Thyroid Hormone Receptor Does Not Heterodimerize with the Vitamin D Receptor but Represses Vitamin D Receptor-Mediated Transactivation
Mihali Raval-Pandya,
Leonard P. Freedman,
Hui Li and
Sylvia Christakos
Department of Biochemistry and Molecular Biology (S.C., M.R.-P.,
H.L.) UMDNJ-New Jersey Medical School Newark, New Jersey
07103-2714
Cell Biology and Genetics Program (L.P.F.)
Memorial Sloan-Kettering Cancer Center New York, New York 10021
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ABSTRACT
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The 9,000 Mr
calcium-binding protein calbindin-D9k
(CaBP9k) is markedly induced by
1,25-dihydroxyvitamin D3
[1,25-(OH)2D3] in
mammalian intestine. However, although a vitamin D response element
(VDRE) has been reported in the promoter of the rat
CaBP9k gene (at -490/-472), the
CaBP9k promoter is weakly transactivated by
1,25-(OH)2D3. Previous
studies indicated that when MCF-7 cells are transfected with the
rat CaBP9k VDRE ligated to the thymidine kinase
promoter and treated with both
1,25-(OH)2D3 and
T3 there is an enhancement of the response
observed with
1,25-(OH)2D3 alone,
suggesting direct cross-talk between thyroid hormone and the vitamin D
endocrine system and activation via the formation of vitamin D receptor
(VDR)-thyroid hormone receptor (TR) heterodimers. To determine whether
the weak response of the rat CaBP9k natural
promoter to
1,25-(OH)2D3 could
be enhanced by T3,
CaBP9k promoter/reporter chloramphenicol
acetyltransferase constructs were transfected in MCF-7 cells,
and the cells were treated with the two hormones alone or in
combination. No induction with T3 alone and no
enhancement of reporter activity in the presence of both hormones was
observed. To determine whether a lack of effect by
T3 was specific for the
CaBP9k promoter and to further examine the
possibility of cross-talk between the TR- and VDR-signaling pathways,
the 1,25-(OH)2D3-responsive rat 24
hydroxylase [24(OH)ase] promoter and the rat osteocalcin VDRE
(-457/-430), both fused to reporter genes were similarly examined in
MCF-7 cells. Again, no enhancement of the response to
1,25-(OH)2D3 was
observed in the presence of T3. In addition, a
similar lack of response to T3 but
responsiveness to
1,25-(OH)2D3 was
observed when UMR10601 osteosarcoma cells [which, like MCF-7 cells,
express VDR, TR, and the retinoid X receptor (RXR) endogenously] were
transfected with a
1,25-(OH)2D3 responsive
mouse osteopontin promoter reporter. In vitro DNA
binding assays were carried out using purified human VDR, human RXR
,
and chick T3R
and 24(OH)ase, osteocalcin, osteopontin, and
CaBP9k VDRE oligonucleotide probes. No VDR-TR
heterodimer binding on any of these VDREs was observed, although, as
expected, there was binding by the VDR-RXR complex and strong TR-RXR
binding to a consensus thyroid hormone response element.
Simultaneous gel retardation assays using similar and lower
concentrations of TR with RXR showed strong binding of TR-RXR on a
32P-labeled thyroid response element. Studies
using the yeast two-hybrid system also did not provide evidence for the
formation of a VDR-TR protein-protein interaction. In addition,
in vivo data showed that transfection of TR, in fact,
repressed VDR-mediated transcription and that the repression could be
reversed by the addition of RXR. Thus, in vitro and
in vivo experiments do not support ligand-sensitive
transactivation mediated by VDR-TR heterodimer formation but rather
suggest that TR expression can repress
1,25-(OH)2D3-induced
transcription predominantly by sequestering RXR.
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INTRODUCTION
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Vitamin D is important for intestinal calcium absorption and for
the development and maintenance of bone. In addition, vitamin D is
involved in a number of diverse cellular processes including effects on
cell proliferation and differentiation and on hormone secretion
(1, 2). The genomic mechanism of action of the secosteroid
hormone, 1,25-dihydroxyvitamin D3
[1,25-(OH)2D3], the biologically active
metabolite of vitamin D, is mediated by the vitamin D receptor (VDR)
(3, 4), a member of the steroid/nuclear receptor super family. VDR,
similar to the thyroid hormone receptor (TR) and the retinoic acid
receptors (RARs), forms a heterodimeric complex with the retinoid X
receptor (RXR) (5, 6, 7). This heterodimer binds to specific DNA sequences
with high affinity and activates or represses the transcription of
specific target genes (5).
Recent studies have begun to address the mechanisms involved in
mediating transcription by these nuclear hormone receptors. It has been
reported that VDR, similar to other nuclear receptors, can interact
with the general transcription factors TFIIB and TFIIA, which may
result in the recruitment of the basal components into the
preinitiation complex (8, 9, 10, 11). In addition, TAFII135 has
been reported to potentiate VDR as well as TR- and RAR-mediated
transcription (12). Recent evidence has indicated that, in addition to
the general transcription factors, another class of factors, known as
coactivators, are needed for nuclear hormone receptor-mediated
transcription. SRC-1 and its homologs have been reported to bind
directly to VDR as well as to many other nuclear receptors (13, 14, 15) and
a related protein, GRIP1, can potentiate VDR transactivation (16).
Thus, heterodimerization of RXR with VDR, TR, and RAR, the interaction
of the components of the preinitiation complex and coactivators with a
number of nuclear hormone receptors, as well as some overlap in DNA
recognition (17, 18, 19), suggest cross-talk of nuclear hormone
receptor-signaling pathways. We are only now beginning to understand
the numerous possibilities for interaction among different hormone
systems and therefore for hormonal coregulation of genes. However, the
physiological significance of many of the findings related to the
convergence of the hormone-signaling pathways remains to be
determined.
Although numerous genes have been reported to be regulated by
1,25-(OH)2D3, at this time only a small number
have been reported to contain vitamin D responsive elements (VDREs)
(2). The gene for 24(OH)ase, the enzyme thought to be involved in the
catabolism of 1,25-(OH)2D3, is strongly
transcriptionally responsive to 1,25-(OH)2D3
(20, 21). It is the first vitamin D-stimulated gene to be controlled by
two independent VDREs [at -151/-137 and -259/-245 for the rat
24(OH)ase gene (20)]. VDREs have also been reported in the promoters
for the genes encoding the cell cycle inhibitor p21, the bone proteins,
osteocalcin (OC) and osteopontin (OP), and the calcium-binding
proteins, calbindin-D28k (CaBP28k) and
CaBP9k (2). CaBP28k and CaBP9k are
vitamin D-dependent calcium-binding proteins that are thought to
facilitate transcellular calcium transport primarily in mammalian
kidney and intestine, respectively. Although a VDRE has been identified
in the promoter of the CaBP9k gene (at -490/-472) (22),
this promoter, as well as the CaBP28k promoter (23), is
weakly transactivated by 1,25-(OH)2D3. Recent
studies by Schräder et al. (24) indicated that when
MCF7 cells were transfected with VDREtkCAT constructs and treated with
T3 in combination with
1,25-(OH)2D3, a significant enhancement of the
response observed with 1,25-(OH)2D3 alone was
obtained, suggesting cross-talk between the thyroid hormone and the
vitamin D endocrine system and activation by VDR-TR heterodimers.
Understanding the cross-talk between VDR and TR would have
physiological significance since it would suggest modulation by
T3 of VDR-mediated transcription of target genes in
tissues that express both receptors, such as intestine, bone, and
pituitary (25, 26, 27). To further understand the potential interaction
between TR and VDR, we examined whether the weak response of the
CaBP9k promoter to 1,25-(OH)2D3
could be enhanced by T3. In addition, to determine whether
cross-talk between T3 and the vitamin D endocrine system
may be target gene specific, natural promoter/chloramphenicol
acetyltransferase (CAT) constructs for rat 24(OH)ase and mouse OP as
well as the rat OC VDRE (-457/-430) thymidine kinase (tk)CAT
construct were similarly examined. Using both the natural promoters and
the VDRE CAT construct, we consistently found no enhancement of the
1,25-(OH)2D3 response in the presence of
T3. Studies using gel retardation assays as well as the
yeast two-hybrid system also did not provide evidence for the formation
of VDR-TR heterodimers. However, transfection of TR was found to
repress VDR- mediated transactivation, and this inhibition was reversed
by the addition of RXR. These findings suggest that cross-talk between
the VDR- and TR-signaling pathways can occur indirectly due to
sequestration by TR of RXR rather than by direct heterodimerization of
VDR and TR, and in this sense TR may have an important role in
modulating VDR-mediated transactivation.
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RESULTS
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The Rat CaBP9k Promoter Is Weakly
Transactivated by
1,25-(OH)2D3
In mammalian intestine one of the most pronounced effects of
1,25-(OH)2D3 is increased synthesis of
CaBP9k (2). However, when the CaBP9k promoter
CAT constructs -1009/+61 and -580/+61 (which contain the previously
identified VDRE at -490/-472) were cotransfected with human VDR
(hVDR) in CV1 cells and treated with
1,25-(OH)2D3, weak transactivation was observed
(Fig. 1A
). Under the same conditions, a
21-fold and a 15-fold induction in CAT expression was detected using
the rat 24(OH)ase promoter construct (-1367/+74, containing VDREs at
-259/-245 and -151/-139) and the OC VDRE (-457/-430) tkCAT
construct, respectively (Fig. 1A
). To test the possibility that
cell-specific factors may be important for
1,25-(OH)2D3-induced transactivation of the
CaBP9k gene, CaBP9k promoter CAT constructs
were transfected in MDBK bovine kidney cells and in Caco-2 human
intestinal cells that contain endogenous VDR (28, 29). Weak
responsiveness of the rat CaBP9k promoter to
1,25-(OH)2D3 was again observed (Fig. 1B
).
Similar results were observed when VDR levels were increased above
endogenous levels by transfection of the VDR expression plasmid pAVhVDR
in MDBK or Caco-2 cells (not shown). Although the CaBP9k
promoter was weakly responsive to 1,25-(OH)2D3,
when the CaBP9k promoter CAT construct -1009/+106,
containing the reported functional estrogen- responsive element at
+50/+65 (30), was cotransfected with the hER expression vector
(pSG5-HEO) in T47-D breast cancer cells, an induction in CAT activity
(up to 6-fold) was observed after estradiol treatment
(10-9-10-7 M) (not shown).

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Figure 1. Weak Responsiveness of the CaBP9k
Promoter to 1,25-(OH)2D3
A, CV1 cells were cotransfected with 6 µg of the VDR expression
vector pAVhVDR and 46 µg CAT reporter plasmid containing the
CaBP9k gene constructs (-590/+61 or -1009/+61), the rat
24(OH)ase promoter construct (-1367/+74) or multiple copies of the rat
OC VDRE (-457/-430). Transfected cells, incubated in hormone-stripped
medium, were either vehicle treated (Basal) or treated with
10-7 M 1,25-(OH)2D3
(cells transfected with CaBP promoter constructs) or with
10-8 M 1,25-(OH)2D3
(cells transfected with the rat 24(OH)ase promoter construct or with
the OC VDRE tkCAT construct) for 24 h. After treatment, cells were
harvested and lysed and CAT activity, measured as described in
Materials and Methods, was normalized to
ß-galactosidase activity. B, MDBK bovine kidney cells and Caco-2
human intestinal cells were transfected with CaBP9k
promoter CAT constructs (-590/+61 or -1009/+61). Transfected cells
were either vehicle treated (Basal) or treated with 10-7
M 1,25-(OH)2D3 (+D) for 24 h.
For panels A and B the mean CAT activity ± SE (n = 46 observations per group)
was quantitated by comparison to basal levels. In CV1 cells (A) the
24(OH)ase promoter and the OC VDRE were significantly responsive to
1,25-(OH)2D3 (P < 0.01).
The CaBP9k promoter construct (-1009/+61) was induced
1.6-fold by 1,25-(OH)2D3 in CV1 cells (A) and
in Caco-2 intestinal cells (B) (P < 0.05).
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1,25(OH)2D3-Mediated
Transactivation Is Not Enhanced by T3
Previous studies by Schräder et al. (24, 31)
indicated when MCF-7 cells (which contain endogenous VDR and TR) or the
Drosophila cell line SL-3 transfected with VDR and TR, was
transfected with different VDREs, including the CaBP9k
VDRE, an enhancement of the response observed with
1,25-(OH)2D3 alone was observed in the presence
of T3 + 1,25-(OH)2D3, suggesting
activation via VDR-TR heterodimers and cross-talk between thyroid
hormone and the vitamin D endocrine system. To determine whether the
weak response of the CaBP9k promoter to
1,25-(OH)2D3 could be enhanced by
T3, the CaBP9k promoter CAT construct
-1009/+61 was transfected in MCF-7 cells, and the cells were treated
with 1,25-(OH)2D3 (10-7
M), T3 (10-7 M) or
1,25-(OH)2D3 + T3 for 24 h. No
induction with T3 alone and no enhancement of CAT activity
in the presence of both hormones was observed (Fig. 2
). Similar results were obtained using
the CaBP9k promoter CAT construct -590/+61 (data not
shown). To determine whether a lack of effect of T3 was
specific for the CaBP9k promoter, the rat OC VDRE
(-457/-430) tkCAT construct and the rat 24(OH)ase promoter construct
-1367/+74, which are strongly responsive to
1,25-(OH)2D3 (Fig. 2
), as well as the
CaBP28k promoter construct -390/+34 (VDRE at -200/-169)
(not shown), were similarly examined in MCF-7 cells. Using these
constructs, under these conditions, neither an activation with
T3 nor an enhanced response in the presence of both ligands
was observed (Fig. 2
). In addition, a similar lack of response to
T3 but responsiveness to
1,25-(OH)2D3 was also observed when a mouse OP
promoter CAT construct (-777/+79) containing the mouse OP VDRE
(-757/-743) was transfected in UMR10601 osteosarcoma cells, which,
like MCF-7 cells, express VDR, TR, and RXR endogenously (not shown). As
a positive control the thyroid hormone response element (TRE)
inverted repeat AGGTCATGACCT CAT construct was responsive to
T3 in these cells (Fig. 2
).

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Figure 2. Neither Transcriptional Activation with
T3 Alone nor a T3-Mediated Enhancement of
1,25-(OH)2D3-Induced Transcription Is Observed
MCF-7 cells were transfected with reporter CAT constructs containing
the indicated vitamin D-responsive promoter OC VDRE tkCAT or a
TRE-inverted repeat CAT construct (MTV-TREpCAT; TRE). Transfected cells
were either vehicle treated (Basal) or treated with 10-8
M 1,25-(OH)2D3 (+D),
10-7 M T3, or a combination of
1,25-(OH)2D3 and T3 for 24 h.
Cells transfected with MTV-TREpCAT were treated only with
T3 as a positive control. Results of six to eight
independent experiments showed similar results. CAT activity in cells
transfected with reporter CAT constructs CaBP9k -1009/+61,
rat 24(OH)ase 1367/+74, or OC VDRE tkCAT and treated with
T3 alone was not significantly different than basal
(P > 0.1). In the presence of T3 + D,
CAT activity was not significantly different than results obtained in
the presence of D alone (P > 0.1).
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Gel Retardation Analysis Does Not Provide Evidence for VDR-TR
Heterodimeric Binding on VDREs
To determine whether VDR-TR heterodimer formation on DNA occurs
in vitro, gel retardation analyses were performed using
purified receptors. As can be seen in Fig. 3
, there was strong binding of the TRE
inverted repeat by the TR-RXR complex. However, VDR-TR heterodimer
binding on the rat 24(OH)ase VDRE could not be demonstrated, although a
prominent shifted complex was observed in the presence of VDR and RXR.
Similar observations were made when the hOC VDRE, the mouse OP VDRE,
and the rat CaBP9k VDRE were used for gel retardation
assays (Fig. 3
). Note in Fig. 3
, as previously reported (32, 33),
purified VDR can bind to the OP VDRE as a homodimer in the absence of
RXR. Thus, for the OP VDRE, the band of retarded mobility observed in
the presence of VDR/TR comigrates with the band observed in the
presence of VDR alone, suggesting that it is due to VDR
homodimerization. The binding observed in the presence of VDR/RXR to
the CaBP9k VDRE was weak compared with VDR/RXR binding to
the 24(OH)ase, OC, and OP VDREs. Results of these gel retardation
assays, which do not provide evidence of binding of VDR-TR heterodimer
to VDREs, are consistent with the lack of functional activity (Fig. 2
).

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Figure 3. Gel Retardation Analysis Does Not Indicate VDR-TR
Heterodimer Binding on VDREs
Gel shift experiments were carried out with purified hVDR, cTR , and
FLAG-hRXR and 32P-labeled VDREs as described in
Materials and Methods. First panel (TRE):
Labeled probe was incubated with 10, 20, or 30 ng cTR and 6.5 ng
FLAG-hRXR . Second panel (24(OH)ase): VDRE-Labeled
probe was incubated with 20 ng VDR alone or 10 and 20 ng VDR in
combination with 6.5 ng FLAG-hRXR or 20 ng VDR in combination with
30 ng cTR . Third panel (OC VDRE): Labeled probe was
incubated with 10 ng VDR alone or 10 and 20 ng VDR in combination with
6.5 ng FLAG-hRXR or 20 ng VDR in combination with 20 and 30 ng
cTR . In the last two lanes experiments were done in the presence of
20 ng VDR and 6.5 ng FLAG-hRXR , labeled probe, and 100 fold molar
excess cold unlabeled OC VDRE or TRE. The complex formed in the
presence of VDR and RXR was competed with OC VDRE but not with TRE.
Although the complex formed in the presence of cold TRE appears less
intense than the complex formed in the absence of cold TRE, there was
no significant difference in intensity when results of three separate
gel shift experiments were compared. Fourth panel (OP
VDRE): Labeled probe was incubated with the same amount of purified
receptor protein used for gel shift analysis of binding to OC VDRE
(third panel). In the last two lanes experiments were
done in the presence of 20 ng VDR and 6.5 ng FLAG-hRXR , labeled
probe, and 100 fold molar excess cold unlabeled OP VDRE or TREp. Note
as previously reported (32 33 ) purified VDR can bind to the OP VDRE in
the absence of RXR, and the band of retarded mobility observed in the
presence of VDR/TR comigrates with the band observed with VDR alone.
Also note, the complex formed in the presence of VDR and RXR was
competed with OP VDRE but not with TRE. Fifth panel
(CaBP9k VDRE): Labeled probe was incubated with 10 and 20
ng VDR alone or 10 ng VDR in combination with 6.5 ng FLAG-hRXR or
20, 50, and 100 ng VDR in combination with 13 ng FLAG h-RXR or 100
ng VDR in combination with 30 ng cTR . The complex formed in the
presence of 100 ng VDR and 13 ng RXR could be competed with 100-fold
excess cold CaBP9k VDRE (not shown). Similar findings were
observed under the same conditions in two additional gel shift
experiments.
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VDR Does Not Interact with TR in a Yeast Two-Hybrid Assay
A yeast two-hybrid assay was established in our laboratory to
identify proteins that interact with VDR in vivo. One hybrid
construct contained the GAL4 DNA-binding domain in pGBT9 fused to the
ligand-binding domain of VDR (93427; GBT9VDR). As a control
interacting protein, a second hybrid construct contained the GAL4
activation domain in pGAD424 fused to the ligand-binding domain of
hRXR
(223462; GADRXR
). TR fusion was generated by ligating
chick TR
(cTR
) (119438) to the GAL4 DNA-binding domain as well
as to the GAL4 activation domain (GBT9TR and GADTR, respectively).
Interaction was characterized by monitoring ß-galactosidase
expression and growth on selective media lacking tryptophan (Trp) and
leucine (Leu). To characterize the specificity of the two-hybrid assay,
as negative controls, the yeast strain SFY56 was transformed with
GBT9VDR and the empty vector pGAD424 or with pGBT9 empty vector and
GADRXR
. No ß-galactosidase staining was observed (Fig. 4A
, VDR-pGAD and pGBT9-RXR). As positive
controls, GBT9VDR and GADRXR
, as well as GBT9TR and GADRXR
, were
transformed (see Fig. 4A
, VDR-RXR and TR-RXR, note positive
interaction). The interaction between VDR and TR was tested by
simultaneous transformation of GBT9VDR and GADTR. Although the VDR-RXR
and TR-RXR interactions were positive for ß-galactosidase, no
interaction between VDR and TR was observed in either orientation (Fig. 4A
; VDR-TR and TR-VDR). The transformation was repeated in yeast strain
Y190 where growth on plates lacking Leu, Trp, and histidine (His) is
dependent on protein interactions. When lysates were assayed for
ß-galactosidase activity, similar results, indicating an interaction
of VDR and TR with RXR but not an interaction between VDR and TR, were
obtained (Fig. 4B
).

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Figure 4. VDR Does Not Interact with TR in a Yeast Two-Hybrid
System
A, Various two-hybrid combinations were examined in the yeast strain
SFY526, and interaction in the presence of ligand was characterized by
ß-galactosidase expression and growth on Trp and Leu-deficient media
containing 10-7 M T3 and
10-8 M 1,25-(OH)2D3.
Specific interaction is observed between TR and RXR and between VDR and
RXR but not between empty vector (pGBT9) and RXR or between empty
vector (pGAD) and VDR (negative controls). An interaction was also not
observed between VDR and TR. B, Transformation was repeated in yeast
strain Y190. Relative growth on Trp-, Leu-, and His-deficient plates
containing 10-7 M T3,
10-8 M 1,25-(OH)2D3,
and 30 mM 3-aminotriazole was assessed after 4 days,
and ß-galactosidase expression was quantitated in liquid cultures as
described in Materials and Methods. Similar results were
observed for VDR and TR in the reverse orientation (not shown). Results
are presented as mean (±SE) of triplicate cultures from
two separate experiments.
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TRs Repress VDR-Mediated Transcriptional Activation
Although in vivo and in vitro studies did
not provide evidence for the formation of VDR-TR heterodimers, we
further investigated the possibility of cross-talk between the TR and
VDR hormone-signaling pathways by examining the effect of
overexpression of TR
on VDR-mediated transactivation. A constant
amount of VDR expression vector, which conferred close to maximal
induction of CAT activity, was cotransfected in CV1 cells with 1 µg
cTR
expression vector and either the 24(OH)ase -1369/+74 promoter
CAT construct (Fig. 5A
) or the OC VDRE
tkCAT construct (Fig. 5B
). In the presence of 1 µg cTR
expression
vector, 1,25-(OH)2D3-induced transactivation
was completely inhibited (Fig. 5
). However, at this concentration of
pEX-cTR
, 1,25-(OH)2D3- induced
transactivation was not completely inhibited in the presence of
T3. In the presence of 1,25-(OH)2D3
(10-8 M) and T3 (10-7
M), a 1.6- to 2.5-fold increase in CAT activity above basal
levels was observed in CV1 cells transfected with pAVhVDR, 1 µg
pEX-cTR
, and the rat 24(OH)ase CAT construct or OC VDREtkCAT (not
shown). Experiments done with both the 24(OH)ase and the OC VDRE CAT
reporter plasmids using higher concentrations of cTR
(3, 6, and 9
µg) also resulted in complete inhibition of VDR-mediated
transactivation.

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Figure 5. TR Represses VDR-Mediated Transcriptional
Activation
CV1 cells were cotransfected with CAT reporter plasmid [6 µg
rat 24(OH)ase promoter construct -1367/+74 (A) or 4 µg rat OC VDRE
tkCAT (B)] and 6 µg pAVhVDR (first two lanes; VDR).
Cells transfected with reporter plasmid and pAVhVDR were also
transfected with 1 µg pEX-cTR (last two lanes;
VDR+TR). The cells were incubated for 24 h in hormone-stripped
medium without or with 1,25-(OH)2D3
(10-8 M). CAT activity was assayed as
described in Materials and Methods using TLC. Similar
results were observed in a second experiment using 1 µg cTR
expression vector.
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Coexpression of RXR Alleviates TR-Dependent Repression
Dose-dependent inhibition of VDR-mediated transcription using
lower concentrations of cTR
expression vector (0.10.5 µg) is
shown in Fig. 6
(left panel).
pEXPRESS vector alone (0.5 µg) had no effect on
1,25-(OH)2D3- induced transactivation (not
shown). Since it had previously been suggested that TR isoforms can
repress transcription by the formation of heterodimers (34), we
investigated the possibility that the repression by TR is mediated by
heterodimer formation by cotransfection experiments in the presence of
RXR
. In the presence of 2 µg hRXR
expression vector, the
TR-negative effect on 1,25-(OH)2D3 mediated
transcription could be reversed (Fig. 6
). After cotransfection of a
cTR
mutant that lacks the DNA-binding domain (1120), inhibition of
1,25-(OH)2D3-dependent CAT activity was still
observed (Fig. 6
, left panel), suggesting that inhibition by
TR does not primarily involve contact with DNA. Reversal of repression
by 0.5 µg pEX-cTR
of 1,25-(OH)2D3- induced
24(OH)ase transcription was also observed using lower concentrations of
hRXR
(0.25, 0.5, and 1 µg; not shown). At higher concentrations of
pEX-cTR
(1 µg), 2 µg hRXR
expression vector also resulted in
an 80% reversal of the repression of
1,25-(OH)2D3-induced 24(OH)ase
transcription. However, at 1 µg pEX-cTR
, cells transfected with
the OC VDREtkCAT construct were less sensitive to reversal of
repression by RXR
(not shown). Repression of
1,25-(OH)2D3-induced transcription was also
observed when cTR
expression vector and the OP promoter CAT
construct (-777/+79) were cotransfected in UMR osteoblastic cells that
contain endogenous VDR [relative CAT activity (fold induction compared
with basal) in the presence of 1,25-(OH)2D3
(10-8 M) and in the absence of pEX-cTR
=
5.6 ± 0.7. In the presence of 0.25 µg pEX-cTR
,
1,25-(OH)2D3 induction of OP transcription was
reduced to 1.6 ± 0.1 fold (P < 0.01). In the
presence of 0.25 µg cTR
and 2 µg hRXR
,
1,25-(OH)2D3-induced transcription of the OP
gene 4.2 ± 0.6 fold (n = 45)]. These findings, obtained
using the 24(OH)ase, OP, and OC CAT reporter plasmids suggest that TR
exerts its effect predominantly by sequestering RXR and reducing the
available RXR needed to heterodimerize with VDR, thereby resulting in
repression of 1,25-(OH)2D3-induced
transactivation.

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Figure 6. Dose-Dependent Repression of VDR-Mediated
Transcription by TR and Reversal of TR-Dependent Repression by RXR
Left panel, CV1 cells were cotransfected with CAT
reporter plasmid [6 µg rat 24(OH)ase promoter construct -1367/+74
(top) or 4 µg rat OC VDRE thCAT
(bottom)], 6 µg VDR expression vector pAVhVDR, and
increasing amounts of pEX-cTR (0.1 ng-0.5 µg). In the presence of
0.5 µg cTR or 0.25 µg cTR expression vector, cells
transfected with the 24(OH)ase promoter CAT construct or OC VDREtkCAT,
respectively, were also transfected with 2 µg hRXR expression
vector (RXR ). Cells transfected with CAT reporter plasmids and
pAVhVDR were also transfected with 0.5 mg pEX-DBD-cTR
(mutant pEX-cTR ), which directs the expression of cTR with the
DNA-binding domain deleted. The cells were incubated in the presence or
absence of 1,25-(OH)2D3 (10-8
M), and CAT activity was normalized to ß-galactosidase
activity. Results, reported as fold induction (mean CAT activity
± SE; three to four observations per group) were
quantitated by comparison to untreated transfected cells. Right
panel, Representative autoradiograms: The rat 24(OH)ase
promoter CAT construct (top) was cotransfected in CV1
cells with the following expression plasmids: 6 µg VDR and 0.5 µg
cTR (VDR+TR) or 6 µg VDR, 0.5 µg cTR , and 2 µg hRXR
(VDR+TR+RXR). The cells were incubated in the presence or absence of
1,25-(OH)2D3 (10-8 M).
The OC VDRE CAT construct (bottom) was cotransfected in
CV1 cells with the following expression plasmids: 6 µg VDR and 0.25
µg cTR (VDR+TR) or 6 µg VDR, 0.25 µg cTR , and 2 µg RXR
(VDR+TR+RXR). The cells were incubated in the presence or absence of
1,25-(OH)2D3 (10-8
M).
|
|
 |
DISCUSSION
|
---|
Findings from our in vitro and in vivo
experiments do not support ligand-sensitive transactivation mediated by
VDR-TR heterodimer formation. Similar to our findings, Yen et
al. (35) also indicated that gel retardation assays did not
provide evidence for the formation of VDR/TR heterodimers on different
hormone response elements (direct repeats with three, four, or five
nucleotide gaps or the F2 TRE) as well as on the CaBP9k and
CaBP28k VDREs. Our observations differ from the findings
reported by Schräder et al. (24, 31), who proposed
TR-VDR heterodimerization and showed, using VDRE tkCAT constructs, that
in the presence of T3 there is a further enhancement of the
response observed with 1,25-(OH)2D3 alone.
Differences in the results we observed and those reported by
Schräder et al. (24, 31) may be due to the use of
natural promoters in most of our functional studies as well as the
performance of gel retardation assays in our laboratory using purified
receptors rather than in vitro translated proteins.
Experiments by Schräder et al. (24, 31) were carried
out in MCF-7 cells as well as in the Drosophila cell line
SL-3 transfected with VDR and TR. Although differences in cell type,
constructs, and protocols may account for some of the differences
observed, our data nevertheless invariantly and clearly indicate that
RXR, and not TR, forms heterodimers with VDR.
Although our data do not support direct heterodimerization of VDR and
TR, our findings do demonstrate that cross-talk between the vitamin D
and T3 signaling pathways can intersect indirectly through
competition by VDR and TR for a common limiting partner, namely RXR.
This results in an attenuation of
1,25-(OH)2D3-induced transactivation when TR is
overexpressed. In addition to repressing VDR-mediated transcription, TR
has also been reported to block transcription mediated by other nuclear
receptors (36, 37, 38, 39, 40, 41, 42, 43, 44, 45). Different mechanisms have been proposed to explain
TRs function as a repressor. One mechanism involves competitive DNA
binding (36, 41). TR can also reduce hormone-dependent gene activation
by receptors that interact with RXR by titrating out the common partner
RXR, resulting in modulation and diversity of hormone responses
(38, 39, 40). A third mechanism proposed for TR repression involves the
direct interaction of unliganded TR with a nuclear receptor corepressor
(43, 44) or with general initiation factors (45, 46). Since RXR was
found to reverse the TR-negative effect on
1,25-(OH)2D3-mediated transcription and TR
lacking its DNA-binding domain was also found to be an effective
inhibitor (Fig. 6
), our findings suggest that the mechanism of TR
repression of VDR-mediated transcription is predominantly due to
sequestration by TR of RXR, similar to the mechanism proposed for TR
inhibition of RAR- and RXR-dependent activation of an RARE or an RXRE,
respectively (38, 40). Modulation of VDR-mediated transcription by TR
depends on the concentration of VDR, TR, and RXR and the dimerization
status of RXR. In addition to sequestration of RXR by TR, it is
possible that part of the TR inhibition of the
1,25-(OH)2D3 dependent transcriptional response
may also involve other mechanisms. For example, since both VDR and TR
interact with transcription factor TFIIB (8, 9, 10), unliganded TR may act
as a transcriptional silencer in part by binding to TFIIB, preventing
the formation of the preinitiation complex and precluding TFIIB from
associating with liganded VDR. Further studies are needed to determine
additional mechanisms that may be involved in TR silencing of
VDR-mediated transcription.
VDR-TR cross-talk was also suggested in recent studies by Yen et
al. (35), which indicated that in the presence or absence of
1,25-(OH)2D3, VDR can repress
T3-mediated transcription. The mechanism proposed for
VDR-mediated repression was competition by transcriptionally inactive
VDR/RXR heterodimers with TR/RXR for TRE binding as well as titration
of a common associated protein or coactivator(s) important for
T3-mediated transcription. A VDR DNA binding mutant had
some dominant negative activity, which was less than the dominant
negative activity of wild-type VDR. Unlike our findings, the common
associated protein did not appear to be RXR since cotransfection of RXR
did not reverse VDR dominant negative activity. Thus, VDR-TR cross-talk
can occur by VDR inhibition of T3 transactivation as well
as by TR repression of VDR- mediated transcription.
In this study TR-mediated repression of VDR-mediated transcription was
observed by examining the transcription of the 24(OH)ase, OP, and OC
genes. We were unable to similarly examine VDR-TR cross-talk in studies
related to CaBP9k gene transcription due to the weak
responsiveness of the CaBP9k promoter to
1,25-(OH)2D3. Although one of the most
pronounced effects of 1,25-(OH)2D3 in mammalian
intestine is increased synthesis of CaBP9k, we found that
the CaBP9k promoter is consistently weakly transactivated
by 1,25-(OH)2D3. The weak transcriptional
response reflects in vivo studies indicating that
1,25-(OH)2D3 induces the expression of the CaBP
gene by a small rapid transcriptional stimulation followed by a large
accumulation of calbindin mRNA long after
1,25-(OH)2D3 treatment (47, 48). Thus, the
large induction of CaBP9k mRNA may be due primarily to
posttranscriptional mechanisms. Our findings do not indicate that the
presence of thyroid receptors and T3 can enhance this weak
transcriptional responsiveness. Recent studies using transgenic mice
have shown that responsiveness to 1,25-(OH)2D3
in the duodenum is contained in a 4.4-kb CaBP9k promoter
construct and that a proximal element (from -117 to +400) as well as a
distal element (from -3741 to -2894), together but not separately,
can confer a 1,25-(OH)2D3-induced response (49, 50). Thus, the control of the transcription of the CaBP9k
gene by 1,25-(OH)2D3 appears to be complex and
may involve nonclassical pathways as well as specific intestinal
transacting factors. Unlike studies related to the regulation of CaBP,
studies concerning the regulation by
1,25-(OH)2D3 of OC and OP have resulted in the
most information concerning transcriptional activation by
1,25-(OH)2D3 (2), and the 24(OH)ase gene is the
gene that is most transcriptionally responsive to
1,25-(OH)2D3 (20). It is likely that further
studies using the OC, OP, and 24(OH)ase genes, in which VDREs that
contribute to 1,25-(OH)2D3 responsiveness in
their natural promoters have been identified, will be important
to better understand not only how T3, but also how other
hormones and signaling pathways, are involved in the modulation of
transcriptional regulation by 1,25-(OH)2D3.
1,25-(OH)2D3 has been reported to be
involved in a number of diverse cellular processes (1, 2), and
receptors for 1,25-(OH)2D3 have been identified
not only in tissues involved in maintaining mineral homeostasis but
also in many other tissues, including pancreas, thyroid, brain, and
pituitary (51). Thus, cross-talk between VDR and TR may have
significance in a number of different physiological systems. Previous
reports have suggested cross-talk between vitamin D and thyroid hormone
in studies in pituitary cells (GH4C1) as well as in bone.
Receptors for 1,25-(OH)2D3 have been reported
in pituitary (51, 52) where high levels of thyroid receptors are
expressed (53). 1,25-(OH)2D3 was found to cause
a dose-dependent down-regulation of TRs in GH4C1 pituitary
cells and to block GH induction by T3 in these cells (27).
It is possible that to increase GH synthesis and secretion, high levels
of TR found endogenously in the pituitary would inhibit the action of
1,25-(OH)2D3 perhaps, as suggested by our
studies, by sequestering RXR. The presence of T3 would then
result in enhanced TR-mediated transcription of the GH gene. In bone,
both T3 and 1,25-(OH)2D3 have been
reported to be important for normal skeletal development and to affect
bone turnover (2, 54, 55). Vitamin D deficiency is associated with
rickets (56), and syndromes of resistance to thyroid hormone are
associated with delayed skeletal maturation as well as growth
retardation (57). Although osteoblasts are established target cells for
1,25-(OH)2D3 action and
1,25-(OH)2D3 responsive genes, including OP and
OC, have been identified in osteoblasts (2), the molecular basis for
T3 action in bone is less clearly defined, and genes
responsive directly to T3 in bone have not been
definitively identified. Previous studies indicated the presence of
thyroid receptors in bone (58, 59). Recent studies by Williams et
al. (26) reported the existence of different expression levels of
TR
1 and TRß1 in three osteosarcoma cell lines (ROS25/1, UMR106,
and ROS17/2.8) that express fibroblast-like, preosteoblast, and mature
osteoblast phenotypes, respectively. The authors also studied the
effect of hormones (1,25-(OH)2D3,
T3, 9-cis-RA, and RA) on the induction of
osteoblast phenotypic genes in these cells (60). Similar to our
findings, under conditions in which
1,25-(OH)2D3 induced the expression of a target
gene such as OC or OP, T3 was not found to have a
significant additive effect over the effect observed with
1,25-(OH)2D3 alone, also suggesting lack of
evidence for transactivation via a VDR/TR heterodimer. In addition to
studies with cell lines, it will be of interest in future studies to
determine the relative levels of VDR, TR, and RXR in bone at different
stages of skeletal development to determine whether the concentration
of the different receptors provides a molecular basis for the different
action of the respective hormones at the various stages of development.
Perhaps at different developmental stages when TR is overexpressed
compared with VDR, not only in bone but also in other tissues, TR may
have a repressor function preventing activation of
1,25-(OH)2D3 target genes under physiological
conditions that do not require them to be expressed. In summary,
although the physiological significance of VDR-TR cross-talk is
speculative at this time, our findings nevertheless suggest an indirect
interaction between TR- and VDR-signaling pathways that is dependent in
part on the concentration of the receptors and modulation of the
regulation of the expression of 1,25-(OH)2D3
target genes by competition for a common dimerization target.
 |
MATERIALS AND METHODS
|
---|
Materials
32P-radiolabeled and
[14C]chloramphenicol (50 mCi/mmol) were from DuPont/New
England Nuclear Corp (Boston, MA). DNA restriction and modifying
enzymes as well as media for cell culture were obtained from
GIBCO/BRL-Life Technologies (Gaithersburg, MD). Sera for cell culture
were purchased from Gemini (Calabasas, CA). Oligonucleotides were
synthesized by the UMD Molecular Resource Facility (Newark, NJ). The
plasmids and yeast strains comprising the two-hybrid system as well as
the yeast YPD medium (2% peptone, 1% yeast extract, 2% glucose) and
synthetic minimal medium were from CLONTECH (Palo Alto, CA).
1,25-(OH)2D3 was a generous gift from Dr. M.
Uskokovic (Hoffmann-La Roche. Co., Nutley, NJ). Acetyl-coenzyme A and
T3 as well as general regents of molecular biology grade
were purchased from Sigma Chemical Co. (St. Louis, MO).
Plasmids Used for Mammalian Cell Transfection
CaBP9k Promoter CAT Constructs
A rat genomic
gt11 library (CLONTECH) was screened using a 170-base
CaBP9k cDNA (61) as well as a synthetic 42-base cDNA probe
starting from the initiation site of the rat calbindin-D9k
gene (62). EcoRI-PstI fragments of phage DNA from
positive plaques were subcloned into the SmaI site of
pBluescript SK (+) vector and sequenced by the dideoxynucleotide chain
termination method (63). EcoRI-AvrII fragments in
pBluescript SK(+) coordinated
1.1 kb (-1009/+106) of the
5'-flanking region of the rat calbindin-D9k gene. This
region is identical to the rat calbindin-D9k sequence
-1009/+106 previously reported (22, 62). The 3'-end deletion mutant
(+61 from the cap site) was obtained using the PCR. For the 3'-end
deletion two 20-bp primers corresponding to region -1009/-989 of the
rat CaBP9k promoter and to +42/+61 of its complementary
strand were synthesized and used for amplification by PCR using the
Gene Amp PCR Reagent kit (Perkin-Elmer Corp., Norwalk, CT). The
5'-deletion mutant -590/+61 was generated using the restriction
endonuclease SspI. The CaBP9k gene construct -1009/+106
and deletion mutants (-1009/+61 and -590/+61), both containing the
reported VDRE (-489/-445) (22), were fused at the SmaI
site of pHCAT (which is derived from pSV2 CAT by deleting the simian
virus 40 promoter; pHCAT was a gift from M. Tocci, Merck, Sharpe and
Dohme). Insertion and orientation of inserts were confirmed by DNA
sequencing.
Other Plasmids
The plasmid OC VDREtkCAT, previously described (64), contains
multiple copies of the VDRE (-457/-430) found in the rat OC gene. The
mouse OPN promoter CAT construct spans nucleotides -777/+79 (65) and
was generously provided by Dr. D. Denhardt (UMDNJ-Robert Wood Johnson
Medical School, Piscataway, NJ). The rat 24(OH)ase promoter CAT
construct -1367/+74 phCAT was sent to us by Dr. John Omdahl
(University of New Mexico, Albuquerque, NM) (20). The pAVhVDR directs
expression of the full- length human VDR (66) and was a gift from
J. W. Pike (University of Cincinnati, Cincinnati, OH).
pCMX-hRXR
was provided by Drs. Ronald Evans (Salk Institute of
Biological Sciences, San Diego, CA) and David Mangelsdorf (University
of Texas Southwestern Medical Center, Dallas, TX). The hER expression
vector (pSG5-HEO) was a gift from Dr. P. Chambon (Institut de Genetique
et de Biologie Moleculaire et Cellulaire, Strasbourg, France).
MTV-IR-CAT contains one copy of the inverted repeat AGGTCA TGACCT with
no nucleotide gap cloned upstream of a mouse mammary tumor viral
long-terminal repeat-CAT vector [referred to as IR or TREp
(67)]. This construct as well as pEXPRESS-cTR
(or pEX-cTR
),
which efficiently directs the expression of full-length chick TR
(1408) (68), and pEX-DBD-cTR
(120408),
which directs the expression of cTR
with DNA-binding domain deleted,
were provided by Dr. Herbert Samuels (New York University Medical
Center, New York, NY). The pCH110 plasmid (Pharmacia, Piscataway, NJ)
contains the LacZ gene, which encodes ß-galactosidase. This
expression vector was used to normalize for variation in transfection
efficiency.
Mammalian Cell Culture Cell Transfection and CAT Assays
The African green monkey kidney cell line CV1, as well as Caco-2
cells (human colon carcinomas), MDBK cells (Madin Darby bovine kidney
cells), and MCF-7 cells (human breast cancer cells) were obtained from
ATCC (Rockville, MD) and maintained in DMEM supplemented with 10% FBS.
Caco-2 cells are also supplemented with bovine insulin (10 ng/ml). For
experiments examining CaBP9k transcription, MDBK cells and
Caco-2 cells were used since bovine kidney contains both
CaBP9k and CaBP28k (Ref. 28 and C. Fullmer,
personal communication), and CaBP9k mRNA has been reported
to be present in Caco-2 intestinal cells (69). Studies in MCF-7 cells
were done in the presence or absence of 5 ng transfected cTR
expression vector that was used to supplement endogenous TR levels.
Similar results for vitamin D-responsive CAT constructs were obtained
in the presence or absence of 5 ng cTR
expression vector. UMR106
osteosarcoma cells, also from ATCC, were maintained in DMEM F-12 medium
supplemented with 5% FBS. All cells were cultured at 37 C and 5%
C02/95% air. Twenty four hours before transfection, cells
were plated at 6070% confluence in 100-mm tissue culture dishes.
Transfection was carried out by the calcium phosphate DNA precipitation
method (70) using the specified reporter plasmid, receptor expression
vector, and the ß-galactosidase expression vector pCH110 as an
internal control. Eighteen hours after transfection, cells were shocked
for 1 min with 10% dimethylsulfoxide-PBS, washed with PBS, and then
cultured in medium supplemented with 2% charcoal-stripped serum. Cells
were harvested 24 h after treatment with vehicle (ethanol in 10
µl), T3, 1,25-(OH)2D3, or both
hormones by trypsinization. Cells were pelleted, washed with PBS,
resuspended in 50 µl 0.25 M Tris-HCl (pH 8.0), and lysed
by pulse sonication three times at 10-sec intervals. Cell debris was
spun down, and supernatants were saved for CAT assays (71), which were
carried out as previously described (64). CAT assays were normalized by
ß-galactosidase activity for each sample. CAT activity was
quantitated by densitometric scanning of TLC autoradiograms (Shimadzu
CS900 U densitomer, dual wavelength flying spot scanner, Shimadzu
Scientific Instruments, Inc. Princeton, NJ). For some experiments
several autoradiographic exposure times were needed for densitometric
analysis to estimate changes in CAT activity. CAT activity was also
quantitated by scanning TLC plates with the PACKARD instant imager
system (Packard Instrument Co., Meriden, CT). Multiple experiments were
performed and the results are reported as the mean ±
SE. Significance was determined by Students t
test or Dunnets multiple comparison t statistic.
Electrophoretic Mobility Shift Assays
The top strand of the oligonucleotide containing the
rat 24(OH)ase proximal VDRE (at -150/-136, half-site
recognition elements underlined) used as a probe is
as follows:
5'-CTAGAGGATGGAGTCAGCGAGGTGAGTGAGGGCGCCGGGGCCTC-3'.
The TREp probe (5'-AGCTTAGGTCATGACCTA-3'; TREp
underlined) was obtained from Dr. H. Samuels (67).
Overlapping forward and reverse strands of these response element
probes were heat denatured and allowed to anneal overnight. Fifty
nanograms of the annealed probes were end filled in the presence of
[
-32P]dCTP and the Klenow fragment of DNA polymerase I
(GIBCO/BRL). The other probes used for gel shift experiments containing
sequences of the rat calbindin-D9k gene (-489/-445), the
human OC gene (-511/-483), and the mouse OPN gene (-761/-738),
which include the reported VDREs (2), were the same as previously
described (22, 72, 73). Complementary strands of these oligonucleotide
probes were annealed, and 50 ng of duplex oligo were end-labeled with
-[32P] ATP using T4 polynucleotide kinase.
Overexpression and purification of hVDR were performed as described
previously (74, 75). FLAG-hRXR
(epitope-tagged hRXR
expressed
from the polyhedrin promoter) was overexpressed by baculovirus
infection and purified from insect cells as described previously (11).
cTR
was purified by diethylaminoethyl-Sephadex chromatography,
heparin-agarose chromatography, and size exclusion chromatography as
previously reported (68) and was a gift from Dr. H. Samuels. Purified
receptor was incubated with
0.5 ng of labeled probe at room
temperature for 20 min in a reaction system containing 0.5 ng poly
(deoxyinosinic-deoxycytidylic)acid and a dilution of binding
buffer that resulted in a final concentration of 20 mM
Tris-HCl (pH 7.9), 1 mM EDTA, 50 mM NaCl, 10%
glycerol, 0.1% NP40, and 1 mM dithiothreitol. Where
appropriate, double-stranded cold competitor oligonucleotide was added
for 20 min at room temperature before addition of labeled probe.
Complexes were resolved by electrophoresis on 8% low-ionic strength
native polyacrylamide gels run at 26 V/cm at 4 C. Gels were dried and
exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) at -80
C.
The Yeast Two-Hybrid System
For the yeast two-hybrid system the hinge region and the
ligand-binding domain of the hVDRcDNA (93427) were amplified as
previously described (76) using VDR gene-specific primers containing
EcoRI and BamHI linkers (5'tcctgaa
TTCATTCTGACAGATGAGGA-3' and 5'-acttggaTCCTAGTCAGGAGATCTCAT-3',
respectively). The amplified product was digested with EcoRI
and BamHI and inserted into the pGBT9 plasmid (pGBT9 is used
to generate a fusion between the GAL4 DNA-binding domain and the hinge
region and ligand-binding domain of hVDR). As a control interacting
protein, the HincII and EcoRI fragment of hRXR
cDNA (223462; hRXR
cDNA was a gift of Dr. David Mangelsdorf and
Dr. Ronald Evans) was excised and ligated to SmaI-digested
pGAD424. pGAD424 was used to generate a fusion between the Gal4
activation domain and hRXR
(223462). To test the interaction
between TR and RXR (positive control) as well as a possible interaction
between TR and VDR, TR fusion was generated between cTR
cDNA
(119438) and the GAL4 DNA-binding domain as well as the GAL4
activation domain by ligating the
880-bp fragment of a
NcoI/BamHI digest to
SmaI/BamHI-digested pGBT9 and pGAD424. The
product was end filled with Klenow and religated. A fusion between the
Gal4 DNA-binding domain and the HincII/EcoRI
digest of hRXR
cDNA was also generated by ligation of the hRXR
insert to the SmaI digest of pGBT9. The fusions were checked
for the presence of insert by hybridization. The orientation and
reading frame were confirmed by sequencing. The yeast strain SFY526
(MATta,ura352, his3200, ade2101, lys2801, trp 1901, leu23,
112, canr, gal4542, gal80538,
URA3::GALUAS-GAL1TATA-lacZ) was used.
SFY526, which contains the lacZ reporter gene under the control of the
Gal4-responsive promoter, made competent with lithium acetate (77), was
grown in YPD medium and then transformed with plasmid DNA using
the Yeastmaker Yeast Transformation System (CLONTECH). Transformants
were plated on media (SD synthetic medium; CLONTECH) without Trp and
Leu and were grown for 4 days at 30 C. SFY526 is auxotrophic for Trp
and Leu; thus, plating the transformed yeast culture on medium without
Trp and Leu selects for colonies containing both types of plasmids
(pGBT9 and pGAD424), which carry the wild-type genes for Trp and Leu,
respectively. Interaction of two proteins in this two-hybrid system
results in Gal4-dependent transcription of the lacZ reporter sequences
integrated into the yeast genome and expression of
ß-galactosidase. Colonies were assayed for ß-galactosidase
expression using a colony lift filter assay (78) modified as
described in the Matchmaker Two-Hybrid System (CLONTECH). To
verify the results observed using SFY526 yeast in another yeast strain,
experiments were also done using Y190 yeast (MATa, ura352,
his3200, ade2101, lys2801, tryp1901, leu23, 112, gal4
,
gal80
, cyhr2,
LYS2::GAL1UAS-GAL1TATA-lacZ), and
transformants were assayed for ß-galactosidase activity on liquid
cultures. The indicated plasmid pairs encoding the hybrid constructs
were transformed into Y190 yeast, plated on media lacking Leu, Trp, and
histidine (His) containing 30 mM aminotriazole
(inhibitor of His synthesis), and incubated for 4 days at 30 C.
Triplicate colonies were grown overnight at 30 C in
Leu-Trp-His- media. The culture
was diluted 1:4 into fresh medium and further grown 35 h to midlog
phase (A600 = 0.51.0). Cell pellets from 1.5-ml cultures
were permeabilized by three cycles of freeze/thaw and assayed for
ß-galactosidase activity using o-nitrophenyl
ß-D-galactopyranoside (ONPG) as a substrate as described
(79), modified as indicated in the Yeast Matchmaker Two-Hybrid System
(CLONTECH). For calculation of ß-galactosidase units, 1 unit of
ß-galactosidase is defined as the amount that hydrolyzes 1 µmol
ONPG to o-nitrophenol and D-galactose per h at
30 C.
 |
ACKNOWLEDGMENTS
|
---|
We gratefully acknowledge Dr. Paul MacDonald for experimental
suggestions related to studies done using the yeast two-hybrid system.
We also thank Terri Towers for advice concerning gel shift experiments.
In addition, we acknowledge helpful discussions with Dr. Herbert
Samuels and Bruce Raaka as well as the secretarial assistance of Mrs.
Valerie Brooks and Ms. Connie Sheffield.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Sylvia Christakos, Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103-2714. E-mail: christak{at}umdnj.edu
This work was supported by NIH Grants DK-38961 (to S.C.) and DK-45460
(to L.P.F.).
Received for publication February 17, 1998.
Revision received May 1, 1998.
Accepted for publication May 20, 1998.
 |
REFERENCES
|
---|
-
Suda T, Shinki T, Takahashi N 1990 The role of vitamin D
in bone and intestinal differentiation. Annu Rev Nutr 10:195211[CrossRef][Medline]
-
Christakos S, Raval-Pandya M, Wernyj RP, Yang W 1996 Genomic
mechanisms involved in the pleiotropic actions of 1,25dihydroxyvitamin
D3. Biochem J 316:361371[Medline]
-
Darwish H, DeLuca HF 1993 Vitamin D regulated gene
expression. Crit Rev Eukaryot Gene Expr 3:89116[Medline]
-
McDonald PN, Dowd DR, Haussler MR 1994 New insight into the
structure and functions of the vitamin D receptor. Semin Nephrol 14:101118[Medline]
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G,
Umesono K, Blumberg, B, Kastner P, Mark M, Chambon P, Evans RM 1995 The
nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
-
Whitfield GK, Hsieh J-C, Nakajima S, MacDonald PN, Thompson
PD, Jurutka PW, Haussler CA, Haussler MR 1995 A highly conserved region
in the hormone binding domain of the human vitamin D receptor contains
residues vital for heterodimerization with retinoid x receptor
and for transcriptional activation. Mol Endocrinol 9:11661179[Abstract]
-
Jin CH, Kerner SA, Hong MH, Pike JW 1996 Transcriptional
activation and dimerization functions in the human vitamin D receptor.
Mol Endocrinol 10:945957[Abstract]
-
Blanco JCG, Wang I-M, Tsai SY, Tsai, M-J, OMalley BW,
Jurutka PW, Haussler MP, Ozato K 1995 Transcription factor IIB and the
vitamin D receptor cooperatively activate ligand-dependent
transcription. Proc Natl Acad Sci USA 92:15351539[Abstract]
-
Masuyama H, Jefcoat SC, MacDonald PN 1997 The N-terminal
domain of transcription factor IIB is required for direct interaction
with the vitamin D receptor and participates in vitamin D mediated
transcription. Mol Endocrinol 11:218228[Abstract/Free Full Text]
-
Ing NH, Beekman JM, Tsai, SY, Tsai M-J, OMalley BW 1992 Members of the steroid hormone receptor superfamily interact with TFIIB
(S300-II). J Biol Chem 267:1761717623[Abstract/Free Full Text]
-
Lemon BD, Fondell JD, Freedman LP 1997 Retinoid x
receptor: Vitamin D3 receptor heterodimers promote stable
preinitiation complex formation and direct 1,25-dihydroxyvitamin
D3-dependent cell free transcription. Mol Cell Biol 17:19231937[Abstract]
-
Mengus G, May M, Carre L, Chambon P, Davidson I 1997 Human
TAFII135 potentiates transcriptional activation by the AF-2
s of the retinoic acid, vitamin D3 and thyroid hormone
receptors in mammalian cells. Genes Dev 11:13811395[Abstract]
-
Masuyama H, Brownfield, CM, St Arnaud R, MacDonald PN 1997 Evidence for ligand-dependent intramolecular folding of the AF-2 domain
in vitamin D receptor-activated transcription and coactivator
interaction. Mol Endocrinol 11:15071517[Abstract/Free Full Text]
-
Onate SA, Tsai SY,Tsai M-J, OMalley BW 1995 Sequence
and characterization of a coactivator for the steroid hormone
receptor superfamily. Science 270:13541357[Abstract]
-
Chen H, Lin RJ, Schiltz L, Chakravarti D, Nash A, Nagy L,
Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor coactivator
ACTR is a novel histone acetyltransferase and forms a multimeric
activation complex with P/CAF and CBP/p300. Cell 90:569580[Medline]
-
Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional
coactivator in yeast for hormone binding domains of steroid receptors.
Proc Natl Acad Sci USA 93:49484952[Abstract/Free Full Text]
-
Williams GR, Harney JW, Moore DD, Larsen PR, Brent GA 1992 Differential capacity of wild-type promoter elements for binding and
transactivation by retinoic acid and thyroid hormone receptors. Mol
Endocrinol 6:15271537[Abstract]
-
Parker MG 1993 Steroid and related receptors. Curr Opin Cell
Biol 5:499504[Medline]
-
Scott REM, Wu-Peng XS, Yen PM, Chin WW, Pfaff DW 1997 Interactions of estrogen and thyroid hormone receptors on a
progesterone receptor estrogen response element (ERE) sequence: a
comparison with the vitellogenin A2 consensus ERE. Mol Endocrinol 11:15811592[Abstract/Free Full Text]
-
Kerry DM, Dwivedi PP, Hahn CN, Morris HA, Omdahl JL, May BK 1996 Transcriptional synergism between vitamin D-responsive elements in
the rat 25 hydroxyvitamin D3 24-hydroxylase (CYP24)
promoter. J Biol Chem 271:2971529721[Abstract/Free Full Text]
-
Chen KS, DeLuca HF 1995 Cloning of the human 1
25-dihydroxyvitamin D3 24-hydroxylase gene promoter and
identification of two vitamin D responsive elements. Biochim Biophys
Acta 1263:19[Medline]
-
Darwish HM, DeLuca HF 1992 Identification of a
1,25dihydroxyvitamin D3 response element in the 5' flanking
region of the rat calbindin-D9k gene. Proc Natl Acad Sci
USA 89:603607[Abstract]
-
Gill RK, Christakos S 1993 Identification of sequence elements
in mouse calbindin-D28k gene that confer
1,25-dihydroxyvitamin D3 and butyrate inducible responses.
Proc Natl Acad Sci USA 90:29842988[Abstract]
-
Schräder M, Müller KM, Nayeri S, Kahlen J-P,
Carlberg C 1994 Vitamin D3-thyroid hormone receptor
heterodimer polarity directs ligand sensitivity of transactivation.
Nature 370:382386[CrossRef][Medline]
-
Forrest D, Sjoberg M, Vennstrom B 1990 Contrasting
developmental and tissue-specific expression of
and ß thyroid
hormone receptor genes. EMBO J 9:15191528[Abstract]
-
Williams GR, Bland R, Sheppard MC 1994 Characterization of
thyroid hormone (T3) receptors in three osteosarcoma cell
lines of distinct osteoblast phenotype: interaction among
T3, vitamin D3 and retinoid signaling.
Endocrinology 135:23752385[Abstract]
-
Kaji H, Hinkle PM 1989 Attenuation of thyroid hormone action
by 1,25dihydroxyvitamin D3 in pituitary cells.
Endocrinology 124:930936[Abstract]
-
Gagnon AM, Simboli-Campbell M, Welsh JE 1994 Induction of
calbindin-D28k in Madin-Darby bovine kidney cells by
1,25(OH)2D3. Kidney Int 45:95102[Medline]
-
Giuliano AR, Franceschi RT, Wood RJ 1991 Characterization of
the vitamin D receptor from Caco-2 human colon carcinoma cell line:
effect of cellular differentiation. Arch Biochem Biophys 285:261269[Medline]
-
Darwish H, Krisinger J, Furlow JD, Smith C, Murdoch FE, DeLuca
HF 1991 An estrogen-responsive element mediates the transcriptional
regulation of calbindin-D9k gene in rat uterus. J Biol
Chem 266:551558[Abstract/Free Full Text]
-
Schräder M, Müller K, Carlberg C 1994 Specificity
and flexibility of vitamin D signaling. J Biol Chem 269:55015504[Abstract/Free Full Text]
-
Nishikawa J, Matsumoto M, Sakoda K, Kitaura M, Imagawa M,
Nishihara T 1993 Vitamin D receptor zinc finger region binds to a
direct repeat as a dimer and discriminates the spacing number between
each half-site. J Biol Chem 268:1973919743[Abstract/Free Full Text]
-
Freedman LP, Arce V, Perez Fernandez R 1994 DNA sequences that
act as high affinity targets for the vitamin D3 receptor in
the absence of the retinoid x receptor. Mol Endocrinol 8:265273[Abstract]
-
Forman BM, Yang CR, Au M, Casanova J, Ghysdael J, Samuels HH 1989 A domain containing leucine-zipper like motifs mediate novel
in vivo interactions between the thyroid hormone and
retinoic acid receptors. Mol Endocrinol 3:16101626[Abstract]
-
Yen PM, Liu Y, Sugawara A, Chin WW 1996 Vitamin D receptors
repress basal transcription and exert dominant negative activity on
triidothyronine-mediated transcriptional activity. J Biol Chem 271:1091010916[Abstract/Free Full Text]
-
Graupner G, Wills KN, Tzukerman M, Zhang X-k, Pfahl M 1989 Dual regulatory role for thyroid-hormone receptors allows control of
retinoic-acid receptor activity. Nature 340:653656[CrossRef][Medline]
-
Damm K, Thompson CC, Evans RM 1989 Protein encoded by v-erb A
functions as a thyroid hormone receptor antagonist. Nature 339:593597[CrossRef][Medline]
-
Barettino D, Bugge TH, Bartunek P, Vivanco Ruiz MdM,
Sonntag-Buck V, Beug H, Zenke M, Stunnenberg HG 1993 Unliganded
T3R but not its oncogenic variant, v-erb A, suppresses
RAR-dependent transactivation by titrating out RXR. EMBO J 12:13431354[Abstract]
-
Hollenbeck PL, Phyillaier M, Nikodem VM 1993 Divergent effects
of 9-cis-retinoic acid receptor on positive and negative thyroid
hormone receptor-dependent gene expression. J Biol Chem 268:38253828[Abstract/Free Full Text]
-
Lehmann, JM, Zhang X-k, Graupner G, Lee M-O, Hermann T,
Hoffmann B, Pfahl M 1993 Formation of retinoid x receptor
homodimers leads to repression of T3 response: hormonal
crosstalk by ligand induced squelching. Mol Cell Biol 13:76987707[Abstract]
-
Graupner G, Zhang X-k, Tzukerman M, Wills K, Hermann T, Pfahl
M 1991 Thyroid hormone receptors repress estrogen receptor activation
of a TRE. Mol Endocrinol 5:365372[Abstract]
-
Spanjaard RA, Nguyen VYP, Chin WW 1995 Repression of
glucocorticoid receptor-mediated transcriptional activation by
unliganded thyroid hormone receptor(TR) is TR isoform-specific.
Endocrinology 136:50845092[Abstract]
-
Don Chen, J, Evans RM 1995 A transcriptional co-repressor that
interacts with nuclear hormone receptors. Nature 377:454457[CrossRef][Medline]
-
Horlein, AJ, Naar, AM, Heinzel T, Torchia J, Gloss B, Kurokawa
R, Ryan A, Kamei Y, Soderstrom M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated
by a nuclear receptor co-repressor. Nature 377:397404[CrossRef][Medline]
-
Fondell JD, Roy AL, Roeder RG 1993 Unliganded thyroid hormone
receptor inhibits formation of a functional preinitiation complex:
implications for active repression. Genes Dev 7:14001410[Abstract]
-
Baniahmad A, Ha I, Reinberg D, Tsai S, Tsai M-J, OMalley BW 1993 Interaction of human thyroid hormone receptor ß with
transcription factor TFIIB may mediate target gene depression and
activation by thyroid hormone. Proc Natl Acad Sci USA 90:88328836[Abstract]
-
Dupret J-M, Brun P, Perret C, Lomri N, Thomasset M,
Cuisinier-Gleizes P 1987 Transcriptional and post transcriptional
regulation of vitamin D-dependent calcium binding protein in rat
duodenum by 1,25-dihydroxycholecalciferol. J Biol Chem 262:1655316557[Abstract/Free Full Text]
-
Varghese S Deaven LL, Huang Y-C, Gill RK, Iacopino AM,
Christakos S 1989 Transcriptional regulation and chromosomal assignment
of the mammalian calbindin-D28k gene. Mol Endocrinol 3:495502[Abstract]
-
Romagnolo B, Cluzeaud F, Lambert M, Colnot S, Porteu A, Molina
T, Thomasset M, Vandewalle A, Kahn A, Perret C 1996 Tissue-specific and
hormonal regulation of calbindin-D9k fusion genes in
transgenic mice. J Biol Chem 271:1682016826[Abstract/Free Full Text]
-
Colnot S, Romagnolo B, Lacourte P, Thomasset M, Kahn A, Perret
C 1997 Analysis of calbindin-D9k gene regulation by
1,25(OH)2D3 using transgenic mice In: Norman
AW, Bouillon R and Thomasset M (eds) Vitamin D: Chemistry, Biology and
Clinical Applications of the Steroid Hormone. Printing and
Reprographics, University of California, Riverside, CA, pp 312313
-
Walters MR 1992 Newly identified actions of the vitamin D
endocrine system. Endocr Rev 13:146
-
Kashio Y, Iwasaki J, Chihara K, Kaji H, Kita T, Okimura Y,
Fujita T 1985 Pituitary 1,25-dihydroxyvitamin D3 receptors
in hyperthyroid and hypothyroid rats. Biochem Biophys Res Commun 131:122128[Medline]
-
Oppenheimer JH 1979 Thyroid hormone action at the cellular
level. Science 203:971979[Medline]
-
Allain TJ, McGregor AM 1993 Thyroid hormones and bone. J
Endocrinol 139:918[Medline]
-
Stern PH 1996 Thyroid hormone and bone. In: Bilezikian JP,
Raisz LG, Rodan GA (eds) Principles of Bone Biology. Academic Press,
New York, pp 521531
-
DeLuca HF 1984 The metabolism, physiology and function of
vitamin D. In: Kumar R (ed) Vitamin D: Basic and Clinical Aspects.
Martinus Nijhoff, Boston, pp 110
-
Refetoff S, Weiss RE, Usala SJ 1993 The syndromes of
resistance to thyroid hormone. Endocr Rev 14:348399[Medline]
-
Krieger NS, Stappenbeck TS, Stern PH 1988 Characterization of
specific thyroid hormone receptors in bone. J Bone Miner Res 3:473478[Medline]
-
Rizzoli R, Poser J, Burgi U 1986 Nuclear thyroid hormone
receptors in cultured bone cells. Metabolism 35:7174
-
Williams GR, Bland R, Sheppard MC 1995 Retinoids modify
regulation of endogenous gene expression by vitamin D3 and
thyroid hormone in three osteosarcoma cell lines. Endocrinology 136:43044314[Abstract]
-
Mifflin TE, Pearson WR, Reinhardt J, Bruns DE, Bruns ME 1988 Molecular cloning and sequencing of calbindin-D9k
cDNA from mouse placenta. In: Norman AW, Schaefer K, Grigoleit HG,
Herrath DV (eds). Vitamin D: Molecular, Cellular and Clinical
Endocrinology. Walter de Gruyter, Berlin, pp 507508
-
Krisinger J, Darwish H, Maeda N, DeLuca HF 1988 Structure and
nucleotide sequence of the rat intestinal vitamin D-dependent calcium
binding protein gene. Proc Natl Acad Sci USA 85:89888992[Abstract]
-
Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with
chain-terminating inhibitors. Proc Natl Acad Sci USA 74:54635467[Abstract]
-
Matkovits T, Christakos S 1995 Ligand occupancy is not
required for vitamin D receptor and retinoid receptor mediated
transcriptional activation. Mol Endocrinol 9:232242[Abstract]
-
Noda M, Vogel RL, Craig AM, Prahl J, DeLuca HF, Denhardt DT 1990 Identification of a DNA sequence responsible for binding of the
1,25-dihydroxyvitamin D3 receptor and 1,25-dihydroxyvitamin
D3 enhancement of mouse secreted phosphoprotein 1 (Spp-1 or
osteopontin) gene expression. Proc Natl Acad Sci USA 87:99959999[Abstract]
-
Liao J, Ozono K, Sone T, McDonnell DP, Pike JW 1990 Vitamin D
receptor interaction with specific DNA requires a nuclear protein and
1,25 dihydroxyvitamin D3. Proc Natl Acad Sci USA 87:97519755[Abstract]
-
Forman BM, Casanova J, Raaka BM, Ghysdael J, Samuels HH 1992 Half-site spacing and orientation determines whether thyroid hormone
and retinoic acid receptors and related factors bind to DNA response
elements as monomers, homodimers or heterodimers. Mol Endocrinol 6:429442[Abstract]
-
Forman B, Samuels HH 1991 pEXPRESS: a family of expression
vectors containing a single transcription unit active in prokaryotes,
eukaryotes and in vitro. Gene 105:915[CrossRef][Medline]
-
Fleet JC, Wood RJ 1994 Identification of
calbindin-D9k mRNA and its regulation by
1,25dihydroxyvitamin D3 in Caco-2 cells. Arch Biochem
Biophys 308:171174[CrossRef][Medline]
-
Gorman C, Padamanbhan R, Howard B 1983 High efficiency
DNA-mediated transformation of primate cells. Science 221:551553[Medline]
-
Gorman CM, Moffat LF, Howard BH 1982 Recombinant genomes which
express chloramphenicol acetyl transferase in mammalian cells. Mol Cell
Biol 2:10441051[Medline]
-
Liu M, Freedman LP 1994 Transcriptional synergism between the
vitamin D receptor and other nonreceptor transcription factors. Mol
Endocrinol 8:15931604[Abstract]
-
Freedman LP, Towers TL 1991 DNA binding properties of the
vitamin D3 receptor zinc finger region. Mol Endocrinol 5:18151826[Abstract]
-
Cheskis B, Freedman LP 1994 Ligand modulates the conversion of
DNA-bound vitamin D3 receptor (VDR) homodimers into
VDR-retinoid x receptor homodimers. Mol Cell Biol 14:33293338[Abstract]
-
Towers TL, Luisi BF, Asianov A, Freedman LP 1993 DNA target
selectivity by the vitamin D3 receptor: mechanism of dimer
binding to an asymmetric repeat element. Proc Natl Acad Sci USA 90:63106314[Abstract]
-
MacDonald PN, Sherman DR, Dowd DR, Jefcoat SC, DeLisle RK 1995 The vitamin D receptor interacts with general transcription factor
IIB. J Biol Chem 270:47484752[Abstract/Free Full Text]
-
Geitz D, Jean AS, Woods RA, Schiestl RH 1991 Improved method
for high efficiency transformation of intact yeast cells. Nucleic Acids
Res 20:1425[Medline]
-
Hannon GJ, Demetrick D, Beach D 1993 Isolation of the
Rb-related p130 through its interaction with CDK2 and cyclins. Genes
Dev 7:23782391[Abstract]
-
Estojak J, Brent R, Golemis EA 1995 Correlation of two-hybrid
affinity data with in vitro measurements. Mol Cell Biol 15:58205829[Abstract]