From the Department of Biochemistry, College of Medicine, The University of Arizona, Tucson, Arizona 85724
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ABSTRACT |
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Gel mobility shift analysis was utilized to
investigate the molecular function of 1,25-dihydroxyvitamin
D3 (1,25-(OH)2D3) and
9-cis-retinoic acid (9-cis-RA) ligands in the
binding of the vitamin D receptor (VDR) and retinoid X receptor (RXR)
to mouse osteopontin and rat osteocalcin vitamin D-response elements
(VDREs). At physiological ionic strength and reduced concentrations of expressed proteins, efficient binding to either VDRE occurs as a
VDR·RXR heterodimer, not as a VDR homodimer.
1,25-(OH)2D3 dramatically enhances
heterodimer-VDRE interaction, whereas somewhat higher concentrations of
9-cis-RA inhibit this association, perhaps related to the
role of this retinoid in facilitating RXR homodimer formation. Interestingly, if VDR is occupied by
1,25-(OH)2D3 prior to complexing with RXR, the
resulting heterodimer is relatively resistant to dissociation and
diversion to other pathways by 9-cis-RA. Therefore, a
proposed molecular action of 1,25-(OH)2D3 is to
generate an allosteric switch in VDR to a form that not only binds to
the VDRE with high affinity and specificity as a heterodimer with RXR,
but also interacts with the RXR partner to conformationally restrict
the action of its cognate ligand.
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INTRODUCTION |
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The active metabolite of vitamin D, 1,25-dihydroxyvitamin
D3
(1,25-(OH)2D3),1
exerts its hormonal effects predominantly on intestine, bone, and
kidney, where it plays a crucial role in calcium and phosphate homeostasis, bone mineralization, and the prevention of rickets (1).
1,25-(OH)2D3 also has effects on many
non-classical target tissues (2), such as those of the bone
marrow/immune system, where it has been reported to be involved in a
wide variety of differentiation processes (3, 4). The cellular
responses to 1,25-(OH)2D3 are mediated by the
vitamin D receptor (VDR) (1), which is a member of a superfamily of
nuclear transcription factors that include the steroid, retinoid, and
thyroid hormone receptors (5). Transcriptional control of specific
1,25-(OH)2D3-responsive genes is achieved by
binding of the receptor, via its DNA binding domain, to a specific
vitamin D-response element (VDRE) located in the 5' promoter region of
the regulated gene. VDREs have been identified in positively controlled
genes such as human and rat osteocalcin (6-8), mouse osteopontin (9),
avian integrin
3 (10), and rat vitamin D 24-hydroxylase
(11, 12), and comparison of these sequences indicates that they
generally consist of a hexanucleotide direct repeat with a spacer of
three nucleotides (DR+3).
In many studies with natural DR+3 VDREs, VDR has been reported to bind as a heterodimer with the retinoid X receptor (RXR) (reviewed in Ref. 13). Thyroid hormone receptor (TR) and retinoic acid receptor (RAR) also form RXR-containing heterodimers on positively controlled DR+4 and DR+5 response elements, respectively (14). Evidence has been provided, however, implying that VDR may also bind as a homodimer to certain DR+3 elements, primarily those containing the osteopontin-like half-site, AGTTCA (15, 16).
In addition to this issue of VDR homodimeric binding to VDREs, controversy exists as to the precise molecular role (if any) of 1,25-(OH)2D3 in VDR·RXR heterodimer (or homodimer) formation and DNA binding. 1,25-(OH)2D3 has been proposed to elicit high affinity association of the VDR with its heterodimeric partner (17, 18), as well as induce a conformational change in VDR·RXR inferred from an increase in the mobility of the VDR·RXR·VDRE complex (8). Other studies indicated that the presence of both RXR and 1,25-(OH)2D3 is required for optimal binding of VDR to a DR+3 from the osteocalcin VDRE (19) and to a 24-OHase VDRE (20). In contrast, it has been proposed that initial VDR·RXR DNA interaction occurs in a ligand-independent fashion (21). Still another variation was postulated by Freedman and co-workers (22), in which the role of 1,25-(OH)2D3 was hypothesized to be the dissociation of the DNA-bound homodimer by decreasing the rate of conversion of DNA-bound monomer to homodimer, and ultimately promoting the formation of a DNA-bound VDR·RXR heterodimer.
There are also conflicting experimental data on the role played by 9-cis-retinoic acid (9-cis-RA), the cognate ligand for RXR, in the binding and transcriptional regulation of 1,25-(OH)2D3-responsive genes by the VDR·RXR heterodimer. 9-cis-RA has been observed to repress the 1,25-(OH)2D3-dependent accumulation of osteocalcin mRNA in osteoblast-like ROS 17/2.8 cells (19). Further analysis of VDRE binding in mobility shift assays (19) demonstrated that heterodimeric interactions between VDR and RXR were enhanced by 1,25-(OH)2D3, and were inhibited by high concentrations of 9-cis-RA, consistent with the observation that RXR-specific ligands can induce RXR homodimer formation in solution (23). In contrast, treatment of a variety of transfected cell types with both 1,25-(OH)2D3 and 9-cis-RA was reported to enhance the expression of an osteopontin VDRE-linked reporter gene over levels observed with 1,25-(OH)2D3 alone (24, 25). An apparent synergistic co-stimulation by 1,25-(OH)2D3 and 9-cis-RA in transfection systems employing AGGTCA synthetic DR+3 constructs has also been reported (26, 27), and in at least one instance, 9-cis-RA was observed to have no effect on 1,25-(OH)2D3-stimulated transcription (28). Because of the wide range of cell types (insect to mammalian) utilized in these transfection studies plus the untold effects of variable receptor/coreceptor expression, as well as different levels of reporter plasmid, it is difficult to interpret the general significance of these findings.
Therefore, to define at the biochemical level how 1,25-(OH)2D3, VDR, RXR and DNA interact ultimately to stimulate the transcription of hormone-responsive genes, the following three areas of conflicting experimental data were addressed: (i) the relative importance of VDR homodimer versus VDR·RXR heterodimer binding to DR+3 VDREs, including elements consisting of osteopontin half-sites; (ii) the effect of 1,25-(OH)2D3 on VDR binding to RXR or VDR·RXR binding to DNA; and (iii) the role, if any, of 9-cis-RA on VDR·RXR DNA binding. To resolve these issues, we have utilized an electrophoretic mobility shift assay system that includes physiological salt and limited receptor concentrations to investigate the effects of cognate ligands on possible VDR homodimer and VDR·RXR heterodimer binding to their target response elements.
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EXPERIMENTAL PROCEDURES |
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Enriched Receptors--
The human RXR (hRXR
), murine
RXR
(mRXR
), and hVDR cDNAs were incorporated into a
baculovirus expression vector system (BEVS) as described previously (8,
19). All recombinant baculoviruses were isolated and plaque-purified
using standard procedures (29), then used to infect Sf9 cultures
(Spodoptera frugiperda ovary cells). After 48 h, whole
cell extracts were prepared by sonication in KETZD-0.3 (0.3 M KCl, 1 mM EDTA, 10 mM Tris-HCl,
pH 7.8, 0.3 mM zinc acetate, 5 mM
dithiothreitol), followed by centrifugation at 200,000 × g for 30 min. Supernatants containing hVDR, hRXR
, or
mRXR
were enriched to at least 70% purity by blue dextran Sepharose
chromatography as described in detail for VDR (8), then snap-frozen and
stored at
70 °C. Human RXR
was also expressed in
Escherichia coli and enriched to approximately 70% purity
as described previously (30). Human RXR
(hRXR
) was obtained from Affinity BioReagents (Golden, CO).
Antibodies--
A monoclonal antibody to the vitamin D receptor,
9A7 (31), an hRXR
polyclonal antibody (32), a monoclonal antibody
against mRXR
, designated MOK 13.17 (33), and an RXR
polyclonal
antibody obtained from Affinity BioReagents were utilized.
Reagents--
Klenow enzyme was purchased from Promega Corp.
(Madison, WI). Poly(dI-dC) was obtained from Boehringer Mannheim.
Synthetic oligonucleotides corresponding to the respective hormone
response elements were synthesized by Integrated DNA Technologies Inc. (Coralville, IA). [-32P]dCTP (3000 Ci/mmol) was
purchased from NEN Life Science Products.
Oligonucleotide Labeling-- Double-stranded versions of each of the tested response elements (direct repeat hexanucleotides are underlined) with HindIII overhangs (lowercase) were created by annealing the following single-stranded synthetic oligonucleotides together with their complements (not shown). For the mouse osteopontin VDRE (9), the oligonucleotide was 5'-agctACAAGGTTCACGAGGTTCACGTTCT-3'. For the rat osteocalcin VDRE (7), the oligonucleotide was 5'-agctGCACTGGGTGAATGAGGACATTACA-3'. For the rat cellular retinol-binding protein (CRBP) II RXRE (34), with a potential third half element indicated by a double underline, the oligonucleotide was 5'-agctGCTGTCACAGGTCACAGGTCACAGTTCA-3'.
The double-stranded oligomers were labeled with [Ligand-dependent Gel Mobility Shift
Assays--
Previous VDR·RXR gel shift protocols were altered to
reduce concentrations of receptors to approximate physiologic levels. Accordingly, hVDR (10-100 ng = 5-50 nM) was added to
a mixture containing 10 mM Tris-HCl, pH 7.6, 150 mM KCl, 1.0 mM dithiothreitol, 15% glycerol, 1 mg/ml acetylated bovine serum albumin, and 50 µg/ml poly(dI·dC),
followed immediately by an amount of RXR or RXR
equivalent to
that of hVDR. Prior to the addition of receptor(s), 1,25-(OH)2D3 was added to the reaction mixture
in ethanol vehicle to give a final ligand concentration of
10
10 to 10
5 M. In experiments
that involved the additional inclusion of 9-cis-RA (10
10 to 10
5 M), ethanol
vehicle controls were included and the binding portion of the reaction
(45 min at 22 °C) was shielded from light. One µl of radioactively
labeled DNA probe (containing 0.5 ng DNA, or approximately 0.8 nM, and 50,000-100,000 cpm) was added to give a final
volume of 40 µl, then incubated for an additional 30 min. Thereafter,
the entire reaction was subjected to nondenaturing electrophoresis on a
4% polyacrylamide gel. The dried gels were used to expose Kodak X-Omat
film at
70 °C. The results of a number of assays were quantitated
by densitometric analysis using an Apple OneScanner with OfotoTM
scanning software developed by Light Source, Inc.
Preparation of Nuclear Extract from ROS 17/2.8 Cells-- Cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 medium (1:1) supplemented with 5% fetal bovine serum and 5% newborn calf serum. Log phase cultures were harvested by trypsinization and collected by centrifugation at room temperature for 10 min at 1000 × g, followed by washing in five volumes of phosphate-buffered saline at 4 °C. The nuclear extract was then prepared as described previously (36), with all steps performed at 4 °C.
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RESULTS |
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VDR Ligand Enhances Binding of RXR·VDR Heterodimers to the Mouse
Osteopontin and Rat Osteocalcin VDREs--
Fig.
1A depicts a gel mobility
shift experiment using the mouse osteopontin (MOP) VDRE and
baculovirus-expressed VDR in the presence and absence of hRXR or
mRXR
(20 ng of each receptor). At limiting concentrations, neither
the VDR alone (lanes 1 and 2) nor RXR alone (data
not shown) (35) produce an observable DNA binding complex either in the
absence or presence of 1,25-(OH)2D3. The only
major DNA binding complex observed in this experiment is that produced
by incubation of VDR plus either RXR
or RXR
, and, by
densitometric analysis, this complex is enhanced 23-fold in the
presence of hormone (compare lane 3 to lane 4,
and lane 7 to lane 8). The average degree of
binding enhancement elicited by 1,25-(OH)2D3
observed from more than 30 such experiments carried out at limiting
receptor concentrations was 20-fold, although effects of ligand
apparently as low as 2.5-fold have been recorded (see Fig. 4). The
hormone effect is selective for 1,25-(OH)2D3, since the relatively inactive natural vitamin D metabolite
24(R)-25-(OH)2D3, its epimer,
24(S)-25-(OH)2D3, as well as the
25-(OH)D3, 1
-(OH)D3, and vitamin
D3 hormone precursors, do not enhance DNA-binding complexes
at 10
8 M (data not shown). The
hormone-stimulated, heterodimeric bands seen in lanes 4 and
8, are eliminated (lanes 5 and 9) by
inclusion of the VDR-specific monoclonal antibody 9A7, which recognizes an epitope within the DNA binding domain. Moreover, the addition of an
RXR
-specific polyclonal antibody (lane 6) caused a
majority of the complex to supershift, confirming the presence of
RXR
in the complex. Interestingly, the RXR
-specific monoclonal
antibody MOK 13.17 (lane 10) showed little interaction with
the VDR·RXR
DNA binding complex. As discussed below, the MOK 13.17 antibody may not be able to access its epitope when RXR
is
heterodimerized with VDR.
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Concentration Dependence of the
1,25-(OH)2D3 Effect on VDR·RXR Heterodimer
Binding to the MOP and ROC VDREs--
Fig.
2 depicts one of three replicate
experiments testing the influence of different concentrations of
1,25-(OH)2D3 on VDR·RXR binding to the MOP
(Fig. 2A) or ROC (Fig. 2B) VDRE. Binding to the
MOP VDRE (Fig. 2A) exhibits a maximum at 105
M 1,25-(OH)2D3, diminishing to a
minimal but significant enhancement at 10
10 M
1,25-(OH)2D3. The ROC VDRE (Fig. 2B)
displays a slightly different pattern with an optimum at
10
6 M hormone, followed by a more gradual
decrease in binding as the ligand concentration is lowered.
Densitometric scanning of these two gels, however, revealed that the
ED50 of 1,25-(OH)2D3 for both VDREs
was approximately 10
9 M, representing a
13-fold increase in receptor heterodimer binding to the ROC VDRE and a
15-fold increase on the MOP element. Accordingly, the concentrations of
1,25-(OH)2D3 chosen for most subsequent experiments were either 10
9 or 10
8
M.
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Effect of 1,25-(OH)2D3 to Reduce
Dissociation of the VDR·RXR Heterodimer from a VDRE--
Labeled MOP
VDRE was incubated with VDR and RXR in the absence and presence of
1,25-(OH)2D3, followed by inclusion of a
100-fold excess of competing unlabeled MOP VDRE at time zero. As shown in Fig. 3A, the presence of
ligand not only greatly potentiates initial (time 0) DNA binding, as
seen previously, but also results in an apparent slower rate of
dissociation of the heterodimeric receptor complex from the labeled
response element. Densitometric analysis of this experiment (Fig.
3B) indicates that the liganded VDR·RXR complex exhibits a
half-life of
11 min (by extrapolation), whereas the unliganded
VDR·RXR heterodimer dissociates with an estimated half-life of only
3.0 min.
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Higher Receptor Levels Generate Apparently Artifactual VDR
Homodimers and Attenuate the VDR Ligand Dependence of VDR·RXR Binding
to the ROC and MOP VDREs--
The experiment depicted in Fig.
4 was performed using both 20 ng and 100 ng of VDR, to determine if homodimeric binding, which was not
observable using limiting receptor concentrations (Fig. 1A),
could be elicited at receptor concentrations significantly in excess of
physiologic levels. On the weaker ROC response element (Fig. 4,
left panel), neither 20 ng (lanes 1 and
2) nor 100 ng (lanes 3 and 4) of VDR
exhibited any observable monomeric or homodimeric DNA binding species,
even in the presence of 1,25-(OH)2D3. We have
previously observed VDR homodimer binding to the ROC VDRE at very high
VDR amounts of 260 ng (35), but the stability of such VDRE complexes is
quite minimal, with an apparent half-life of less than 0.5 min when
assayed in a mobility shift dissociation assay similar to that shown in
Fig. 3.2 On the stronger MOP
response element (Fig. 4, right panel), 100 ng of VDR
produced a faint DNA binding complex, apparently corresponding to a
homodimeric species (lanes 3 and 4). Moreover, by
densitometric analysis, the putative homodimeric binding seen in
lane 3 (right panel) displayed virtually no
enhancement (1.3-fold) in the presence of hormone (lane 4 versus
lane 3). As a positive control in these experiments, 20 ng of VDR
in combination with 20 ng of RXR produced DNA binding heterodimeric
species on the ROC and MOP VDREs which were enhanced by ligand 10-fold
and 2.5-fold, respectively (Fig. 4, lane 6 versus lane 5 in
each panel). At higher, nonphysiological amounts (100 ng of each
receptor), however, the binding of this heterodimeric species was not
significantly enhanced (1.3-fold for both ROC and MOP) by the presence
of 1,25-(OH)2D3 (Fig. 4, lane 8 versus
lane 7 in each panel).
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Effect of 9-cis-RA on RXR Binding to an RXRE as well as on
RXR·VDR Association with a VDRE--
9-cis-RA reportedly
can augment the DR+1 binding efficiency of RXR produced by in
vitro transcription/translation, although no RXR ligand effect was
observed with receptor produced in E. coli, Sf9, or
COS-1 cells (37). Accordingly, we probed the possible influence of
9-cis-RA on the binding of RXR homodimers to a natural RXRE,
a DR+1 element from the rat CRBP II gene (34). Using our conditions of
reduced amounts of receptor and physiological salt concentrations,
binding of BEVS mRXR
(40 ng) as a homodimer to the CRBP II RXRE was
enhanced 16-fold by the presence of 10
6 M
9-cis-RA (Fig. 5A,
lane 2 versus lane 1). Although it was noted in Fig.
1A that the MOK 13.17 RXR
antibody had relatively little effect on VDR·RXR
binding to a VDRE, an apparent stabilization as
well as robust supershift of the DNA-bound, unliganded, and ligand-enhanced RXR homodimeric species by this antibody was observed (Fig. 5A, lanes 3 and 4). Thus, RXR
appears to be more accessible to the MOK 13.17 antibody when configured
in its homodimeric form than when it exists in a heterodimer with VDR.
As expected, the ligand-dependent RXR homodimer complex
with the RXRE was neither inhibited nor supershifted in the presence of
a VDR antibody (Fig. 5A, lanes 5 and
6). Fig. 5B depicts the RXRE binding displayed by
E. coli-expressed hRXR
in the presence of varying
concentrations of 9-cis-RA. At concentrations of
10
5, 10
6, and 10
7
M, the presence of 9-cis-RA enhances homodimeric
binding to the RXRE 5.1-, 4.0-, and 1.4-fold, respectively, as
determined by densitometric scanning.
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Effect of Different Orders of Receptor-Ligand Assembly--
In a
typical experiment depicted in Fig. 7, we
employed baculovirus expressed mRXR as the heterodimeric partner for
VDR. In lanes 1-3, the individual receptors and the
indicated ligands were added simultaneously to the reaction mixture.
Lanes 4-6 contain reactions in which VDR and RXR were first
allowed to associate for 30 min and were then added to a reaction
mixture containing either 1,25-(OH)2D3
(lane 5) or a combination of
1,25-(OH)2D3 and 9-cis-RA
(lane 6). Finally, each receptor was individually preincubated with the indicated ligands and, after a 30-min period, the
respective receptor partner was added to the reaction (lanes 7-10). Densitometric analysis of four such independently
performed experiments indicated 9-cis-RA inhibits
1,25-(OH)2D3-mediated VDRE binding by an
average of 78% when each component is added simultaneously to the
reaction (Fig. 7, lane 3 versus lane 2). Similar repression
of heterodimeric binding (85%) is observed when the preincubated
VDR·RXR mixture is combined with ligands (Fig. 7, lane 6 versus
lane 5), and a significant inhibitory effect (60%) also is
observed when RXR is incubated initially with 9-cis-RA to
apparently form homodimers (Fig. 7, lane 10 versus lane 8). However, the 9-cis-RA inhibitory effect is nearly prevented
(an average of only 14% reduction) whenever VDR is allowed to
preincubate in a mixture containing
1,25-(OH)2D3 and 9-cis-RA, with RXR
being added to the system after 30 min (Fig. 7, lane 9 versus
lane 8). Further experiments (data not shown) indicated that if a
preformed heterodimer is incubated with either
1,25-(OH)2D3 alone for 30 min or with
1,25-(OH)2D3 followed by the addition of
9-cis-RA, or even first with 9-cis-RA alone for
30 min followed by exposure to 1,25-(OH)2D3,
this heterodimer is still strikingly susceptible to inhibition by
9-cis-RA (approximately 88% to 95% reduction). Thus, the
situation in which VDR is allowed to bind
1,25-(OH)2D3 before heterodimer
formation is unique in that the resulting heterodimer complex is
largely resistant to dissociation by 9-cis-RA.
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DISCUSSION |
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An experimental system has been devised to investigate the
interaction of VDR, RXR, 1,25-(OH)2D3, and
9-cis-RA with DNA, and to assess the contribution of each
component to overall complex formation. Previous studies investigating
the role of ligand in the interaction of VDR with DNA have produced
somewhat conflicting results and conclusions (see Introduction).
Indeed, even in our hands, VDR complex formation on a VDRE can become
ligand- and RXR-independent either when salt concentrations are
non-stringent (<100 mM KCl; data not shown) or when
receptor levels are increased to 50 nM (Fig. 4). The
parameters used in the present gel mobility shift assay system were
carefully chosen to approach those found within the cell, including 150 mM KCl and receptor concentrations in the range of 5-10
nM. Under these conditions, association between VDR and the
MOP or ROC VDREs occurs only in the presence of RXR, and is greatly
enhanced by 1,25-(OH)2D3 (Fig. 1, A
and B). Further, the fact that both types of VDRE display an
average of >10-fold ligand-dependent enhancement of
receptor-DNA binding under near-physiologic conditions suggests that
this effect is not gene- or element-specific. Endogenous VDR and RXR
from a ROS 17/2.8 cell nuclear extract (Figs. 1C and
6B) also exhibit a ligand-dependent enhancement of binding to the VDRE, although the magnitude of the effect is lower
(2-3-fold). This lower degree of ligand stimulation possibly reflects
the presence of proteins that nonspecifically bind to 1,25-(OH)2D3 or interfering DNA-binding
proteins in the cruder nuclear preparations. It is also conceivable
that ligand acts on overexpressed receptors to facilitate their folding
into a native conformation, thereby enhancing
hormone-dependent DNA binding. Nevertheless, the fact that
we observe a consistent effect of ligand using endogenous VDR in ROS
17/2.8 cell nuclear extracts argues that the enhancement by ligand of
receptor binding to VDREs is biologically relevant. This conclusion is
independently supported by in vivo footprinting experiments
(38), which reveal that 1,25-(OH)2D3 is
required for VDR occupation of the VDRE in the native rat osteocalcin
promoter.
The present results indicate that VDR homodimerization is not a physiologically relevant event on either the ROC or MOP response elements. Previous studies utilizing an artificial osteopontin-like VDRE (15, 22), and the actual mouse osteopontin VDRE (16), suggested that these elements represented a target for VDR homodimeric binding, although the salt concentrations used during gel mobility shift assays were not specified in every case, and very high (micromolar) concentrations of VDR were utilized in gel filtration experiments (22). We have used both the natural MOP and ROC VDREs (Fig. 4), which typify the two main subtypes of VDREs reported to date. ROC is an imperfect direct repeat with a G present as the third base in each half element, whereas MOP is a perfect direct repeat with a T present in this position. It is this latter feature of the MOP element that was proposed to distinguish it for VDR homodimeric binding (15). The current results (Fig. 4) employing limited amounts of receptor (20 ng or 10 nM) fail to show binding of any homodimeric form of VDR to either VDRE, arguing against any distinction between these positive VDREs in their binding of VDR homo- versus heterodimers. Only at supraphysiological levels of receptor (approximately 50 nM) can a faint homodimeric species be observed to form on the MOP VDRE; further, this weak binding is relatively unaffected by the presence of 1,25-(OH)2D3 (see Fig. 4, lanes 3 and 4 in the right panel). Finally, MacDonald et al. (39) have demonstrated that VDR does not interact with itself in the yeast two-hybrid system, arguing against homodimerization of VDR in solution as well.
Although the heterocomplex of liganded VDR with RXR appears to constitute the physiologically significant DNA binding species, readily detectable VDRE association does occur in the absence of 1,25-(OH)2D3 when both receptors are present in excess, in vitro (Fig. 4, lanes 7 and 8 in each panel). Loss of the 1,25-(OH)2D3 requirement at higher receptor levels probably explains previous reports that VDR·RXR, and perhaps other heterodimeric DNA complexes such as those with TR·RXR and RAR·RXR, do not require ligand for specific DNA binding. We propose a model (see Fig. 8) in which VDR·RXR heterodimers exist in equilibrium between two distinct states, with the addition of 1,25-(OH)2D3 shifting the equilibrium of the heterodimer to a form that binds to the VDRE with not only specificity, but also greater affinity (Fig. 3). A supraphysiologic concentration of receptor would, by mass action, artifactually increase the amount of receptor species already in the allosteric state that binds DNA, thus explaining the lack of hormone effect under conditions of vast VDR and RXR excesses.
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The ligand-dependent gel mobility assay system has also
been utilized to examine the ligand responsiveness of RXR, the
heterodimeric partner of VDR, in binding as a homodimer to its cognate
response element. Employing an RXRE of the DR+1 type (34), we find that RXR and RXR
3 homodimers
form a DNA-bound complex preferentially in the presence of
9-cis-RA (Fig. 5, A and B). In
contrast to 1,25-(OH)2D3, the concentrations of
9-cis-RA (10
5 to 10
6
M) required to produce a striking
ligand-dependent effect are higher than what are normally
considered physiological concentrations (low nanomolar) of this
retinoid. However, it should be noted that the Kd
(1.2 × 10
8 M) for 9-cis-RA
binding to RXR (40) is 2 orders of magnitude higher than that for
1,25-(OH)2D3 binding to VDR. It is also
possible that the extreme photolability of 9-cis-RA may
lower the effective concentration present in these in vitro
reactions, and in vivo, target tissues may require local
concentrations that are much higher than circulating levels for a
hormonal response. Nevertheless, our results agree qualitatively with a
previous report that 9-cis-RA enhances the binding of RXR
homodimers to a DR+1 element (41).
A persistent area of controversy has been the effect of
9-cis-RA on VDR·RXR heterodimer binding to DNA and, hence,
regulation of gene transcription. Previous studies from a number of
laboratories have produced conflicting results ranging from evidence
that 9-cis-RA can destabilize VDR·RXR heterodimers and
presumably induce RXR homodimer complex formation (19), to a conclusion
that this ligand interacts synergistically with
1,25-(OH)2D3 to transcriptionally activate the
VDR·RXR heterodimer (24, 25). Our data from the gel mobility shift
assay system tend to confirm that the effect of 9-cis-RA is
to dissociate the VDR·RXR heterodimer and divert RXR to form
homodimers. In combination with 109 M
1,25-(OH)2D3, 9-cis-RA at
concentrations of 10
5 M to 10
8
M, equal to or slightly lower than those that elicit
homodimerization of RXR (Fig. 5), cause significant dissociation of the
VDR·RXR
heterodimer (Fig. 6A). The inhibitory effect of
9-cis-RA is seen even in the absence of an RXRE (data not
shown), consistent with previous reports that RXR homodimerization
takes place in solution (23). Importantly, using endogenous receptors
present in a ROS 17/2.8 cell nuclear extract, 10
6
M 9-cis-RA inhibits
1,25-(OH)2D3 stimulated binding to the VDRE by
80% (Fig. 6B). The fact that this inhibitory effect of
9-cis-RA can be overcome by the subsequent addition of
excess RXR (Fig. 6C) argues against the possibility that
9-cis-RA may be altering the affinity of the heterodimer for
the VDRE and suggests that this inhibition is due to ligand-induced
diversion of limiting RXR from the VDR·RXR complex toward homodimers.
These findings can be generalized to propose the existence of hormonal
crosstalk between metabolites of vitamins A and D in controlling
VDR-mediated transcriptional regulation.
The present results further suggest that, in addition to the availability of target sequences, receptors, and hormonal ligands, another parameter regulating the transcriptional response to hormone may be the sequential order in which these components assemble. As can be seen from Fig. 7, the ability of 9-cis-RA to shift the equilibrium from DNA-bound VDR·RXR heterodimer to RXR homodimer formation depends either on 9-cis-RA first occupying RXR monomers to facilitate their homodimerization, or on VDR and RXR forming an apoheterodimer. Evidence that VDR and RXR can form apoheterodimers in solution is supported by yeast two-hybrid interaction studies (39), and protein-protein association assays using either VDR or RXR expressed as glutathione S-transferase fusion proteins (data not shown). Taken together, these findings suggest the existence of at least two allosteric pathways for VDR·RXR association and response to ligand, as presented in Fig. 8. This model predicts two allosterically distinct forms of the 1,25-(OH)2D3-occupied heterodimer: (i) a preformed VDR·RXR heterodimer that subsequently binds 1,25-(OH)2D3 and hence is induced to bind its specific DNA target sequence, but can be dissociated by the addition of 9-cis-RA (Fig. 8, pathway A) and (ii) a pre-liganded form of VDR that creates a heterodimer, which is largely resistant to 9-cis-RA-induced dissociation (Fig. 8, pathway B). This model may explain why 9-cis-RA is observed only to partially suppress 1,25-(OH)2D3 induced transcription in transfection studies (19), since the intact target cell likely contains a mixed population of the proposed two allosteric forms of the heterodimer.
Recently, a type of converse regulation has been reported with RXR homo- and heterodimers (42). It was found that unliganded RAR and TR suppressed RXR-mediated transcriptional response to an RXR specific-ligand, indicating that RAR and TR function as attenuating partners of RXR. Further, TR, when bound to its cognate ligand, completely suppresses RXR ligand binding, similar to what is observed with pre-liganded VDR (Fig. 8, pathway B). However, in contrast to both VDR·RXR and TR·RXR heterodimers, 9-cis-RA binding to RXR is restored when its partner RAR binds its cognate ligand, resulting in a synergistic response to both ligands. It was therefore proposed (42) that the addition of the RAR ligand transmits a positive allosteric signal to the RXR heterodimeric partner, whereas similar addition of ligand to TR prior to heterodimerization transmits a negative allosteric signal to RXR, which prevents it from binding 9-cis-RA. As summarized in Fig. 8, the current experimental evidence for VDR suggests that only the pre-liganded form of VDR can effectively mediate a negative allosteric change in RXR, such that it can neither bind 9-cis-RA nor readily dissociate to form RXR homodimers.
In conclusion, we propose that a role of 1,25-(OH)2D3 is to drive VDR·RXR heterodimers to an allosteric form which displays high affinity and specificity for its target response element. We also have presented evidence that the sequential order of assembly of ligand, receptor, and DNA apparently can result in distinct allosteric forms of the heterodimer which respond differently to the presence of 9-cis-RA, and perhaps reflect allosteric changes in VDR that are conveyed to its heterodimeric partner. This proposed conformational switch may be fundamental to transcriptional regulation by the VDR subfamily of nuclear receptors.
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ACKNOWLEDGEMENTS |
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We thank Milan Uskokovic of
Hoffmann-LaRoche Inc. for kindly providing 1,25-dihydroxyvitamin
D3 and other metabolites for our studies and Arthur Levin,
also of Hoffmann-LaRoche, for the kind gift of
9-cis-retinoic acid. We are grateful to Drs. Jacqueline Dyck
and Ronald Evans for supplying us with anti-RXR polyclonal antibody
and to Dr. Keiko Ozato for providing anti-RXR
monoclonal antibody.
We are indebted to Dr. Jui-Cheng Hsieh for the preparation of E. coli expressed RXR
. We express appreciation to Sanford H. Selznick for technical expertise and patience in the preparation of
figures for this manuscript. We also thank Michelle Thatcher, Michael
Galligan, and Lenore Remus for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AR-15781 and DK-33351 (to M. R. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biochemistry, College of Medicine, The University of Arizona,
Tucson, AZ 85724. Tel.: 520-626-6033; Fax: 520-626-9015; E-mail:
haussler{at}u.arizona.edu.
1
The abbreviations used are:
1,25-(OH)2D3, 1,25-dihydroxyvitamin
D3; VDR, vitamin D receptor; VDRE, vitamin D response
element; TR, thyroid hormone receptor; RAR, retinoic acid receptor;
RXR, retinoid X receptor; m, mouse; h, human; 9-cis-RA,
9-cis-retinoic acid; BEVS, baculovirus expression vector
system; CRBP II, cellular retinol-binding protein II; RXRE, retinoid X
receptor response element; ROC, rat osteocalcin; MOP, mouse
osteopontin.
2 P. N. MacDonald and M. R. Haussler, unpublished observation.
3
We consistently observe that
baculovirus-expressed mRXR, in either homo- or heterodimerization
with VDR, exhibits a greater responsiveness to its ligand than does
E. coli-expressed hRXR
.
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REFERENCES |
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