Enhancement of VDR-Mediated Transcription by Phosphorylation: Correlation with Increased Interaction Between the VDR and DRIP205, a Subunit of the VDR-Interacting Protein Coactivator Complex
Frank Barletta,
Leonard P. Freedman and
Sylvia Christakos
Department of Biochemistry and Molecular Biology (F.B., S.C.),
University of Medicine and Dentistry of New Jersey-New Jersey
Medical School and Graduate School of Biomedical Sciences, Newark, New
Jersey 07103; and Cell Biology and Genetics Program (L.P.F.), Memorial
Sloan-Kettering Cancer Center, New York, New York 10021
Address all correspondence and requests for reprints to: Dr. Sylvia Christakos, Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103. E-mail:
christak{at}umdnj.edu
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ABSTRACT
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When UMR-106 osteoblastic cells, LLCPK1 kidney cells, and VDR
transfected COS-7 cells were transfected with the rat 24-hydroxylase
[24(OH)ase] promoter (-1,367/+74) or the mouse osteopontin (OPN)
promoter (-777/+79), we found that the response to
1,25dihydroxyvitamin D3
[1,25-(OH)2D3] could be significantly
enhanced 2- to 5-fold by the protein phosphatase inhibitor, okadaic
acid (OA). Enhancement of 1,25-(OH)2D3-induced
transcription by OA was also observed using a synthetic reporter gene
containing either the proximal 24(OH)ase vitamin D response element
(VDRE) or the OPN VDRE, suggesting that the VDRE is sufficient to
mediate this effect. OA also enhanced the
1,25-(OH)2D3-induced levels of 24(OH)ase and
OPN mRNA in UMR osteoblastic cells. The effect of OA was not due to an
up-regulation of VDR or to an increase in VDR-RXR interaction with the
VDRE. To determine whether phosphorylation regulates VDR-mediated
transcription by modulating interactions with protein partners, we
examined the effect of phosphorylation on the protein-protein
interaction between VDR and DRIP205, a subunit of the vitamin D
receptor-interacting protein (DRIP) coactivator complex, using
glutathione-S-transferase pull-down assays. Similar to
the functional studies, OA treatment was consistently found to enhance
the interaction of VDR with DRIP205 3- to 4-fold above the interaction
observed in the presence of 1,25-(OH)2D3 alone.
In addition, studies were done with the activation function-2
defective VDR mutant, L417S, which is unable to stimulate
transcription in response to 1,25-(OH)2D3
or to interact with DRIP205. However, in the presence of OA, the mutant
VDR was able to activate 24(OH)ase and OPN transcription and to recruit
DRIP205, suggesting that OA treatment may result in a conformational
change in the activation function-2 defective mutant that creates an
active interaction surface with DRIP205. Taken together, these findings
suggest that increased interaction between VDR and coactivators such as
DRIP205 may be a major mechanism that couples extracellular signals to
vitamin D action.
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INTRODUCTION
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STEROID HORMONE ACTION is mediated through
intracellular receptors that function as transcription factors in
target cells (1). These receptors are comprised of modular
domains that possess ligand binding, DNA binding, dimerization, and
transactivation activities that are essential to their function. The
binding of ligand results in a conformational change of the steroid
receptor into an active form (2, 3, 4). The ligand-receptor
complex binds DNA sequences within target gene promoters and modulates
transcription (5). In addition to ligand binding, a number
of steroid receptors are activated by different signaling pathways:
growth factors [epidermal growth factor and IGF (6, 7)],
the neurotransmitter dopamine (8), activators of PKA
(9), and the protein phosphatase inhibitor, okadaic acid
(10, 11). Phosphorylation has been implicated in
transcriptional activation as well as repression (12). It
has been suggested that the phosphorylation of nuclear hormone
receptors may play a role in nuclear localization, hormone binding, DNA
binding, and cofactor recruitment (12).
1,25-Dihydroxyvitamin D3
[1,25-(OH)2D3] is a key
regulator of calcium homeostasis and has been reported to have
additional functions, including effects on differentiation, immune
function, and cell proliferation (13). The action of
1,25-(OH)2D3, the active
form of vitamin D, is mediated through the VDR. VDR is a member of the
superfamily of nuclear receptors, the activity of which is also
affected by phosphorylation-dependent signaling pathways
(14, 15, 16). VDR, like the PR (17), GR
(18, 19), AR (20, 21), ER (22, 23), and the RARs (RAR
, -ß, and -
) (24, 25, 26), is a
phosphoprotein. The majority of the phosphorylation of VDR is on serine
residues (27). VDR becomes hyperphosphorylated upon
1,25-(OH)2D3 treatment
(28, 29). It has been demonstrated that VDR can be
phosphorylated by PKA, PKC, and casein kinase II (30, 31, 32).
VDR, similar to other steroid receptors, exerts its transcriptional
activity by interaction with proteins of the preinitiation complex and
by interaction with coactivators that may bridge the interaction
between VDR and the basal transcription machinery (5, 33).
Steroid receptor coactivators (SRCs) include SRC-1/NcoA1,
GR-interacting protein 1 (GRIP-1)/transcriptional intermediary factor
2, and activator of thyroid and RARs (ACTR)/p300-CBP
cointegrator-associated protein (pCIP). These coactivators
exhibit histone acetylase (HAT) activity and can associate with
additional proteins that possess HAT activity, such as CREB binding
protein (CBP)/p300. HAT activity is thought to destabilize the
interaction between DNA and the histone core, liberating DNA for
transcription (34, 35). In addition to the SRC family of
coactivators, VDR-mediated transcription is also modulated by a
coactivator complex, DRIP [VDR-interacting protein, also called TRAP
and ARC, (33, 35)]. The DRIP complex does not possess HAT
activity but rather functions, at least in part, through recruitment of
RNA polymerase II (36). Due to the fact that steroid
receptors are phosphorylated, and given the interplay between these
steroid receptors and other proteins that may also be affected by
phosphorylation (37), it was important to examine the role
that phosphorylation plays in vitamin D-mediated gene expression. The
results of this investigation show that okadaic acid (OA), an inhibitor
of protein phosphatase 1 and 2A (PP-1 and PP-2A), enhances
1,25-(OH)2D3-dependent
transcription and suggest that this enhancement may be due, at least in
part, to an increased interaction between the VDR and the coactivator
protein DRIP205.
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RESULTS
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Enhancement of 1,25-(OH)2D3-Induced
24-Hydroxylase [24(OH)ase] Transcription by Inhibition of PP-1 and
PP-2A
To investigate the role of phosphorylation in
1,25-(OH)2D3-dependent
transcriptional activation, OA (an inhibitor of PP-1 and PP-2A) was
used to enhance overall cellular phosphorylation. Enhancement of
cellular phosphorylation resulted in an increase in
1,25-(OH)2D3-induced
24(OH)ase transcription. LLCPK1-, UMR-106-, or human VDR
(hVDR)-transfected COS-7 cells were transfected with the rat 24(OH)ase
promoter construct (-1,367/+74) and treated for 24 h with vehicle
(-D), 10-8 M
1,25-(OH)2D3 (+D), 50
nM OA (OA), or
1,25-(OH)2D3 and 50
nM OA (OA+D) (Fig. 1
).
Treatment with OA alone was unable to significantly increase
transcription over the levels observed with vehicle treatment. However,
the response to
1,25-(OH)2D3 was
significantly enhanced 2- to 5-fold by cotreatment with OA in all three
cell lines. Enhancement of
1,25-(OH)2D3-induced
transcription was also observed using 25 or 30 nM OA, and
maximal enhancement of chloramphenicol acetyltransferase
(CAT) activity was observed at 50 nM (not shown).

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Figure 1. Enhancement of
1,25-(OH)2D3-Induced 24(OH)ase Transcription by
Inhibition of PP-1 and PP-2A
LLCPK1, UMR-106, or VDR expression vector (pAVhVDR) transfected COS-7
cells were transfected with 3 µg of the CAT construct of the rat
24(OH)ase promoter -1,357/+74 (which contains both VDREs at
-258/-244 and at -151/-137). Transfected cells were treated with
vehicle (-D), 10-8 M
1,25-(OH)2D3 (+D), 50 nM OA (OA),
or 1,25-(OH)2D3 plus 50 nM OA (OA +
D) for 24 h. After treatments, cells were harvested and lysed, and
CAT activity was measured as described in Materials and
Methods. Shown are representative autoradiograms and the
graphic representation of three or more separate experiments for each
cell line. Longer radiographic exposure time was needed to visualize
CAT activity under basal conditions. The results are expressed as the
percent of maximal response ± SE after normalization
for ß-galactosidase activity. Transfection of the -1,357/+74 rat
24(OH)ase promoter construct and treatment with 10-8
M 1,25-(OH)2D3 resulted in a
16.5 ± 2.5-fold increase in CAT activity in LLCPK1 cells and a
22.7 ± 4- and a 24.5 ± 2-fold increase in CAT activity in
UMR and COS cells, respectively. In all three cell lines the
1,25-(OH)2D3 and OA cotreatment led to
significant enhancement of CAT activity compared with treatment with
1,25-(OH)2D3 (P <
0.05).
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Enhancement of 1,25-(OH)2D3-Induced
Osteopontin (OPN) Transcription by Phosphorylation
To determine whether the enhancement of
1,25-(OH)2D3-induced
transcription is specific for 24(OH)ase, we tested the ability of
OA to enhance
1,25-(OH)2D3-induced OPN
transcription. COS-7 cells were transfected with a mouse OPN promoter
construct (-777/+79) and pAVhVDR expression vector. After
transfection, the cells were incubated with vehicle (-D),
10-8 M
1,25-(OH)2D3 (+D), 50
nM OA (OA), or
1,25-(OH)2D3 plus 50
nM OA (OA + D) for 24 h followed by analysis of CAT
expression (Fig. 2
). Similar to the
experiments using the 24(OH)ase reporter construct (Fig. 1
), the
cotreatment with OA resulted in a 2- to 3-fold enhancement of
1,25-(OH)2D3-induced OPN
transcription. Unlike VDR-mediated 24(OH)ase transcription, OA, in the
absence of ligand, was found to activate VDR-mediated OPN transcription
(Fig. 2
). The transcription of the pRSV-CAT reporter construct, which
places CAT under the control of the Rous sarcoma virus (RSV) long
terminal repeat enhancer/promoter, was not affected by OA (Fig. 3
). The activity of a thymidine kinase
(tk) CAT construct, containing the minimal promoter region of the
herpes virus thymidine kinase gene and cotransfected with hVDR in COS-7
cells, was also not affected by 50 nM OA treatment (not
shown). The inability of OA to enhance transcription of the minimal
promoter constructs and experiments using the OPN promoter construct
indicate that the enhancement of
1,25-(OH)2D3-induced
24(OH)ase transcription by inhibition of PP-1 and 2A is neither
specific for 24(OH)ase nor due to a general increase in
transcription.

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Figure 2. Enhancement of 1,25-(OH)2D3
Induced OPN Transcription by OA
COS-7 cells were cotransfected with 500 ng of the VDR expression vector
pAVhVDR and 3 µg of a CAT reporter plasmid containing the -777/+79
region of the mouse OPN promoter (VDRE at -757/-743). Transfected
cells were treated with vehicle (-D), 10-8 M
1,25-(OH)2D3 (+D), 50 nM OA (OA),
or 1,25-(OH)2D3 plus 50 nM OA (OA +
D) for 24 h. After treatment, cells were harvested and lysed, and
CAT activity was measured as described in Materials and
Methods. A representative autoradiogram is shown. After
normalization for ß-galactosidase activity, the results are expressed
as the percent of maximal response and represent the mean of three
separate experiments ± SE. Treatment with
10-8 M 1,25-(OH)2D3
resulted in a 2.9 ± 0.4-fold increase in CAT activity over basal
(-D) levels. Cotreatment with 1,25-(OH)2D3
plus OA results in a significant enhancement of
1,25-(OH)2D3 induced OPN transcription
(P < 0.05).
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Figure 3. Enhancement of
1,25-(OH)2D3-Induced Transcription by
Inhibition of PP-1 and -2A is Not Due to a General Increase in
Transcription
UMR osteoblast-like cells, that contain endogenous VDR, were
transfected with 4 µg of a control reporter plasmid pRSV-CAT, which
places CAT under the control of Rous sarcoma virus long terminal
repeat. Cells were treated with vehicle or 50 nM OA for
24 h, harvested, and lysed, and CAT activity was measured. Shown
is a representative autoradiogram. Similar results were observed in
three experiments. OA treatment did not affect the transcription of the
pRSV-CAT reporter construct (P > 0.5; basal
vs. 50 nM OA treatment).
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Effect of OA on the Endogenous Expression of 24(OH)ase and OPN
mRNAs in UMR Cells
Northern blot analysis of mRNA from UMR-106 cells was performed to
assess the effect of enhanced phosphorylation on the endogenous
expression of 24(OH)ase and OPN mRNAs (Fig. 4
). UMR-106 cells were treated with
vehicle (-D), 1,25-(OH)2D3
(+D), OA alone (OA), or
1,25-(OH)2D3 and OA (OA +
D) for 24 h. 24(OH)ase and OPN mRNA levels were significantly
increased upon 1,25-(OH)2D3
treatment, and cotreatment with the phosphatase inhibitor led to an
enhancement over the
1,25-(OH)2D3-induced
levels. Similar to the findings using the OPN and 24(OH)ase promoter
constructs (Fig. 4
), OA alone resulted in a significant increase in OPN
mRNA but not in 24(OH)ase mRNA levels in UMR cells (Fig. 4
).

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Figure 4. OA Treatment Results in an Enhancement of
1,25-(OH)2D3-Induced 24(OH)ase and OPN mRNA
Levels
Northern analysis was performed using 8 µg poly (A+) RNA per lane
from UMR cells that had been treated with vehicle (-D),
1,25-(OH)2D3 (10-7 M;
+D), OA alone (50 nM), or
1,25-(OH)2D3 plus OA (50 nM) for
24 h. Right panel, Representative autoradiogram.
The filter was hybridized with 32P-labeled rat 24(OH)ase
cDNA and then stripped and rehybridized sequentially with
32P-labeled mouse OPN and ß-actin cDNAs. Left
panel, Graphic representation of the data normalized on the
basis of results obtained after rehybridization with ß-actin cDNA.
The results of four separate experiments are expressed as percent
maximal response ± SE. A significant induction of
24(OH)ase and OPN mRNA levels compared with
1,25-(OH)2D3-induced levels was observed with
cotreatment with OA + 1,25-(OH)2D3
(P < 0.05). 24(OH)ase mRNA was not observed under
basal conditions (-D) or after treatment with OA alone even after
longer autoradiographic exposure time. OA alone significantly induced
OPN mRNA levels above the levels observed under basal (-D) conditions
(P < 0.05).
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Mechanism of the Effect of Phosphorylation on
1,25-(OH)2D3-Induced Transcription
To determine a mechanism by which OA enhances
1,25-(OH)2D3-induced
VDR-mediated transcription, we examined the effect of OA treatment on
VDR expression. Northern (Fig. 5A
) and
Western blot (Fig. 5B
) analyses were performed using mRNA and protein
from UMR-106 cells. VDR mRNA and protein levels were induced upon
treatment with
1,25-(OH)2D3. However,
there was no increase in the expression of VDR mRNA or VDR protein
after treatment with
1,25-(OH)2D3 plus OA as
compared with levels observed after treatment with
1,25-(OH)2D3 alone (Fig. 5
, A and B). There was also no change in VDR mRNA or protein levels when
compared with basal (-D) levels after treatment with OA alone. These
findings indicate that the OA-mediated enhancement of transcription is
not due to an increase in VDR expression.

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Figure 5. Enhancement of
1,25-(OH)2D3-Induced Transcription by
Inhibition of PP-1 and PP-2A is Not Due to an Increase in VDR
Expression
A, Northern analysis was performed using 8 µg poly (A+) RNA per lane
from UMR cells that had been treated with vehicle (-D),
1,25-(OH)2D3 (10-7 M;
+D), OA (50 nM), or 1,25-(OH)2D3
plus OA (OA + D) for 24 h. A representative autoradiogram is
shown. The filter was hybridized with 32P-labeled VDR cDNA.
The blot was stripped and rehybridized with 32P-labeled
ß-actin cDNA. Treatment with 1,25-(OH)2D3
plus OA did not result in any significant changes in VDR mRNA levels
compared with treatment with 1,25-(OH)2D3 alone
(+D, 1.6 ± 0.2, OA + D 1.5 ± 0.2-fold induction over basal;
P > 0.5; results of three experiments). B, Western
blot analysis was performed using 50 µg of nuclear protein prepared
from UMR cells treated with vehicle (-D),
1,25-(OH)2D3 (+D), OA, or
1,25-(OH)2D3 plus OA (OA + D) as described in
panel A for 24 h. Detection was by immunoblotting with a
monoclonal anti-VDR antibody. An increase in VDR protein levels was not
observed when comparing 1,25-(OH)2D3 and
1,25-(OH)2D3 plus OA-treated cells (80% of the
VDR protein levels observed after treatment with
1,25-(OH)2D3 was observed after treatment with
OA and 1,25-(OH)2D3).
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Gel shift analysis was performed to determine whether OA may modify the
binding of VDR/RXR to the vitamin D response element (VDRE) (Fig. 6
). Binding reactions were done
using nuclear extracts prepared from UMR cells and an
oligonucleotide containing the proximal 24(OH)ase VDRE. Protein-DNA
interaction was induced using nuclear extracts from
1,25-(OH)2D3-treated UMR
cells. No further increase in DNA binding activity was observed using
nuclear extracts prepared from
1,25-(OH)2D3 plus
OA-treated cells (Fig. 6
). When nuclear extracts were prepared from
cells treated with OA alone, the protein-DNA interaction was similar to
that obtained under basal (-D) conditions (Fig. 6
; -D and OA). These
results suggest that the effect of OA is not due to an increase in
VDR/RXR binding to the VDRE.

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Figure 6. OA Treatment Does Not Enhance VDR-RXR Interactions
with the VDRE
Gel mobility shift assay using 3 µg of nuclear protein prepared from
UMR cells treated with vehicle (-D),
1,25-(OH)2D3 (+D), OA, or
1,25-(OH)2D3 plus OA (OA +D) for 24 h and
32P-labeled oligonucleotide containing the 24(OH)ase VDRE
(-150/-136). Preincubation with cold VDRE or monoclonal VDR antibody
(anti-VDR) depleted the binding of the VDR/RXR to the labeled probe.
Data are representative of three experiments.
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To test whether the enhancement by OA occurs through the VDRE and not
via other sequences in the promoter, we examined the effect of OA using
the rat proximal 24(OH)ase VDRE-tk CAT construct or the mouse OPN
VDRE-tk CAT construct (Fig. 7
). The
cotreatment of 1,25-(OH)2D3
and OA enhanced the
1,25-(OH)2D3-induced
transcription of the VDRE-tk CAT constructs, similar to the effect seen
with the larger promoter constructs, indicating that the VDRE is
sufficient to mediate the effect of phosphatase inhibition. Similar to
the activity of the -1,367/+74 24(OH)ase promoter CAT construct (Fig. 1
), 24(OH)ase VDRE-tk CAT activity was not significantly affected by
treatment with OA alone when compared with basal (-D) levels (Fig. 7
;
OA, left panel). Although the response to OA alone using the
-777/+79 OPN promoter construct was equivalent to the
1,25-(OH)2D3 response (Fig. 2
), the response of the OPN VDRE-tk CAT construct to OA alone was
1.5 ± 0.3-fold over basal [P > 0.1; basal (-D)
vs. OA treatment] and not equivalent to the
1,25-(OH)2D3 response
(5.6 ± 0.9-fold induction). The inability of OA alone to increase
the transcription of the OPN-tk CAT construct suggests that the
previously observed activation by OA (Fig. 2
) may be mediated through
promoter elements other than the VDRE. These findings suggest that
sequences in the OPN promoter contribute to the ligand-independent
induction of OPN expression and transcription.

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Figure 7. Enhancement of
1,25-(OH)2D3-Induced VDRE-tk CAT Transcription
by OA
COS-7 cells were cotransfected with 500 ng of the VDR expression vector
pAVhVDR and 3 µg of a CAT reporter plasmid containing multiple copies
of the rat 24(OH)ase VDRE (-150/-136) or the mouse OPN VDRE
(-757/-743). Transfected cells were treated with vehicle (-D),
10-8 M 1,25-(OH)2D3
(+D), OA (50 nM), or 1,25-(OH)2D3
plus 50 nM OA (OA + D) for 24 h. After treatments
cells were harvested and lysed, and CAT activity was measured as
described in Materials and Methods. Representative
autoradiograms are shown. The results of three separate experiments are
expressed as the percent of maximal response ± SE
after normalization for ß-galactosidase activity. Cells transfected
with the 24(OH)ase VDRE-tk CAT construct or the OPN VDRE-tk CAT
construct and treated with 10-8 M
1,25-(OH)2D3 exhibited a 9.4 ± 0.2-fold
and a 5.6 ± 0.9-fold induction in CAT activity, respectively.
Longer radiographic exposure time was needed to visualize CAT activity
under basal conditions. Cotreatment with
1,25-(OH)2D3 plus OA results in an enhancement
of 1,25-(OH)2D3-induced VDRE-tk CAT
transcription (P < 0.05).
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Previous studies have indicated that OA can induce phosphorylation of
VDR (14, 43). To examine whether phosphorylation of VDR is
a mechanism involved in the enhancement of
1,25-(OH)2D3-induced
transcription by OA, we used VDR mutants that have mutations in two
major phosphorylation sites, serine 208 [phosphorylated by casein
kinase II (32)] and serine 51 [phosphorylated by PKC
(31)]. Using mutant VDR expression vector constructs S51A
and S208A (mutation of serine to alanine), the rat 24(OH)ase
-1,367/+74 promoter construct and transfection in COS-7 cells, a
3.9 ± 0.5- and a 3.8 ± 0.2-fold enhancement, respectively,
of 1,25-(OH)2D3-induced
transcription was still observed in the presence of OA, suggesting that
the enhancement is not due to phosphorylation of these specific
sites.
To further investigate the mechanism involved in the
phosphorylation-dependent enhancement of VDR-mediated transcription, we
used glutathione-S-transferase (GST) pull-down assays to
assess any effect that OA may have on the binding of VDR to cofactor
proteins. The binding of VDR to two cofactors, DRIP205 and GRIP-1, was
analyzed (Fig. 8
). The interaction of VDR
to each cofactor was increased when nuclear extracts were used from
1,25-(OH)2D3-treated
VDR-transfected COS cells, consistent with previous results indicating
that VDR cofactor interactions are
1,25-(OH)2D3 dependent
(36, 38). The cotreatment of
1,25-(OH)2D3 and OA was
unable to increase GRIP-1 and VDR interaction over that seen with
1,25-(OH)2D3 alone (Fig. 8A
). However, the interaction between VDR and DRIP205 was significantly
increased when nuclear extracts were used from
1,25-(OH)2D3- and
OA-treated cells as compared with extracts treated with
1,25-(OH)2D3 alone
(P < 0.05) (Fig. 8B
). Treatment with OA alone resulted
in a VDR-DRIP interaction that was 32.5% of the interaction observed
with 1,25-(OH)2D3 and OA
treatment. Additional GST pull-down experiments using VDR that was
in vitro phosphorylated by purified casein kinase II did not
exhibit increased interaction with in vitro translated
DRIP205 (not shown), suggesting that phosphorylation of VDR on serine
208 is not involved in the OA-mediated enhancement of VDR-DRIP205
interaction.

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Figure 8. Phosphorylation-Mediated Enhancement of VDR-DRIP205
Interaction
A, GST pull-down assay was performed using 15 µg of GST-GRIP-1 fusion
protein and equal amounts of VDR protein from nuclear extracts of VDR
expression vector, pAVhVDR, transfected COS-7 cells treated as
described in Fig. 7 with vehicle (-D),
1,25-(OH)2D3 (+D), OA, or
1,25-(OH)2D3 plus OA (OA +D) for 24 h.
Western blot analysis with a monoclonal anti-VDR antibody was performed
to visualize VDR binding. B, GST pull-down assays were performed as in
panel A except using GST-DRIP205 (527970) fusion protein instead of
GST-GRIP-1. Representative Western blots are shown (left
panels, A and B). The results of three to six separate
experiments are expressed as relative signal intensity (100 =
maximal intensity) ± SE after normalization to VDR
input (right panels, A and B).
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Additionally, we evaluated the role of the activation function-2 (AF-2)
domain of VDR in transcriptional activation by OA. The
C-terminal helix 12, the core AF-2 domain, contains residues that
function as an interaction surface for coactivators (33).
We used the AF-2 defective mutant L417S (the leucine residue 417 at the
C terminus of the predicted helix 12 in VDR was mutated to serine).
When COS-7 cells were cotransfected with the L417S mutant VDR and the
rat proximal 24(OH)ase VDRE-tk CAT construct or the mouse OPN
VDRE-tk CAT construct,
1,25-(OH)2D3-dependent
transcriptional activation was not observed [Fig.
9, A and B (-D, +D)]. The failure of
the AF-2 defective VDR to induce transcription in response to
1,25-(OH)2D3 correlates
with the inability of the L417S mutant VDR to interact with DRIP205 in
the presence of
1,25-(OH)2D3 (Fig. 9C
, -D,
+D). In contrast, in the presence of OA, the AF-2 defective mutant was
able to activate the VDRE-tk CAT constructs in a ligand-independent
manner (3- to 4-fold induction compared with basal, P
< 0.05). Similar findings were observed using the -1,367/+74 rat
24(OH)ase promoter construct or the -777/+79 mouse OPN promoter
construct. The activity of a tk CAT construct cotransfected with L417S
VDR was not affected by OA treatment (not shown). Thus, activation of
VDR-mediated transcription by phosphorylation can be independent of the
conserved leucine in the AF-2 domain. In addition, phosphorylation
rescued the recruitment of DRIP205 to the AF-2 defective mutant (Fig. 9C
). These results suggest, similar to the findings shown in Fig. 8
, that stimulation of transcription by inhibition of phosphatase involves
increased interaction between VDR and the DRIP coactivator
complex.

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Figure 9. Activation of AF-2 Defective VDR Mutant by
Phosphorylation
COS cells were cotransfected with AF-2 defective VDR (L417S VDR) and
either the 24(OH)ase VDRE-tk CAT construct (A) or the OPN VDRE-tk CAT
construct (B). Transfected cells were treated as previously described
with vehicle (-D), 1,25-(OH)2D3 (+D), OA, or
1,25-(OH)2D3 plus OA (OA + D) for 24 h.
After treatment, cells were harvested and lysed, and CAT activity was
measured as described in Materials and
Methods. Representative autoradiograms are shown. The
results of three separate experiments are expressed as the percent of
maximal response ± SE after normalization for
ß-galactosidase activity. C, GST pull-down assay was performed using
15 µg of GST-DRIP205 fusion protein and equal amounts mutant VDR
(L417S) protein from nuclear extracts of transfected COS-7 cells
treated as previously described with vehicle (-D),
1,25-(OH)2D3 (+D), OA, or
1,25-(OH)2D3 plus OA (OA +D) for 24 h.
Western blot analysis was performed with a monoclonal anti-VDR antibody
to visualize VDR binding. Representative VDR Western blots are shown
(left panel). The results of three or more separate
experiments are expressed as relative signal intensity (100 =
maximal intensity) ± SE after normalization to VDR
input.
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|
 |
DISCUSSION
|
---|
The results of this study suggest that inhibition of cellular
phosphatases can enhance
1,25-(OH)2D3-induced
transcription and mRNA expression of 24(OH)ase and OPN, at least in
part, by enhancing cofactor recruitment. This is the first report of a
mechanism involved in the enhancement of
1,25-(OH)2D3-induced
transcription by inhibition of protein phosphatases. The binding of
DRIP205 to VDR but not the VDR/GRIP-1 interaction was enhanced. The
DRIP complex lacks HAT activity characteristic of GRIP-1 and other p160
coactivators, and the individual DRIP subunits, including DRIP205, are
not homologous to other known coactivators (36). In
addition, GRIP-1 is structurally unrelated to DRIP205 except for the
presence of a LXXLL motif that is required for the interaction with the
ligand-binding domain (LBD) of nuclear receptors (35),
and the specific amino acids needed for VDR binding to the two
coactivators may indeed be different (36). The
20-epi-analogs of
1,25-(OH)2D3 are more
potent than 1,25-(OH)2D3
and enhance cellular responses, such as differentiation of myeloid
cells and p21 expression (39). Interestingly, these
analogs also result in enhanced recruitment of DRIP205 but not GRIP-1
to VDR (39). Taken together with our results, this
indicates that the DRIP complex is an important and perhaps preferred
coactivator complex not only for the enhanced response to 20-epi
analogs but also for enhancement of VDR-mediated transcription by
phosphorylation.
OA enhanced
1,25-(OH)2D3-induced
VDR-mediated transcription without increasing VDR levels or the
1,25-(OH)2D3-dependent
binding of VDR-RXR to the VDRE. Enhancement of hormone-activated
receptor-mediated transcription by OA has also been reported using
human and chick PR (10, 40), rat GR (41),
human RAR
and RARß, human RXR
, and mouse RXRß and RXR
(15). Similar to our study, Beck et al.
(40) using the human PR and Moyer et al.
(41) using the rat GR reported that the enhancing effects
of OA are not due to changes in receptor content. Beck et
al. (40) also noted that receptor DNA binding was
unaffected by OA treatment. In these studies OA was found not to alter
receptor phosphorylation. It was suggested that OA could possibly
modulate transcription by altering the interaction with coactivators
that may function as intermediaries between the receptor and the basal
transcription machinery (40).
The phosphorylation of human VDR involves serine residues in distinct
functional domains (30, 31, 32, 42). The phosphorylation of
VDR by casein kinase II at serine 208 in the ligand binding domain has
been reported to account for 60% of the phosphorylation of the
receptor and to play an important role in transcriptional activation
(16). In previous studies, metabolic
32P labeling and measurement of total VDR
phosphorylation using ROS17/2.8 cells or VDR-transfected CV1 cells
indicated that OA can induce phosphorylation of VDR (14, 43). In this study we found that the OA enhancement of
1,25-(OH)2D3-induced
VDR-mediated transcription was not abolished using VDR mutants with
either serine 208 or serine 51 phosphorylation sites mutated to
alanine. In addition, casein kinase II phosphorylation of VDR did not
result in enhanced binding of VDR to DRIP205. Although OA-mediated
transcriptional enhancement was observed using the mutant VDRs, it is
possible that novel sites may be phosphorylated as a result of OA
treatment, resulting in an increased interaction with DRIP205 binding.
Hilliard et al. (42) found that mutations in
serine 208 and 51 could not eliminate VDR phosphorylation and that
these mutations cause subsequent phosphorylation on amino acids that
are not normally modified. It is also possible that OA may be acting by
enhancing the phosphorylation of another protein that may stabilize the
formation of the VDR-DRIP205 complex, resulting in enhanced VDR-DRIP
interaction observed in this study. Mapping of VDR phosphorylation
sites in response to inhibition of the cellular phosphatases will be
required to determine whether OA increases site-specific
phosphorylation of VDR. Knowledge of the crystal structure of VDR
(4) and computer modeling of the conformational changes
upon ligand binding and hyperphosphorylation will facilitate the
identification of the exact mechanism by which phosphorylation enhances
receptor-coactivator interactions and transactivation.
In this study we found that although OA can enhance
1,25-(OH)2D3-induced
VDR-mediated transcription of both 24(OH)ase and OPN, significant
ligand-independent activation by OA was observed only for the OPN
promoter construct -777/+79. OA alone was unable to stimulate
VDR-mediated transcription using the OPN VDRE-tk CAT construct, the
24(OH)ase VDRE-tk CAT construct, or the 24(OH)ase promoter
(-1,367/+74). These findings suggest, similar to what has been
reported for the AR (44, 45), that ligand-independent
activation depends on promoter context. However, the refractory effect
is lost in the context of the AF-2 defective VDR mutant. In contrast to
the action of 1,25-(OH)2D3,
in the presence of OA, the AF-2 defective mutant was able to activate
both VDRE-tk CAT constructs, and OA alone was able to induce an
interaction between DRIP205 and the mutant VDR (Fig. 9
). These results
suggest a subtle conformational effect in the AF-2 defective mutant,
not as yet understood, that is more permissive to ligand-independent
activation by OA. Recent studies have indicated that the Ets-1
transcription factor, a target of the Ras-MAPK signaling pathway, can
also result in a ligand- and AF-2-independent activation of nuclear
receptors, including VDR (46). It was suggested that Ets
acts by inducing a conformational change in the receptor that creates
an active interaction surface with coactivators even in the AF-2
defective mutant (46). The results obtained using Ets-1
(46), as well as our findings using OA and the AF-2
defective VDR mutant, suggest novel mechanisms by which inactive VDR
can be converted to an effective transcriptional activator.
In addition to factors that may induce a conformational change in the
receptor, previous studies have shown that species specificity of the
steroid receptor and cell type are also important in determining
ligand-independent activation (10, 40, 44, 47).
Ligand-independent activation by modulation of cellular phosphorylation
has been reported for chick PR (10) but not for human PR
(40). Also, Chinese hamster ovary cells stably transfected
with human AR do not exhibit ligand-independent transcriptional
activation (44). However, studies performed in CV1 cells
and PC-3 cells indicated ligand-independent activation of hAR
(47). Ligand-independent transcriptional activation of VDR
has been noted using CV1 cells and either the promoter of the human
osteocalcin (OC) gene (14) or multiple copies of the rat
OC VDRE ligated to tk CAT (15). However, when the rat OC
promoter or the rat OC VDRE-tk CAT was used to transfect ROS17/2.8
cells, ligand-independent activation by OA was not observed
(43). The cell type and species specificity of
ligand-independent activation may be due to the relative abundance of
coactivators in different cell types and differences in the interaction
of specific nuclear receptors with coactivators.
Our findings suggest that the enhancement of
1,25-(OH)2D3-induced
transcription by extracellular signals involves
phosphorylation-dependent modulation of cofactor recruitment.
Phosphorylation-mediated cofactor interaction was first shown to be
important for the recruitment of CBP by the cAMP response element
binding protein (CREB) (48). More recent studies
have shown that cofactor recruitment by PPAR
and TR is also affected
by phosphorylation. EGF was found to enhance binding of PPAR
to
the corepressor SMRT (silencing mediator of retinoid and thyroid
hormone receptor) (49). However, phosphorylation of SMRT
by MEKK1 inhibits the binding of SMRT to TR (50).
Phosphorylation of ERß by MAPK leads to ligand-independent activation
and recruitment of the coactivator SRC-1 (51).
Phosphorylation of the orphan receptor SF-1 by MAPK has also been
reported to enhance cofactor recruitment (52). It was
suggested that enhanced cofactor recruitment could then modulate the
coordinate regulation of multiple SF-1 target genes. Most recently,
phosphorylation of SRC-1, induced by cAMP on two MAPK kinase sites
(threonine 1,179 and serine 1,185), was found to be necessary for
optimal transactivation by PR and for optimal interaction between SRC-1
and p300/CBP-associated factor (53). Thus, it is
becoming increasingly evident that phosphorylation-dependent
cofactor recruitment plays an important role in the cross-talk between
steroid receptors and signal transduction pathways. It will be of
interest in the future to further consider the functional significance
of cofactor phosphorylation as well as nuclear receptor phosphorylation
and to examine the levels of various cofactors present endogenously in
specific cell types. Differences in cofactor levels may account for the
differential regulation of steroid-responsive genes in multiple cell
types in response to activation of signaling pathways.
Although a number of previous studies have shown that inhibition of
phosphatases can enhance steroid hormone-induced transcription
(10, 11, 14, 15, 40, 41), our study is the first to show
that endogenous VDR-mediated responses can be affected by OA treatment.
1,25-(OH)2D3 induction of
24(OH)ase and OPN mRNAs was enhanced in UMR osteoblastic cells by
the inhibition of phosphatases. 24(OH)ase hydroxylates
25(OH)D3 and
1,25-(OH)2D3 and is
involved in the catabolism of
1,25-(OH)2D3
(54). Thus, by inducing the 24(OH)ase enzyme,
1,25-(OH)2D3 induces its
own deactivation. The 24(OH)ase gene is the most transcriptionally
responsive vitamin D-inducible gene identified to date
(54). OPN, a phosphorylated protein abundant in the bone
matrix, is also induced by
1,25-(OH)2D3 and has been
reported to modulate both mineralization and bone resorption
(55). Previous studies in endogenous systems,
including osteoblastic cells, have indicated that both OPN and
24(OH)ase can be modulated by factors that affect signal transduction
pathways (55, 56, 57, 58, 59). The physiological significance of the
enhancement of 1,25-(OH)2D3
induction of 24(OH)ase in osteoblastic cells by activation of signaling
cascades may be to contribute to the prevention of elevated
intracellular 1,25-(OH)2D3
levels that can adversely affect mineralization (60).
Enhancement of 1,25-(OH)2D3
induction of OPN expression by activation of signaling cascades can
result in enhanced effects on mineralization or resorption. It will be
important in future studies to identify the phosphorylation sites in
the coactivator and/or the VDR as well as to identify the specific
kinases that catalyze the phosphorylations involved in 24(OH)ase-
and OPN-enhanced transcription.
In summary, our findings suggest that increased interaction between VDR
and specific coactivators is needed for enhanced expression of
1,25-(OH)2D3 target genes
in response to activation of signaling pathways and that differential
cofactor recruitment mediated by phosphorylation may be a major
mechanism involved in the integration of signal transduction pathways
and vitamin D action.
 |
MATERIALS AND METHODS
|
---|
Materials
[14C]Chloramphenicol (50 mCi/mmol),
[
-32P]-dCTP [3,000 Ci (111 TBq)/mmol],
[
-32P]-ATP [3,000 Ci (111 TBq)/mmol],
Easytag L-[35S]-methionine
[>1,000 Ci (37.0 TBq)/mmol], nylon membranes, and enhanced
chemiluminescent (ECL) detection system were purchased from
NEN Life Science Products (Boston, MA). Casein kinase II
and TNT Coupled Reticulocyte Lysate System were from Promega Corp. (Madison, WI). Acetyl coenzyme-A, formamide, and OA
(sodium salt) were obtained from Sigma (St. Louis, MO).
DMEM plus Hams F12 nutrient mixture (DMEM-F12), DMEM, Medium 199,
Random Primers DNA Labeling kit, T4 polynucleotide kinase, and oligo
(dt) cellulose were purchased from Life Technologies, Inc.
(Gaithersburg, MD). RNAzol was obtained from Tel-Test
(Friendswood, TX). Rat monoclonal anti-VDR antibody was purchased from
Affinity BioReagents, Inc. (Neshanic Station, NJ).
1,25-(OH)2D3 was a generous
gift from M. Uskokovic (Hoffmann-LaRoche Inc., Nutley,
NJ).
Cell Culture
COS-7 African green monkey kidney cells, UMR-106 rat
osteoblastic cells, and LLCPK1 porcine kidney cells were obtained from
American Type Culture Collection (ATCC,
Manassas, VA) and were cultured in DMEM supplemented with 10%
heat-inactivated FBS from Gemini Biological Products
(Calabasas, CA), DMEM-F12 supplemented with 5% FBS, and Medium 199
supplemented with 3% FBS, respectively. All cell lines were
grown in a humidified atmosphere of 95% O2-5%
CO2 at 37 C. Cells were grown to 6070%
confluence in 100-mm2 tissue culture dishes, and
24 h before the start of experiments medium was replaced with
medium supplemented with 2% charcoal-stripped serum. Cells were
treated with vehicle or the compounds noted at the concentrations and
times indicated.
Transfection and CAT Assay
Promoter constructs containing the rat 24(OH)ase promoter
(-1,367/+74) (61) or the mouse OPN promoter (-777/+79)
(62) linked to the CAT reporter gene were used. A tk CAT
reporter plasmid containing multiple copies of the rat 24(OH)ase VDRE
(-150/-136) (63) was also used. The mouse OPN VDRE-tk
CAT construct, containing multiple copies of the mouse OPN VDRE
(-757/-743) was prepared in the laboratory of L. P. Freedman.
All cells were cotransfected with the appropriate reporter plasmid and
a ß-galactosidase expression vector (pCH110, Pharmacia Biotech, Piscataway, NJ) as an internal control for transfection
efficiency, using the calcium phosphate DNA precipitation method
(64). In addition to the reporter plasmid and a
ß-galactosidase expression vector, COS-7 cells were also
transfected with the hVDR expression vector pAVhVDR (from J. W.
Pike, University of Cincinnati, Cincinnati, OH), mutated VDR expression
vector constructs [S208A and S51A were also a gift from J. W.
Pike (42)], or the AF-2 defective VDR mutant L417S
expression vector construct (prepared in the laboratory of L. P.
Freedman). The pRSV-CAT expression plasmid (American Type Culture Collection) and the pBL2CAT construct (tk CAT; from J. W.
Pike) were used in certain transfections as negative controls. Sixteen
hours after transfection cells were shocked for 1 min with PBS
containing 10% dimethylsulfoxide, washed with PBS, and treated for
24 h with the indicated treatments
[1,25-(OH)2D3
(10-8 M) or OA (50 nM)]
in the appropriate medium supplemented with 2% of
charcoal-dextran-treated FBS.
Twenty-four hours after treatment cells were harvested by
trypsinization, pelleted, washed with PBS and resuspended in 0.25
M Tris-HCl, pH 8.0, and lysed by freezing and thawing five
times. Cellular extract was collected and used for
ß-galactosidase or protein analysis. CAT assay was performed by
standard protocols on the cell extract normalized to ß-
galactosidase activity or total protein (64, 65).
Autoradiograms were analyzed by densitometric scanning using the
Shimadzu CS9000U Dual-Wavelength Flying spot scanner (Shimadzu
Scientific Instruments, Princeton, NJ). For some experiments several
autoradiographic exposure times were needed for densitometric analysis.
CAT activity was also quantitated by scanning TLC plates using the
Packard Constant Imager System (Packard Instrument Co., Meriden,
CT).
Northern Blot Analysis
Total RNA was prepared from UMR-106 cells with RNAzol RNA
extraction solution following the manufacturers instructions.
Polyadenylated [poly (A+)] mRNA was isolated
from the total RNA using oligo (deoxythymidine)-cellulose column
chromatography. Northern blot analysis was performed as previously
described (66). Briefly, Northern analysis
prehybridization and hybridization were performed in Ultrahyb solution
from Ambion, Inc. (Austin, TX).
32P-labeled cDNA probes were prepared using
Random Primers DNA Labeling Systems (Life Technologies, Inc.) according to the random prime method (64).
The 1-kb mouse OPN cDNA, used for Northern analyses, was generated by
HindIII digestion and was a gift from D. Denhardt (Rutgers
University, Piscataway, NJ) (67). A 1.7-kb rat VDR cDNA
was obtained by digestion of pIBI76 with EcoRI
(68). The 3.2-kb rat 24(OH)ase cDNA was obtained by
EcoRI digestion and was a gift from K. Okuda (Hiroshima
University School of Dentistry, Hiroshima, Japan) (69).
The ß-actin cDNA was purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). The blot was hybridized to the specific
32P-labeled cDNA probe for 16 h at 42 C,
washed, and subjected to autoradiography. Autoradiograms were analyzed
by densitometric scanning using the Shimadzu CS9000U Dual-Wavelength
Flying spot scanner (Shimadzu Scientific Instruments). Results are
expressed as the optical density ratio of test probe to ß-actin
control probe.
VDR Western Blot Analysis
UMR-106 cells were treated with the indicated compounds for
24 h and harvested by trypsinization, and nuclear extracts were
prepared following the method of Dignam et al.
(70). After isolation of nuclear extracts, 50 µg of
protein were separated by 10% SDS-PAGE. Proteins were transferred to a
polyvinylidene difluoride membrane from Bio-Rad Laboratories, Inc. (Hercules, CA). The membrane was blocked with PBST (0.5%
Tween 20 in PBS) containing 5% nonfat milk for at 4 C. After 16 h
of blocking the blot was incubated with rat monoclonal anti-VDR
antibody (Affinity BioReagents, Inc.) for 2 h at 27
C, washed with PBST, and incubated for an additional hour with a goat
antirat IgG conjugated to horseradish peroxidase (Sigma).
Again the membrane was washed with PBST and the enhanced
chemiluminescence Western blotting detection system (NEN Life Science Products) was used to detect the antigen/antibody
complex.
EMSA
Nuclear extracts were prepared by the method of Dignam et
al. (70) from cells incubated with the indicated
treatments for 24 h. A DNA fragment of the rat 24(OH)ase promoter
(-165/-122) containing the proximal VDRE (-150/-136) (Ref.
61 ; prepared by the UMD Molecular Resource Facility,
Newark, NJ) was used as a probe. Overlapping and reverse strands were
heat denatured and annealed overnight. Fifty nanograms of duplex oligo
were end labeled with [
-32P]-ATP using
T4 polynucleotide kinase (Life Technologies, Inc.) and purified using a micro bio-spin p-30
column (Bio-Rad Laboratories, Inc.). The eluted probe was
used for EMSA as described (64). Briefly, aliquots of the
nuclear preparations (35 µg of protein) were incubated for 20 min
at 27 C with 0.30.5 ng of the labeled oligonucleotide probe
(
100,000 cpm) in binding buffer (4 mM
Tris-HCl, pH 7.9, 1 mM EDTA, pH 8.0, 60
mM KCl, 12% glycerol, 12
mM HEPES, 1 mM
dithiothreitol). The samples were separated by electrophoresis on a 6%
nondenaturing polyacrylamide gel. Gels were dried and exposed to
Kodak XAR-5 film (Eastman Kodak Co.,
Rochester, NY).
GST Fusion Protein Pull-Down Assay
Nuclear extracts were prepared from COS-7 cells transiently
transfected with a wild-type or mutated (L417S) VDR expression vector
and incubated with the indicated test compound for 24 h. VDR
protein in each sample was assessed by Western blot analysis using
monoclonal anti-VDR antibody (Affinity BioReagents, Inc.). The GST-DRIP205 (527970) or GST-GRIP-1 fusion
proteins (39), immobilized on Sepharose beads, were
incubated with GST binding buffer (20 mM Tris-HCl, pH 7.9,
180 mM KCl, 0.2 mM EDTA, pH 8.0, 0.05% Nonidet
P-40, 0.5 mM phenylmethylsulfonyl fluoride, 1
mM dithiothreitol) that contained 1 mg/ml BSA for 2 h.
The GST binding buffer was removed and nuclear extract aliquots,
containing equal amounts of VDR protein, were incubated with 15 µg of
the appropriate fusion protein for 16 h at 4 C. Bound proteins
were washed four times with GST binding buffer containing 0.1% Nonidet
P-40 and then incubated with elution buffer [GST binding buffer
containing 0.1% Nonidet P-40 and 0.15% Sarkosyl
(Sigma)] at 4 C for 20 min. The eluted proteins were
analyzed by SDS-PAGE followed by Western blotting with VDR antibody.
Additional GST pull-down assays were performed using GST-VDR-LBD-
(110427) (39) that was phosphorylated in
vitro with casein kinase II. Briefly, 15 µg of GST-VDR-LBD
fusion protein were incubated in assay buffer (25
mM Tris-HCl, pH 7.4, 10 mM
MgCl2, 200 mM NaCl, and 0.1
mM ATP) with 1 U purified casein kinase II for 30
min. GST-VDR-LBD was washed three times with GST binding buffer.
Phosphorylation of GST-VDR-LBD was judged by performing the
phosphorylation reaction in the presence of 25 µCi of
[
-32P]-ATP followed by SDS-PAGE and
autoradiography. GST pull-down assays were performed in the presence or
absence of 1,25-(OH)2D3
(10-8 M) with the
phosphorylated or unphosphorylated GST-VDR-LBD and Easytag
L-[35S]-methionine-labeled
DRIP205 that was prepared using the TNT Coupled Reticulocyte Lysate
System from Promega Corp. After washing, bound DRIP205 was
analyzed by SDS-PAGE and autoradiography.
Statistical Analysis
Significance was determined by t test or Dunnetts
multiple comparison t statistic.
 |
ACKNOWLEDGMENTS
|
---|
We gratefully acknowledge Dr. Wen Yang and Dr. Michael
Huening for helpful suggestions.
 |
FOOTNOTES
|
---|
This work was supported by NIH Grants DK-38961 (to S.C.) and DK-45460
(to L.P.F.).
Abbreviations: AF-2, Activation function-2; CAT,
chloramphenicol acetyltransferase; CBP, CREB binding protein; CREB,
cAMP response element binding protein; DRIP, VDR-interacting protein;
GRIP, GR-interacting protein; GST,
glutathione-S-transferase; HAT, histone acetylase; hVDR,
human VDR; LBD, ligand-binding domain; OA, okadaic acid; OC,
osteocalcin; 24(OH)ase, 24-hydroxylase;
1,25-(OH)2D3, 1,25-dihydroxyvitamin
D3; OPN, osteopontin; PP-1 and PP-2A, protein phosphatase 1
and 2A; RSV, Rous sarcoma virus; tk, thymidine kinase; SMRT, silencing
mediator of retinoid and thyroid hormone receptor; SRC, steroid
receptor coactivator; VDRE, vitamin D response element.
Received for publication January 5, 2001.
Accepted for publication October 1, 2001.
 |
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