(Received for publication, November 6, 1995; and in revised form, January 10, 1996)
From the
We have examined vitamin D receptor (VDR), thyroid hormone
receptor (TR), and retinoid X receptor (RXR
) binding to
vitamin D response elements (VDREs), two thyroid hormone response
elements (TREs) (DR4 and F2), and a retinoic acid response element
(DR5). VDR/RXR bound well to the VDREs and to DR4 and DR5 using the
electrophoretic mobility shift assay. Surprisingly, VDR/RXR also bound
well to F2, which contains half-sites arranged as an inverted
palindrome. In co-transfection experiments using CV-1 cells, we
observed that VDR repressed basal transcription in the absence of
ligand on DR3 and osteopontin VDREs and F2, but had no effect on DR4 or
DR5. VDR selectively mediated ligand-dependent transcription on only
VDREs. VDR also exhibited dominant negative activity as it blocked
triiodothyronine (T
)-mediated transcriptional activity on
DR4 and F2. These results demonstrate that VDR/RXR heterodimers can
bind promiscuously to a wide range of hormone response elements,
including inverted palindromes. Moreover, they show that unliganded
VDRs, similar to TRs and retinoic acid receptors, can repress basal
transcription. Last, they also suggest a novel repressor function of
VDR on T
-mediated transcription which may be significant in
tissues where VDR and TR are co-expressed.
Thyroid hormone receptors (TRs) ()transactivate
hormone response elements (HREs) that contain different half-site
orientations: palindromes, inverted palindromes, and direct repeats
(DRs)(2, 3) . Studies on the spacing of half-sites
arranged as DRs have shown that TRs preferentially mediate
ligand-dependent transactivation via a DR with a four-nucleotide gap
(DR4), whereas vitamin D receptors (VDRs) and retinoic acid receptors
(RARs) transactivate via DRs with three- and five-nucleotide gaps,
respectively (DR3 and DR5)(2, 4) . The transcriptional
specificity for these receptors is thought to depend upon optimal
heterodimerization by these receptors with retinoid X receptors (RXRs)
on these hormone response elements(2, 4) .
In
general, nuclear hormone receptor binding to HREs correlates well with
transcriptional activity, although there are examples of promiscuous
receptor binding with variable effects on transcription. In particular,
TRs can bind to direct repeats and inverted palindromes containing
variable spacing(5, 6, 7, 8) .
TR/RXR heterodimers also can bind to the palindromic vitellogenin
estrogen response element, but have weak transcriptional activity via
this element(9, 10) . Additionally, TR and RAR can
bind to the palindromic HRE, TREpal, and the rat growth hormone HRE,
and both T and retinoic acid can co-regulate transcription
via these elements(11, 12, 13, 14) .
It is likely that heterodimerization with RXR facilitates TR binding to
these HREs. Although not studied extensively, it is possible that
VDR/RXR heterodimerization also may promote similar promiscuous binding
by VDR.
Recently, Schrader et al.(1) demonstrated
that VDR and TR could form heterodimers on the mouse and rat calbindin
HREs which are arranged as DR3 and DR4, respectively. Through
cross-linking studies, they demonstrated that VDR/TR dimers have a 5`
to 3` polarity on these elements similar to that described for TR/RXR
and RAR/RXR heterodimers. In particular, on the rat calbindin HRE
(DR3), VDR bound to the downstream half-site whereas in the mouse
calbindin HRE (DR4), TR bound to the downsteam half-site. Moreover,
both vitamin D and T could co-regulate transcription via
these elements. The demonstration of VDR:TR cross-talk is significant
as it suggests that VDR may modulate T
-mediated
transcription in target genes in cells that express both receptors such
as bone, gut, and skin.
In order to understand further potential
interactions between TR and VDR, we have studied the DNA binding and
transcriptional activity of TR and VDR complexes on different HREs. We
report that VDR can bind promiscuously to DR4 and DR5 HREs as well as
the chicken lysozyme thyroid hormone response element (TRE), F2 (which
contains two half-sites arranged as an inverted
palindrome)(15) . In co-transfection studies, VDR did not
mediate ligand-dependent transcription via reporter plasmid containing
these HREs although unliganded VDR could repress basal transcription
via DR3, osteopontin VDRE, and F2. Interestingly, VDR also could block
T-mediated transcriptional activation on DR4- and
F2-containing reporter plasmids. These findings suggest that, similar
to TRs and RARs, VDRs can repress basal transcription. Furthermore,
VDRs may have an important modulatory role in T
-mediated
transcription.
The method for preparation of the VDR
DNA-binding mutant, DBD, is similar to the method that we
have used previously(23, 24) . Briefly, mutagenesis of
human VDR in pSG was performed using 30-base pair mutagenic
oligonucleotide and restriction site oligonucleotide according to the
manufacturer's instructions (Clontech). Final constructs were
sequenced to verify mutations. DBD
contains a single
nucleotide change in the codon for the fourth coordinating cysteine
(resulting in Cys to Ser change) of the first zinc finger of the
DNA-binding domain which has been shown previously to be important for
TR binding to TREs(24, 25) .
In vitro translated receptor and 10,000 cpm of oligonucleotide probe were mixed and incubated together before being subjected to electrophoresis and autoradiography as described previously(26) .
CV-1 cells were grown in Dulbecco's
modified Eagle's medium, 10% fetal calf serum. The serum was
stripped of T and vitamin D by incubating with charcoal for
12 h at 4 °C, and constant mixing with 5% (w/v) AG1-X8 resin
(Bio-Rad) twice for 12 h at 4 °C before ultrafiltration. The cells
were transfected with expression (0.1 µg) and reporter (2 µg)
plasmids as well as a Rous sarcoma virus-
-galactosidase control
plasmid (1 µg) (27) in 3.5-cm plates using the
calcium-phosphate precipitation method(28) . Cells were grown
for 48 h in the absence or presence of 10
M T
(Sigma) or vitamin D (Biomol), and harvested. Cell
extracts then were analyzed for both luciferase (29) and
-galactosidase (27) activity in order to correct for
transfection efficiency. Except where indicated, the corrected
luciferase activities of untreated samples were normalized to the
luciferase activities of samples containing vector alone in the absence
of ligand (1-fold basal).
We first examined VDR homodimer and heterodimer binding on
DR3, DR4, and DR5 by electrophoretic mobility shift assay. VDR/RXR
heterodimer bound well to labeled DR3, DR4, and DR5 oligonucleotides (Fig. 1, lanes 5, 11, and 17). Two
VDR/RXR bands were observed as in vitro translated RXR
has two major translation products. The rank order of binding was DR3
> DR4 > DR5. Interestingly, addition of vitamin D slightly
decreased VDR/RXR heterodimer binding to these elements. No VDR
homodimer or monomer binding was observed on DR3 or DR5; however, a
faint band was observed on DR4 with the sample containing VDR alone (Fig. 1, lane 9). This band disappeared after addition
of vitamin D (Fig. 1, lane 10), similar to recent
reports of ligand-induced dissociation of TR and VDR
homodimers(26, 31, 32, 33, 34) .
Additionally, anti-VDR antibody could block the formation of this band
(data not shown). These results suggest that VDR weakly formed a
homodimer on the DR4 oligonucleotide. In contrast, no VDR/TR dimers
were observed on these elements ( Fig. 3and data not shown).
Figure 1:
VDR binding to direct repeats. In
vitro translated VDR (3 µl) and RXR (2 µl) were incubated
with P-labeled oligonucleotides in the presence and
absence of 10
M vitamin D and then analyzed
by EMSA as described under ``Materials and Methods.'' Please
note that there are two in vitro translated RXR
products
with the smaller one likely due to translation from an internal
methionine. These, in turn, result in two VDR/RXR
bands on EMSA. HD, VDR/RXR heterodimer; VD, VDR homodimer: rl, reticulocyte lysate; and *, nonspecific
band.
Figure 3:
Formation of VDR/RXR heterodimers on DR4
and the inverted palindrome F2 TRE. In vitro translated
TR, VDR, and RXR
(2-5 µl) were incubated with
P-labeled DR4 or F2 and then analyzed by EMSA as in Fig. 1. Reticulocyte lysate was added to some samples so that
the total volume of reticulocyte lysate was the same for each sample.
Similar amounts of TR
and VDR, as quantitated by
SDS-polyacrylamide gel electrophoresis analyses described under
``Materials and Methods,'' were added. A, DR4. Note
that weak TR
and VDR homodimer bands were seen (lanes 3 and 4) after longer film exposure (48 h) to gel. B, F2. Lanes 10-12, preimmune, anti-RXR
or
anti-VDR antibodies were added after samples incubated with probe.
M, TR
monomer;
D, TR
homodimer; HD, TR/RXR or VDR/RXR heterodimer; and SS,
supershifted complex.
We next examined the transcriptional activity of VDR on these elements by transfecting CV-1 cells with VDR expression plasmid and reporter plasmids containing the DRs (Fig. 2). In the absence of vitamin D, we observed repression of basal transcription on DR3- and the mouse osteopontin VDRE-containing reporter (this osteopontin VDRE contains an imperfect DR3). Little or no basal repression was observed on DR4 and DR5. In the presence of vitamin D, transcriptional activation was observed on DR3 and osteopontin VDRE, but not on DR4 and DR5. These findings suggest that VDR can repress basal repression in the absence of ligand on DR3 and osteopontin VDRE similar to TR on certain TREs(6, 35, 36) . Additionally, despite binding as VDR/RXR heterodimers to DR4 and DR5, VDR had minimal or no effect on basal repression or ligand-dependent transactivation.
Figure 2:
Transcriptional activity of VDR on several
different HREs. VDR transcriptional activity on DR3, DR4, DR5, and
osteopontin. VDR expression vector (0.1 µg), HRE-containing
reporter plasmid (1.7 µg), and -galactosidase control vector
(1.0 µg) were co-transfected in CV-1 cells in the absence or
presence of 10
M vitamin D for 48 h. In
these experiments, treated cells then were harvested and luciferase was
measured. Luciferase activity was normalized to
-galactosidase
activity and then calculated as fold basal luciferase activity with
1-fold basal activity defined as the luciferase activity with control
pSG vector alone in the absence of ligand. Each point represents the mean of four samples, and bars denote S.E.
* denotes significant difference from basal luciferase
activity.
GAL4 fusion protein systems have been used to characterize roles of nuclear hormone receptor heterodimers on transcriptional activation by minimizing potential contributions of endogenous RXR on transcription (17, 37) . In order to further characterize basal repression by VDR, we performed co-transfection studies using GAL4, GAL4-RXR, and VDR expression plasmids and a GAL4 binding site-containing reporter in CV-1 cells. Both VDR and GAL4, or GAL4-RXR alone, slightly decreased transcription both in the presence or absence of vitamin D when compared to GAL4 alone. However, when cells were co-transfected with VDR and GAL4-RXR in the absence of vitamin D, there was greater than 90% repression of basal transcription compared to GAL4 alone (fold basal luciferase activity = 0.07 ± 0.01 S.D.) and 90% repression of basal transcription compared to GAL4-RXR alone (fold basal luciferase activity = 0.10 ± 0.01 S.D.). This repression was reversed with addition of vitamin D, as ligand stimulated transcription greater than 3-fold above basal levels (fold basal luciferase activity = 3.45 ± 0.36 S.D.). These data strongly support an important functional role for VDR/RXR heterodimer in mediating basal repression and transcriptional activation.
We
then compared VDR and TR binding to the consensus TRE, DR4, and
the chicken lysozyme TRE, F2, which contains half-sites arranged as an
inverted palindrome. On DR4, TR
alone bound mostly as a monomer (Fig. 3A, lane 3) whereas both TR
and VDR
bound poorly as a homodimer ( Fig. 1and Fig. 3A, lanes 3 and 4). Both TR/RXR and VDR/RXR heterodimers
bound well to DR4, but no VDR/TR dimers were observed even after a 48-h
exposure of the film to the gel. Surprisingly, when we examined TR and
VDR binding to the inverted palindrome, F2, we also observed strong
VDR/RXR heterodimer binding comparable to that observed for TR/RXR (Fig. 3B, lanes 5 and 6). As observed
for the DRs, vitamin D slightly decreased VDR/RXR heterodimer binding
to F2 (Fig. 3B, lanes 6 and 9).
Anti-RXR
antibody partially supershifted the VDR/RXR bands,
whereas anti-VDR antibody blocked DNA binding by these complexes (Fig. 3B, lanes 11 and 12). No VDR
homodimer or VDR/TR dimers were observed even after a 48-h exposure of
the gel (Fig. 3B, lanes 4 and 7).
These latter findings suggest that VDR not only can bind promiscuously
to DRs with variable spacing between half-sites, but also can bind to
HREs containing different half-site orientations, e.g. inverted palindromes.
Studies comparing the transcriptional
activities on TR and VDR using the DR4- and F2-containing
reporters also were performed. On DR4, basal repression by unliganded
TR
and T
-mediated transcriptional activation were
observed (Fig. 4A) as noted previously(18) .
VDR appeared to have weak repression of basal transcription on DR4,
although this effect did not reach statistical significance (n = 5 experiments and Fig. 1and Fig. 4A). VDR also did not stimulate transcription on
DR4 in the presence of vitamin D. On F2, similar basal repression and
ligand-mediated transcriptional activation for TR
also was
observed. Interestingly, VDR repressed basal transcription in the
absence of vitamin D and derepressed basal transcription in the
presence of vitamin D on F2 (Fig. 4B). However, there
was no significant ligand-dependent transactivation by VDR on
F2-reporter activity.
Figure 4:
Comparison of transcriptional activity of
VDR and TR on DR4 and F2. VDR, TR, or pSG control expression
vector (0.1 µg), DR4- or F2-containing reporter plasmid (1.7
µg), and
-galactosidase control vector (1.0 µg) were
co-transfected in CV-1 cells in the absence or presence of
10
M T
or vitamin D for 48 h.
Treated cells then were harvested and luciferase was measured.
Luciferase activity was normalized to
-galactosidase activity and
then calculated as fold basal luciferase activity with 1-fold basal
activity defined as the luciferase activity with control pSG vector
alone in the absence of ligand. Each point represents the mean
of four samples and bars denote S.D. * denotes significant
difference from basal luciferase activity. A, DR4-containing
reporter; B, F2-containing
reporter.
Since VDR could bind to the DR4 and F2 TREs
but was unable to mediate ligand-dependent transcriptional activation,
we examined whether VDR might instead exert dominant negative activity
on T-mediated transcription. Previously, it had been shown
the natural mutant TR
s from patients with resistance to thyroid
hormone as well as the viral oncogene homolog of TR, v-erbA, possess
dominant negative activity on wild-type TRs. Moreover, this dominant
negative activity depended on mutant TR or v-erbA binding to TREs.
Accordingly, we transfected increasing amounts of VDR expression
plasmid and a fixed amount of TR
expression plasmid with DR4
reporter into CV-1 cells (Fig. 5A). In the absence or
presence of vitamin D, increasing VDR:TR expression plasmid ratio
progressively decreased T
-mediated transcriptional
activation. In fact, a 3:1 ratio of VDR and TR
expression plasmids
completely blocked T
-mediated transcriptional activation to
basal levels. Similar findings were observed for VDR blockade of
T
-mediated transcription on the F2 reporter (Fig. 5B). We also observed similar VDR blockade of
T
-mediated transcription when TR
expression plasmid
was used, indicating VDR did not exhibit TR isoform specificity in its
dominant negative activity (data not shown).
Figure 5:
VDR blocks T-mediated
transcriptional activity on DR4 and F2 reporter. Expression plasmids
encoding TR
, VDR, or pSG control vector (0.1 µg) were
co-transfected with DR4- or F2-containing reporter plasmid (1.7 µg)
and Rous sarcoma virus-
-galactosidase control vector (1 µg).
Cells then were treated with or without 10
M T
± vitamin D for 48 h and analyzed for
luciferase activity. Luciferase activity was normalized to
-galactosidase activity and then calculated as fold basal
luciferase activity with 1-fold basal activity defined as the
luciferase activity with pSG alone in the presence of T
alone or T
+ vitamin D. Each point represents the mean of four samples, and bars denote S.D. A, DR4-containing reporter; B, F2-containing
reporter.
In order to investigate
whether the dominant negative activity depended on VDR binding to TREs,
we created a mutant VDR, DBD, in which the fourth
cysteine of the first zinc finger was mutated to a serine. We and
others have shown that similar mutations in TR abrogate or markedly
reduce TR homodimer and TR/RXR heterodimer binding to TREs without
affecting ligand binding(24, 25) . DBD
was unable to bind as a heterodimer to F2, DR3, and DR4 (Fig. 6A and data not shown). We then examined the
dominant negative activity of DBD
on
T
-mediated transcriptional activity on F2-containing
reporter (Fig. 6B). At a 1:1 expression plasmid ratio,
DBD
had little dominant negative activity in contrast to
wild type VDR. At increasing expression plasmid ratios, however,
DBD
had some dominant negative activity which was less
than wild type VDR dominant negative activity at the same expression
plasmid ratio. Addition of vitamin D did not significantly affect the
dominant activity of DBD
or wild type VDR, and similar
results were observed with the DR4-containing reporter (Fig. 5, A and B, and data not shown). Additionally, we
performed [
H]vitamin D binding studies on nuclear
extracts from CV-1 cells co-transfected with VDR or DBD
and observed similar binding, suggesting similar expression of
these receptors in CV-1 cells (data not shown). Collectively, these
results suggest that DNA binding is not absolutely required for
dominant negative activity by VDR, particularly at high expression
plasmid ratios. However, VDR/RXR heterodimer binding to TREs may be a
major contributor to dominant negative activity at low expression
plasmid ratios ( Fig. 6and Fig. 7).
Figure 6:
DNA binding and dominant negative activity
by DNA-binding mutant, DBD, on F2. A, in
vitro translated VDR, RXR, and DBD
in the presence
and absence of vitamin D were incubated with
P-labeled F2
probe, similar to Fig. 1and Fig. 3. Samples then were
analyzed by EMSA. B, increasing amounts of VDR or DBD
expression vector (0.1, 0.3, 0.5 µg) and a fixed amount of
TR
expression vector (0.1 µg) were co-transfected with
F2-containing reporter plasmid (1.7 µg) and
-galactosidase
control vector (1 µg) in the presence of 10
M T
for 48 h and analyzed for luciferase
activity. In some samples, control pSG vector was added so that the
total amount of expression vector was identical for each sample.
Luciferase activity was normalized to
-galactosidase activity and
then calculated as fold basal luciferase activity with 1-fold basal
activity defined as the luciferase activity with control pSG vector
alone in the absence of ligand. Each point represents the mean
of triplicate samples. Similar results were obtained with
DR4-containing reporter.
Figure 7:
Model for dominant negative activity by
VDR on T-mediated transcription. Abbreviations same as in
text.
, coactivator.
These studies provide new information on transcriptional
mechanisms mediated by VDR and TR and suggest cross-talk between these
receptors. In our studies of transcriptional activity by VDR on
different reporter plasmids, we observed that unliganded VDR can
repress basal transcription on DR3, osteopontin VDRE, and F2. This
phenomenon has not been reported for VDR previously. However, basal
repression or silencing has been well described for
TRs(6, 35, 36) . One mechanism proposed for
basal repression involves the binding of unliganded TR homodimer and
TR/RXR heterodimer to TREs, possibly in conjunction with
co-repressor(s) to TREs. These complexes may then interact with the
basal transcriptional machinery to repress basal
transcription(24, 38) . In the presence of
T, the TR homodimer dissociates from the TRE (inducing
derepression), whereas the liganded TR/RXR assumes an
``active'' conformation such that it now interacts with
co-activator(s) or the basal transcriptional machinery to support
ligand-dependent transcriptional activation. It is likely that VDR may
utilize similar repression and derepression mechanisms as liganded VDR
homodimer dissociates from HREs whereas liganded VDR/RXR heterodimers
remain bound to HREs(34) . However, VDR formed homodimers
weakly on DR4 and poorly on all other elements studied (including DR3
and osteopontin VDRE) suggesting that unliganded VDR/RXR may be the
major complex in VDR-mediated repression of basal transcription. Our
studies with GAL4-RXR and VDR further support the notion that VDR/RXR
heterodimers can mediate basal repression.
Recently, two groups also
have shown that TRs and RARs can interact with co-repressors that may
participate in basal repression of
transcription(39, 40) . This new class of proteins are
called thyroid hormone and retinoic acid receptor associated
co-repressors (TRACs). Interestingly, these proteins bind well to TRs
or RARs in the absence, but not in the presence, of ligand. The
co-repressor interacts with the hinge region of TR and RAR, and a
three-amino acid substitution of amino acids 223, 224, and 227 of
TR abrogates binding with co-repressor(40) .
Interestingly, these amino acids are conserved in the vitamin D
receptor. It remains to be seen whether VDR interacts with these
recently described co-repressors or its own distinct co-repressor.
Our findings also demonstrated that VDR/RXR heterodimers can bind to DRs with gaps greater than three nucleotides. Although these heterodimers can bind to DR4 and DR5, reporter plasmids containing these HREs exhibit little vitamin D-dependent transcriptional activity. This discordance between DNA binding and transcriptional activity likely is due to spacing between VDRE half-sites modulating VDR/RXR heterodimer conformation, which, in turn, may affect its interaction with other associated proteins involved in transcriptional activation. In this connection, recent studies have shown similar discordance between TR/RXR heterodimer binding to certain elements and transcriptional activity(21, 41, 42) . Moreover, binding to different TREs can affect the trypsin sensitivity of TR/RXR heterodimers(42) . Likewise, heterodimer binding was not sufficient for mediating basal repression as we observed basal repression on DR3, osteopontin, and F2 HREs, but not on DR4 or DR5. This selective ability of unliganded VDR to repress and derepress basal transcription on certain HREs is reminiscent of recent studies by Kurokawa et al.(43) in which they observed similar effects for RAR on DR1 and DR5. Thus, DNA sequences as well as spacing between half-sites may modulate receptor complex interactions with associated proteins for both transcriptional activation and basal repression.
We also observed VDR/RXR heterodimer binding on the chicken lysozyme TRE, F2, suggesting that VDRs can bind to HREs in which half-sites are arranged as inverted palindromes. Again, as previously observed for TR/RXR heterodimers(6, 7, 8) , the formation of VDR/RXR heterodimers allows a more flexible and permissive binding to HREs containing different half-site spacing and orientations. Recently, Schrader et al.(44) have reported VDR/RXR heterodimer binding on two other inverted palindromes with a 9-nucleotide spacing, human calbindin and rat osteocalcin HREs. Both vitamin D and 9-cis-retinoic acid can stimulate transcription via these elements. However, in the case of F2, which contains an inverted palindrome with a gap of 6 nucleotides, no vitamin D-stimulated transcription was observed. These results again suggest that spacing between half-sites may dictate transcriptional activity by VDR/RXR heterodimers.
Previously, Schrader et al.(1) reported VDR/TR heterodimer binding to calbindin HREs.
We were unable to observe formation of VDR/TR heterodimers on any of
the different HREs as well as on the mouse and rat calbindin HREs. ()Although differences in EMSA conditions likely account for
these differences, these data nonetheless suggest that RXR
preferentially forms heterodimers with VDR in comparison to TR on most
HREs. Schrader et al.(1) also observed vitamin D,
T
, and dual ligand activation via the calbindin HREs. In
contrast, we have observed that VDR had no ligand-dependent
transcriptional activation on two TREs (DR4 and F2) and a retinoic acid
response element (DR5). Moreover, when VDR was co-transfected with TR,
we observed dominant negative activity on T
-mediated
transcription. Similar results also were observed for VDR on
RA-mediated transcription using the DR5 reporter.
These
findings suggest that VDR modulation of transcriptional activity on
these HREs are fundamentally different than those reported on the
calbindin HREs(1) , both in terms of mechanism and the
functional consequence of blocking hormone-mediated transcriptional
activation.
It previously had been shown that mutation of the
DNA-binding domain abrogated dominant negative activity by natural
mutant TRs and v-erbA (25, 45) suggesting that DNA
binding is essential for dominant negative activity on
T-mediated transcription by these proteins. Our studies
with DBD
, however, indicate that, although DNA binding is
important for full potency of dominant negative activity by VDR, it is
not absolutely required as high concentrations of DBD
also can have dominant negative activity. These findings suggest
that both competition for DNA binding to TREs by transcriptionally
inactive VDR/RXR heterodimers as well as titration of putative common
associated proteins or co-activators may contribute to dominant
negative activity by VDR (Fig. 7). It does not appear that
titration of RXR is the likely mechanism for the latter effect as
co-transfection of RXR
, the major endogenous
T
-receptor auxiliary protein in CV-1 cells(46) ,
does not reverse VDR dominant negative activity on
T
-mediated transcription.
Moreover, in contrast
to TR/RXR heterodimers, VDR/TR heterodimers do not form in solution (47) . (
)
Interestingly, another dominant negative
inhibitor of T action, c-erbA
-2 (the alternative
splice variant of TR
), may have similar mechanisms of
action(48, 49, 50) . Several groups have
shown DNA binding and/or DNA-dependent blockade of
T
-mediated transcription by
c-erbA
-2(48, 49, 50, 52) .
However, DNA binding is not absolutely required as co-transfection of
high amounts of a DNA-binding mutant of c-erbA
-2 still can mediate
dominant negative activity(51) . Recently, Juge-Aubry et
al.(53) showed that peroxisome proliferator-activated
receptor, a member of the nuclear hormone receptor superfamily, also
can block T
-mediated transcription. These studies with
peroxisome proliferator-activated receptor suggest that
heterodimerization with RXR as well as titration of a common
co-activator may be important in mediating dominant negative activity.
Several human VDR mutations have been described which contain either
mutations in the DNA-binding domain or premature stop
codons(54, 55) . Some of these mutants have been shown
to bind DNA poorly or are likely to dimerize poorly since the critical
distal heptad repeats in the ligand-binding domain which are important
for TR and VDR dimerization are eliminated(56) . It is possible
that these mutants may behave similarly to the DBD; that
is, they may exhibit impaired dominant negative activity on
T
-mediated transcription. It thus is possible that
mutations in certain nuclear hormone receptors may have a deleterious
effect on the function(s) of other nuclear hormone receptors, which, in
turn, may contribute to the phenotype of affected patients.
Currently, little is known about vitamin D regulation of TRs and
vice versa. Previously, Kaji and Hinkle (57) showed that
vitamin D decreased [I]T
binding
capacity and TR mRNA in GH4C1 pituitary cells, but the precise
mechanism(s) is not known. Recently, studies by Shrader et al.(1) suggested VDR and TR can form dimers on some HREs that
enable dual-ligand regulation. Our studies suggest that VDR may be
involved in yet another mechanism for VDR:TR cross talk, formation of
VDR/RXR heterodimers on TREs that can modulate T
-mediated
transcription. These findings raise the possibility that VDR can
modulate T
-mediated transcription in tissues where both
receptors are co-expressed (e.g. bone, gut, skin). Although
the physiological significance of this cross-talk currently is not
known, studies on the thyroid hormone status and function in patients
with vitamin D resistance or in VDR knockout animals may shed light on
this issue. In summary, our studies of VDR and TR regulation of
transcription demonstrate the intricacy and complexity of the signaling
pathways and networks that may occur among nuclear hormone receptors.
These, in turn, may enable finely tuned regulation of gene
transcription in target tissues.