well as another
nuclear protein(s) other than RXR (17). On the other hand, Falzon
reported another nVDRE at the
805-bp region of the rat PTHrP gene
(18). This element resembles a prototype DR3 VDREs, and similar nVDREs mimicking DR3 VDRE were reported to lie at the
1100-bp region of the
rat PTHrP gene (19) or at the
80-bp region of the chicken PTH gene
(21). VDR and probably RXR could bind to all of them, although it is
still unknown why this DR3-type structure was able to mediate negative
gene regulation by vitamin D.
Our competition EMSA experiments (Fig. 2) suggested that
nVDREhPTHrP and VDREmop bound to different sets
of MT2 nuclear proteins, although each DNA-MT2 nuclear protein complex
migrated similarly on a gel. Nonetheless, the use of a 200-fold molar
excess of the reciprocal competitors in each EMSA similarly abolished
the binding between MT2 nuclear proteins and each probe (not shown),
suggesting that each complex might share a common protein(s). The
obvious candidate of such a protein is VDR. Our EMSA employing the
anti-VDR and anti-RXR
antibodies revealed that, unlike the classical
interaction between VDR and RXR
found in the binding to the
authentic VDRE, such as VDREmop shown here (Fig. 4),
nVDREhPTHrP recognized only VDR, and the supershift pattern
in EMSA (Fig. 4) predicted that VDR might exhibit a different
conformation from that in the binding to RXR
, suggesting that
another unknown partner (16, 17) might be involved. Although we cannot
rule out the possibility that RXRs other than RXR
are involved here,
the usual partnership composed of VDR and RXR
, which is found in the
binding to the positive VDREmop as shown in Fig. 3, is not
predominant in the binding between nVDREhPTHrP and MT2
nuclear proteins. This notion was also supported by the Southwestern
assay, in which MT2 VDR was immunodepleted by the anti-VDR antibody
(Fig. 5C). In this assay, we demonstrated that
nVDREhPTHrP could not bind to 50-kDa nuclear proteins after
immunodepletion, whereas VDREmop did not bind to this band
irrespective of immunodepletion. This result suggests that
VDREmop, unlike nVDREhPTHrP, can bind to MT2
VDR only when the latter is associated with RXR
.
0n the other hand, our experiments using anti-Ku antigen antibody,
which recognizes one of its subunits, p70, demonstrated that it
supershifted the complex between MT2 nuclear proteins and
nVDREhPTHrP but not VDREmop (Fig. 3).
Furthermore, our experiments using the antisense-oriented Ku antigen
expression vector (Fig. 6) demonstrated that negative regulation by
vitamin D via the nVDREhPTHrP was abrogated by the
introduction of such a vector. Because such an abrogation was not
observed in the case of the VDREmop-TkCAT, we concluded
that Ku antigen was crucially involved in negative gene regulation by
vitamin D in MT2 cells. Although the mode of action of Ku antigen has
been currently under extensive study (32-34), only recently has its
role as a transcription factor been established (29, 33). Ku antigen
consists of two regulatory subunits, p70 and p86, as well as the
catalytic unit of another large protein of DNA-dependent
protein kinase, DNA-dependent protein kinase (32). Its
ability to bind DNA ends nonspecifically could explain its function
such as double strand break repair or DNA recombination. We (29)
recently demonstrated that p70 and p86 subunits of Ku antigen could
interact in association with another nuclear protein, redox factor 1 (1), with one of the negative calcium-responsive elements in a
sequence-specific manner. We showed that such an interaction led to
extracellular calcium-mediated transcriptional inhibition of the genes
bearing negative calcium-responsive elements. Furthermore, of
particular interest, the DNA sequence following the heptamer in the
nVDREhPTHrP, ATTTGGTT, was similar to the partial sequence
of one of the negative calcium-responsive elements, oligo(A) (15, 28),
ATTTGTGT. The latter sequence was protected from DNase digestion in
HeLa cells (15), and we had proposed that Ku antigen along with ref1
protein (29) bound to oligo(A) in a sequence-specific manner.
Therefore, we hypothesized that two different portions within the
nVDREhPTHrP bound to two different nuclear proteins, VDR
and Ku antigen, and that protein-protein interaction between VDR and Ku
antigen might not occur. Such an independent binding of Ku antigen and
another protein(s) to one DNA fragment was reported in mouse mammary
tumor virus long terminal repeat where Ku antigen recognized one
specific internal DNA sequence 20-30 bases away from the
glucocorticoid receptor binding site (35). However, since the
examination of the exact DNA binding manner of Ku antigen requires very
careful preparation of DNA samples due to the the Ku antigen nature of
binding DNA ends nonspecifically, proving this hypothesis has so far
been unsuccessful.
The underlying mechanism by which vitamin D weakens the binding between
nVDREhPTHrP and MT2 VDR as shown in Fig. 5 is currently unclear. Although there have been several reports describing
up-regulatory effects of vitamin D on the amount of VDR protein or on
its binding activity to VDRE (37), the mechanism by which vitamin D
inhibits the binding of VDR to nVDRE has not been addressed. One
attractive hypothesis is that Ku antigen might play some role in such
negative regulation as shown in this manuscript. Particularly, the
recent report suggesting that protein kinase activity of
DNA-dependent protein kinase, the catalytic subunit of Ku
antigen, would modulate glucocorticoid receptor activity after the
specific binding to DNA (35, 36) prompted us to carry out the
Southwestern assay in which the VDR from 1,25-dihydroxyvitamin
D3-administered MT2 cells was dephosphorylated by potato
acid phosphatase (Fig. 8). Although dephosphorylation of VDR did not
affect its binding to nVDREhPTHrP when VDR was obtained
from MT2 cells treated with vehicle alone, it reversed VDR binding to
nVDREhPTHrP pre-attenuated by the treatment with
1,25-dihydroxyvitamin D3. Therefore, we raise the
possibility that phosphorylation of VDR by the Ku antigen, which would
have been triggered by the treatment with 1,25dihydroxyvitamin D3, might weaken its activity to bind to
nVDREhPTHrP followed by vitamin D-mediated gene repression,
although direct evidence to support this contention is still
lacking.
If vitamin D mediates conditional inhibition of PTHrP gene as suggested
here, what is the significance of the constitutively active binding
between nVDREhPTHrP and MT2 nuclear protein(s) in the
unliganded state? We speculate that such a binding might be somewhat
related to constitutively active production of PTHrP in such cell lines
as MT2 cells, leading to humoral hypercalcemia of malignancy, the most
common cause of human hypercalcemia (26). We suggest here that a
therapeutic approach to targeting to the transcriptional apparatus
would be of potential benefit.
In this paper, we focused on the difference between positive and
negative VDREs as described above. Future investigation is directed to
the detailed analyses of the interaction between
nVDREhPTHrP and VDR including mutagenesis of
nVDREhPTHrP as well as characterization of VDR domain
involved here, all of which are now in progress in our
laboratory.
 |
ACKNOWLEDGEMENT |
We thank Dr. K. Ozono for providing us with
the anti-VDR human antibody we used in the initial pilot study and Dr.
A. Suwa for his gift of anti-Ku antigen antiserum.
 |
FOOTNOTES |
*
This work was supported by grants-in-aid for scientific
research from the Ministry of Education, Science, and Culture of Japan and by a grant from Araki Grant of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 81-3-3943-1151;
Fax: 81-3-3943-3102; E-mail: okbgeni-tky{at}umin.u-tokyo.ac.jp.
1
The abbreviations used are: VDR, vitamin D
receptor; PTH, parathyroid hormone; PTHrP, PTH-related peptide; hPTHrP,
human PTHrP; CAT, chloramphenicol acetyltransferase; EMSA,
electrophoretic mobility shift assay; Tk, thymidine kinase; VDRE,
vitamin D response element; VDREmop, VDRE in the mouse
osteopontin gene; RXR
, retinoic acid X receptor
; nVDRE,
negative vitamin D response element; nVDREhPTHrP, nVDRE in
the human PTHrP gene; nVDREhPTH, nVDRE in the human PTH
gene; bp, base pair.
 |
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