(Received for publication, December 3, 1996, and in revised form, April 21, 1997)
From the Department of Retinoid Research, Ligand Pharmaceuticals, Inc., San Diego, California 92121
We have previously shown that RNA levels of
kidney 25-hydroxyvitamin D3-24-hydroxylase
(24(OH)ase), a key metabolic enzyme for 1,25-dihydroxyvitamin
D3 (1,25(OH)2D3), is up-regulated
by retinoids in mice within hours. Deletion analysis of ~5500 base pairs of the human 24(OH)ase promoter showed that the sequence between
316 and
142 contained the information necessary and sufficient for
retinoid-induced activation of the promoter. This region contains two
previously defined vitamin D-responsive elements (VDREs) at
294 to
274 and
174 to
151. Mutation of either VDRE diminished
responsiveness of the
316 to
22 promoter sequence to retinoids or
1,25(OH)2D3, while mutation of both VDREs
essentially abolished the activity of the ligands via the promoter.
Heterologous promoter vectors driven by the VDREs were responsive
to a retinoid X receptor (RXR)-selective ligand (LG100268), a
retinoic acid receptor (RAR)-selective ligand (TTNPB), or
1,25(OH)2D3, while combinations of LG100268
with either TTNPB or 1,25(OH)2D3 resulted in
additive increases in activity. Band shift analyses showed that vitamin
D receptor, RAR, or RXR alone did not bind to the VDREs; however, the
combination of either vitamin D receptor or RAR with RXR led to
retardation of each of the labeled probes. Treatment of nontransfected
CV-1 cells with retinoids or 1,25(OH)2D3 resulted in induction of 24(OH)ase RNA, and ligand combinations led to
increased RNA levels. These data imply that either or both of the
heterodimer partners can be occupied with ligand to induce this enzyme,
with dual receptor occupation leading to increased activation.
The retinoic acid receptors (RARs)1
and retinoid X receptors (RXRs) are nuclear transcription factors that
are activated by their retinoid ligands, all-trans-retinoic
acid (tRA) and 9-cis-retinoic acid (9cRA) (1). While RARs
bind to both tRA and 9cRA with high affinity, RXRs bind to 9cRA but not
to tRA (2-5). Both receptor subfamilies are thought to mediate the
biological actions of retinoids in processes such as cellular growth
and differentiation and development by altering the production of
certain proteins in various cells at the level of gene transcription
(1, 6). The ligand-occupied receptors generally act by binding to
retinoid receptor-responsive elements in the promoter regions of target
genes. Ligand-bound RAR cooperates with RXR to form a heterodimer that
is an efficient and high affinity binder of retinoic acid response
elements, thereby activating transcription of certain tRA-responsive
genes such as RAR (7-9) and cellular retinoic acid binding protein
II (10). RXR is postulated to function by at least two modes of action. RXR has been shown to act as a silent (nonliganded) partner with a
number of other intracellular receptors, including RAR, vitamin D
receptor (VDR), and thyroid hormone receptor, in response to their
respective ligands (11-16). RXR has also been shown to form homodimers
in a complex with DNA upon binding to 9cRA (17). However, evidence for
RXR homodimers as functional units in the transcription of biologically
relevant target genes has yet to be demonstrated.
For example, while RXR binds to, and stimulates transcription from, an element within the cellular retinol binding protein II gene promoter in cotransactivation assays in mammalian cells (18) and in yeast cells (which do not contain RAR; Ref. 2), this gene has not been shown to be regulated by retinoids in nontransfected cells or in the animal. However, evidence is emerging that RXR may not function solely as a silent partner in hormone signaling pathways. Recently, 9cRA has been shown to activate RXR in heterodimeric interactions with two orphan receptors, LXR (19) and NGFI-B (20), to stimulate transcription from synthetic response elements in cotransfections assays. Additionally, RXR ligands have been shown to increase the effects of RAR ligands to induce certain RNA species (21). In these experiments, however, while an RXR ligand modulated the effects of an RAR ligand to activate certain genes, the RXR ligand alone did not induce RNA levels.
We show here that an RXR-selective ligand alone is able to activate transcription of the human 25-hydroxyvitamin D3-24-hydroxylase (24(OH)ase) promoter by binding to the RXR partner of RXR·VDR or RXR·RAR heterodimers and acting through previously defined VDRE sequences (22, 23) within the promoter. Either the RXR ligand alone or the VDR ligand alone leads to stimulation of the promoter, while the presence of both ligands leads to additive or more than additive induction of luciferase activity. These data indicate that ligand occupation of either or both heterodimeric receptor partners leads to a productive transcriptional event at this promoter, with maximal induction observed upon occupation of both receptors. Additionally, ligand-occupied RAR also activates this promoter through these sequences by binding with RXR, either with or with its ligand. Therefore, these two previously defined VDREs within the human 24(OH)ase promoter can also serve as retinoic acid response elements, since they are able to confer responsiveness of the promoter to retinoids.
LG100268 (24), TTNPB (25, 26), and 9cRA (25) were synthesized and purified at Ligand Pharmaceuticals, Inc. (San Diego, CA). 1,25(OH)2D3 was from Solvay Duphar (The Netherlands).
Receptor and Reporter VectorsReceptor expression vectors
(pRShRXR, pRSmRXR
, pRShRAR
, and pRShVDR) were as described
previously (2, 27). 24(OH)ase promoter-driven reporter constructs were
derived from an ~6-kb human genomic clone (23) that included sequence
3
to the start site of transcription. The 6-kb promoter fragment
(~
5500 to +455) was cloned into a promoterless luciferase
expression vector, pLUCpl (2), at SalI and PstI
sites upstream of the translation start site of the luciferase coding
region of the plasmid. Deletion and mutant promoter constructs were
generated from the 6-kb sequence with common 3
ends by digestion with
NsiI at position
22 in the promoter sequence (~
5500 to
22)-LUC. (
1177 to
22)-LUC was generated by digesting (~
5500
to +455)-LUC with Van91I (blunted) and NsiI, and
the resultant fragment was subcloned into pLUCpl digested with
XhoI (blunted) and PstI. (
316 to
22)-LUC,
(
294 to
22)-LUC, (
261 to
22)-LUC, and (
143 to
22)-LUC were
generated by PCR utilizing 5
primers, some with an overhanging
SalI site immediately upstream of the 5
-most base of the
deletion construct (others without an enzyme site) and a common 3
primer encompassing the NsiI site. PCR products were
digested with SalI (or used blunt-ended) and NsiI
and subcloned into pLUCpl digested with XhoI (blunted or
not) at the 5
end and PstI at the 3
end. (
261
1)-LUC
(
168CCC mutated to GTT) was created by
site-specific oligonucleotide-directed mutagenesis of the (
261 to
22) PCR product in M13, digested with SalI and
NsiI, and subcloned into pLUCpl cut with XhoI and PstI. (
316
1)-LUC (
168CCC mutated to
GTT) was made by digestion of (
316 to
22)-LUC with
HindIII/BstEII, digestion of (
261
1)-LUC with
BstEII/BamHI, and ligation of the two fragments
into pLUCpl digested with HindIII/BamHI. (
316
2)-LUC (
289CACC to AAAA)
was created utilizing the MORPHTM site-specific plasmid
DNA mutagenesis kit (5 Prime
3 Prime, Inc., Boulder, CO).
(
316
1
2)-LUC (
168CCC to GTT and
289CACC to AAAA) was generated by
digestion of (
316
2)-LUC with HindIII/BstEII
and (
261
1)-LUC with BstEII/BamHI and
combining the two fragments in pLUCpl digested with
HindIII/BamHI. (
316
3)-LUC (
308GGGAGGCGCGTTCG mutated to
AGAAGGCGCAAATT) was generated by
using PCR; the 5
primer contained the individual base changes as well as an XhoI site immediately upstream of position
316, and
the 3
primer contained the NsiI site at
22. The resultant
PCR product was digested with XhoI and NsiI and
subcloned into pLUCpl digested with XhoI/PstI.
Oligonucleotides corresponding to the individual VDREs (
294 to
274
and
174 to
151) were synthesized with HindIII ends,
annealed, and subcloned into the
MTV-LUC vector. The identities of
all constructs were confirmed by sequencing.
Receptor and
reporter vectors were transfected along with carrier DNA and
-galactosidase internal control plasmid in COS-1 cells by the use of
calcium phosphate and
N,N-bis(2-hydroxyethyl)-2-aminoethane-sulfonic acid-buffered saline. Briefly, transfections were performed in triplicate in 12-well plates with a total of 20 µg/ml DNA (0-0.5 µg of receptor expression vector, 5-10 µg of reporter plasmid, 5 µg of
-galactosidase plasmid, and pGEM carrier plasmid to a total
of 20 µg). Amounts of plasmids transfected in each experiment are
listed in individual figure legends. Fifteen hours later, ligands were
added, followed by an additional 30-h incubation. Cells were lysed, and
measurements of luciferase and
-galactosidase activities were as
described previously (4). Luciferase values were normalized with
-galactosidase values to control for variable transfection
efficiencies. -Fold induction was calculated by dividing the maximal
response by the response elicited with vehicle. Statistical analyses
were performed using analysis of variance and Fisher's protected least
squares determination.
Oligonucleotides
(294 to
274 with BamHI and HindIII sites at
each end and
174 to
151 with HindIII ends) and a PCR
fragment (
314 to
121) spanning the retinoid and
1,25(OH)2D3-responsive region of the human
24(OH)ase promoter were synthesized. Oligonucleotides containing a
consensus DR3 sequence (GGGAGGTCATTTAGGTCAGGG)
or a DR1 motif (AGGTCAGAGGTCA) were synthesized
with HindIII, or SalI overhanging ends,
respectively. The oligonucleotides and PCR fragment were end-labeled
with 32P-labeled nucleotide triphosphates. Protein extracts
were prepared from the yeast strain BJ2168 transformed without or with
VDR, RXR
, or RAR
expression vectors (2). In reactions including antibodies, anti-VDR 9A7
, anti-RXR
, anti-RAR
, or anti-estrogen receptor antibodies (28, 29) were preincubated with crude protein
extracts (~1 µl) for 1 h on ice prior to the addition of DNA
probe. Reactions performed with ligand present included 9cRA (1 µM, 0.1% ethanol) or ethanol vehicle and RXR-containing extracts; incubation was at 4 °C for 1 h prior to the DNA
addition. Protein-DNA binding reactions were carried out in buffer
containing 50-100 mM KCl, 20 mM Hepes, pH 7.4, 20% glycerol, 12.5 mM MgCl2. Oligonucleotide
probe (~20,000 dpm) was added to protein mixtures along with 1 µg
of poly(dI-dC) competitor DNA/reaction and incubated on ice for 20 min,
followed by 2 min at room temperature. Electrophoresis of protein-DNA
complexes was performed on 6% acrylamide, 0.5 × TBE gels (Novex,
San Diego) in 0.5 × TBE running buffer at 4 °C (~15 mA).
Gels were dried and exposed to autoradiographic film for 1-2 h at
80 °C.
CV-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with L-glutamine and 10% fetal bovine serum. Cells at ~40% confluency were incubated in media containing 10% charcoal-stripped fetal bovine serum for 48 h prior to treatment with ligands. Cells were ~80% confluent upon the addition of ligands in 0.1% ethanol vehicle. 1,25(OH)2D3, LG100268, TTNPB, 9cRA, or combinations thereof were added in fresh media containing charcoal-stripped fetal bovine serum and incubated with cells for 6 h prior to harvest. Concentrations of ligands are indicated in the figure legends. Total cellular RNA was extracted, and Northern analysis was performed as per standard methodology. Probes included a 900-base pair EcoRI/XbaI DNA fragment of the rat 24(OH)ase cDNA (30) and a 1.4-kb human glyceraldehyde-3-phosphate dehydrogenase fragment (Clonetech). Hybridization was performed at 65 °C in Quik-Hyb solution (Amersham Corp.) for 2 h followed by washing (0.1 × SSC, 0.1% SDS at 65 °C for 1 h). Quantitation was by PhosphorImager analysis (Molecular Dynamics).
Two regions of the human 24(OH)ase promoter are responsive to
retinoids and 1,25(OH)2D3. The cloning of the
rat (31, 32) and human (22, 23) 24(OH)ase promoters has been reported
previously. Two VDREs have been defined within both the rat and the
human promoters that confer responsiveness of the promoters to
1,25(OH)2D3 (22, 32). We have previously
reported that retinoids induce kidney 24(OH)ase activity in mice (27),
and cursory experiments indicated that the human 24(OH)ase promoter was
stimulated by retinoids in CV-1 cell cotransactivation assays (27). The
in vivo effects were observed with a synthetic RXR-selective
ligand, LG100268 (24), a synthetic RAR-selective compound, TTNPB, and the endogenous retinoid ligands, 9cRA and tRA. To determine the mechanism of retinoid activation of the 24(OH)ase promoter, an in depth
promoter study was undertaken. Various deletions of the previously
cloned ~6-kb human 24(OH)ase promoter (23) were constructed into a
promoterless luciferase reporter vector (2) and tested for the ability
to respond to various ligands in kidney cell lines cotransfected with
retinoid receptors and/or VDR. The promoter fragment containing ~5.5
kb upstream of the start site of transcription ((5500 to
22)-LUC)
yielded 3-fold induction of luciferase activity by the RXR-selective
ligand, LG100268, in either of two kidney cell lines, CV-1 (data not
shown) or COS-1 (Fig. 1A). COS-1 cells were
used for the remainder of the study, since they gave identical results
to CV-1 cells and were more stable in the transfection assays than CV-1
cells. The magnitude of the response (~3-fold) to LG100268 with
(
5500 to
22)-LUC was identical to that previously observed by us in
the mouse (27) and was also identical to that observed with (
1177 to
22)-LUC and (
316 to
22)-LUC. (
261 to
22)-LUC yielded a less
efficacious response (~1.8-fold activation; p < 0.05) with LG100268, and (
143 to
22)-LUC and pLUCpl did not respond
to LG100268 (Fig. 1A). Therefore, two regions within the
promoter conferred responsiveness to the RXR-selective ligand LG100268,
one between
316 and
261 and the other between
261 and
143 (see
Fig. 2). Interestingly, these two regions are also responsive to 1,25(OH)2D3 (Fig. 1B)
and contain previously defined VDREs (see Fig. 2; Refs. 21 and 22).
Sequence upstream of
1177 may contain an additional VDRE (Fig.
1B). The two LG100268-responsive regions also confer
responsiveness of the promoter to TTNPB (Fig. 1C;
p < 0.05) and to 9cRA treatment (Fig. 1D).
COS-1 cells contain endogenous VDR, RAR, and RXR proteins (data not
shown), and ligand-induced luciferase activity driven by the 24(OH)ase
promoter sequences was also observed without transfected receptor
expression vectors. The -fold induction varied from 20 to 80% of that
in the presence of transfected receptors, depending on the ligand (Fig.
3). The endogenous retinoid receptor pan-agonist, 9cRA,
elicited greater induction of luciferase activity through each of the
promoter constructs both in the presence (~6-fold; Fig.
1D) and in the absence (~3-fold; Fig. 3) of transfected
receptors, than either TTNPB or LG100268 alone, implying that occupancy
of both RAR and RXR leads to greater activation of the promoter than
either individual liganded retinoid receptor.
RXR Binds as a Heterodimer with VDR or RAR, but Not as a Homodimer, to Retinoid-responsive Sequences of the Human 24(OH)ase Promoter
Upon identification of the retinoid-responsive regions
of the human 24(OH)ase promoter (see Fig. 2), the ability of those sequences to bind directly to RXR and RAR was tested. It was previously demonstrated that RXR·VDR heterodimers could bind to these regions (22, 23). Oligonucleotides were synthesized spanning the regions from
294 to
274 and from
174 to
151. Wild type nontransformed yeast
extracts did not display DNA binding activity via either of these
sequences (Fig. 4, A-C). Extracts from yeast
transformed with a human RXR
expression vector (2) were able to bind
to each of these sequences in the presence of extracts from yeast expressing human VDR or human RAR
(Fig. 4, A-C) or human
RAR
(data not shown). However, RXR did not bind alone to these
sequences (Fig. 4, A-C) or to the entire responsive region
between
314 and
121 (Fig. 4D) in the absence or presence
of 9cRA (Fig. 4D and data not shown). Conversely, RXR was
able to bind to an oligonucleotide containing a consensus direct repeat
separated by 1 base pair (DR1) (Fig. 4D), as previously
shown (17, 18). Therefore, we conclude that RXR is unable to bind as a
homodimer to the retinoid-responsive elements of the 24(OH)ase promoter
but that it does form heterodimers with either VDR or RAR. Also, VDR
alone or RAR alone or the combination of the two receptors did not bind
to any of the DNA probes (Fig. 4, A-C, and data not shown).
Binding of RXR·RAR heterodimers to the human 24(OH)ase VDRE sequences
was surprising in that the DR3 motifs that they contain are thought to
bind preferentially to RXR·VDR heterodimers, while RXR·RAR
heterodimers have been shown to prefer DR2 and DR5 type motifs (33).
However, the 24(OH)ase promoter VDREs are not perfect consensus DR3
elements (see Fig. 2). Therefore, we tested the ability of RAR to
heterodimerize with RXR on a consensus DR3 element and compared the
binding with that via the 24(OH)ase VDREs. Fig. 4C shows
that RXR-RAR interactions do occur on a consensus DR3-containing
oligonucleotide probe (lane 4); however, this interaction is
much weaker than the RXR-VDR interaction via the DR3 (lane
5). In contrast, the RXR-VDR and RXR-RAR interactions on the
24(OH)ase VDREs were both of similar high affinity (Fig. 4,
A-C).
Mutations within the VDREs Abrogate Responsiveness of the Human 24(OH)ase Promoter to Retinoids
Two approaches were utilized to
further delineate the cis-elements involved in the response
of the promoter to retinoids: mutational analysis of the wild type
promoter and the use of heterologous promoter constructs driven by the
retinoid-responsive sequences. Mutations were made within the two
previously defined VDREs and within DR-like elements between 316 and
291 in the context of the wild type human 24(OH)ase promoter-driven
reporter vector. Point mutations within the 5
half-site of either the
upstream VDRE (
316
2-LUC) or the downstream VDRE (
316
1-LUC)
led to diminished -fold induction of luciferase activity by LG100268 or
1,25(OH)2D3 (Fig. 5,
A and B). Mutation of both VDREs
(
316
1
2-LUC) essentially abolished the response to each of the
retinoids as well as to 1,25(OH)2D3 (Fig. 5,
A-C). The mutations within the sequence between
316 and
294 (
316
3-LUC) had no effect on retinoid or
1,25(OH)2D3 responsiveness (Fig. 5,
A-C). Therefore, the two VDREs confer responsiveness of the
promoter to retinoids as well as to
1,25(OH)2D3. The noninvolvement of the sequence
between
316 and
294 was confirmed by a deletion construct
containing promoter sequence from
294 to
22, which exhibited the
same efficacy of response as the
316 to
22 construct to either
retinoid or 1,25(OH)2D3 (data not shown).
Either VDRE Confers Retinoid Responsiveness to a Heterologous Promoter: LG100268 and 1,25(OH)2D3 Yield Additive Induction via Each VDRE
To confirm that the VDREs were
able to function as cis-acting elements to confer
responsiveness of the promoter to retinoids, oligonucleotides spanning
the regions of activity were cloned into a MTV-LUC (MTV-LUC with the
glucocorticoid response element deleted) reporter vector. Fig.
6 shows that either VDRE alone as a single copy ((
294
to
274)-LUC or (
174 to
151)-LUC) was able to drive increased
luciferase activity in response to 1,25(OH)2D3 (panels A and C) or LG100268 (panels
A-C). 1,25(OH)2D3 treatment resulted in
12- and 11.3-fold induction of luciferase activity from the 3
and 5
VDREs, respectively (Fig. 6A). LG100268 alone yielded 4- and
3.5-fold induction from the 3
and 5
VDREs, respectively, while the
combination of 1,25(OH)2D3 and LG100268
resulted in 17.4- and 16-fold responses from the 3
and 5
VDREs,
respectively, which represent additive increases in luciferase activity
(Fig. 6A). TTNPB also acted through these elements on its
own and additively increased the activation elicited by LG100268 alone
(Fig. 6B). In these experiments (Fig. 6, A and
B), receptor plasmids were used at 0.1 µg/ml, and reporter
constructs were at 5 µg/ml, typical concentrations used in our
cotransfection experiments. To ensure that the additive effects
observed were not due to monomeric receptor activation of individual
reporter templates instead of heterodimer action on common templates,
the amount of reporter used was decreased to 0.1 µg/ml. Fig.
6C shows that, using the VDRE1-
MTV-LUC reporter construct
at this concentration, the overall luciferase values fall
substantially, as expected (Fig. 6, compare panels A and B with panel C). However, the additive effect of
1,25(OH)2D3 with LG100268 or 9cRA is still
observed, implying that the activity is on common templates and most
likely through heterodimers. Therefore, these data show that each of
the human 24(OH)ase VDREs is able to confer retinoid and
1,25(OH)2D3 responsiveness to a heterologous promoter and that saturating concentrations of each ligand in combination elicit greater reporter activity than either compound alone, implying that both receptors can be occupied with ligand to
yield greater activation of the promoter.
24(OH)ase mRNA Is Induced by LG100268, TTNPB, 9cRA, or 1,25(OH)2D3 in Nontransfected CV-1 Cells: Combinations of Ligands Elicit Additive Effects
To determine if
the additive effects of retinoids and
1,25(OH)2D3 in stimulation of the 24(OH)ase
promoter observed in cotransfection assays were borne out in an
endogenous setting, nontransfected CV-1 cells (which contain VDR, RARs,
and RXRs; our data not shown) were treated with various concentrations
of LG100268, TTNPB, 9cRA, or
1,25(OH)2D3, alone and in combination, and
24(OH)ase mRNA levels were assessed. Following ligand treatment for
6 h, cells were harvested, and total RNA was isolated and analyzed
via Northern blotting with a rat 24(OH)ase cDNA probe (30). Fig.
7 illustrates that 1,25(OH)2D3
at 108 M (lane 3) or
10
7 M (lane 4) was an effective
inducer of 24(OH)ase message levels (9.1- and 12.4-fold, respectively),
while vehicle-treated cells expressed very low levels of 24(OH)ase RNA
(lanes 1 and 2). LG100268 (lanes 5 and
6) and TTNPB (lanes 11 and 12) each at
10
7 and 10
6 M, also
up-regulated RNA levels (2.1-3.3-fold and 4.1-5-fold, respectively).
Combinations of 1,25(OH)2D3 and LG100268
yielded increased amounts (additive or superadditive) of 24(OH)ase RNA produced as compared with that resulting from cell treatment with identical concentrations of either ligand alone (compare lanes 3, 5, and 7; lanes 3,
6, and 8; lanes 4, 5, and
9; and lanes 4, 6, and 10).
Similar effects were also observed with the combination of TTNPB and
LG100268 versus either ligand alone (compare lanes 5, 11, and 13; lanes 6,
11, and 14; lanes 5, 12,
and 15; and lanes 6, 12, and
16). The naturally occurring bifunctional retinoid, 9cRA,
was also an efficacious inducer (9-10-fold) of 24(OH)ase RNA
(lanes 17 and 18), an effect that was greater
than that observed with either the RXR-selective (lanes 5 and 6) or the RAR-selective (lanes 11 and
12) ligand alone. 9cRA at 10 nM was as
efficacious as 10 nM 1,25(OH)2D3 in
induction of 24(OH)ase RNA levels in nontransfected CV-1 cells (compare
lanes 3 and 17). Additionally, 9cRA led to increased levels of 24(OH)ase RNA in combination with
1,25(OH)2D3 (lanes 19 and
20). These data corroborate the information elucidated from
the cotransfection/cotransactivation assays, since they indicate that
endogenous 24(OH)ase production in kidney cells can be effected either
by a retinoid alone or by 1,25(OH)2D3 alone at
nanomolar concentrations. Furthermore, the data also show that
combination treatment with retinoids and
1,25(OH)2D3 or two selective retinoids leads to
increased levels of 24(OH)ase RNA, as was also demonstrated in the
cotransfection assays. This information taken together with the
DNA-binding data lead to the conclusion that ligand occupation of
either or both receptor partners results in activation of 24(OH)ase, with increased levels of stimulation observed upon liganding of both
receptors.
The experiments described here indicate that RXR-selective ligands are able to activate the human 24(OH)ase promoter in cotransactivation assays in COS-1 cells through binding to RXR, which has the ability to form heterodimers with either VDR or RAR. These heterodimers form on previously defined VDREs (22, 23), which as demonstrated here, also act as retinoic acid response elements. RXR is not observed to homodimerize on the retinoid-responsive sequences of the promoter as determined by electrophoretic mobility shift assays. Activation via the VDREs is achieved by specific ligands for either receptor, and the presence of both ligands leads to increased stimulation. Pan-agonists such as 9cRA lead to greater activation than either retinoid receptor-specific ligand alone. While we describe ligand-bound RXR interacting with VDR or RAR to activate this promoter, we cannot rule out the possibility of the occurrence of another partner for RXR in vivo, such as an orphan receptor (19, 20). However, the experiments in nontransfected CV-1 cells (Fig. 7) showing that a combination of saturating amounts of 1,25(OH)2D3 and an RXR ligand yields increased levels of 24(OH)ase RNA is difficult to reconcile with the involvement of another partner.
The formation of RXR·VDR and RXR·RAR heterodimers occurs with approximately equal affinity on each of the VDREs within the human 24(OH)ase promoter as determined by electrophoretic mobility shift assays. This was somewhat surprising, since the VDREs contain DR3 motifs that have been shown to be preferential binders of RXR·VDR heterodimers rather than other receptor combinations (33). However, Umesono et al. (33) used consensus DR sequences for their experiments. Upon comparison of consensus DR3-containing oligonucleotides and the nonconsensus 24(OH)ase VDREs, it was apparent that while the perfect DR3 sequence did have a higher affinity for RXR·VDR heterodimers than for RXR·RAR heterodimers, the human 24(OH)ase VDREs bound both heterodimer pairs with approximately equal affinity (Fig. 4). These VDRE sequences also confer responsiveness of the promoter to retinoids and 1,25(OH)2D3 in cotransactivation assays. Therefore, the VDREs within the human 24(OH)ase promoter also function as retinoic acid response elements.
The stimulation of the human 24(OH)ase promoter in COS-1 cell
cotransfection/cotransactivation assays extends our previous work,
which showed that retinoids, including an RXR-specific ligand, induced
kidney 24(OH)ase RNA in mice within hours (27). This effect was
observed in normally fed or vitamin D-deficient mice, implying that
1,25(OH)2D3 was not required for the activation by retinoids. We performed cursory promoter experiments in that report,
which indicated that the activation by retinoids was not dependent on
the 3 VDRE. Our present data show that while stimulation of the human
24(OH)ase (
316 to
22) promoter sequence by retinoids is retained
with mutations in the 3
VDRE, it is diminished, and mutations in both
VDREs essentially destroy the ability of retinoids to initiate
transcription from the promoter. This work on the dissection of the
human 24(OH)ase promoter in cotransfection/cotransactivation assays
corroborates the effects previously observed in vitamin D-deficient and
normally fed mice (27), i.e. an RXR-selective ligand is able
to activate 24(OH)ase in the absence or presence of
1,25(OH)2D3.
The doses of 1,25(OH)2D3 that were administered
to the mice maximized the induction of 24(OH)ase RNA, and the addition
of LG100268 with 1,25(OH)2D3 had no effect on
RNA levels at 32 h postdose (27). To test the effects of
combinations of retinoids and 1,25(OH)2D3 at
shorter times post dosing, we used CV-1 kidney cells as a model. CV-1
cells were found to produce 24(OH)ase RNA, and the levels of the RNA
were modulated by 1,25(OH)2D3 and retinoids. 1,25(OH)2D3 induced 24(OH)ase RNA in CV-1 cells
in a dose-dependent manner (Fig. 7 and data not shown).
Each of the retinoids (LG100268, TTNPB, and 9cRA) tested also induced
24(OH)ase RNA in CV-1 cells. Combinations of
1,25(OH)2D3 with either LG100268 or 9cRA at
saturating doses gave additive or superadditive increases in the amount
of 24(OH)ase RNA that was produced. Additionally, 9cRA yielded a greater induction of RNA than either receptor-selective retinoid alone
at the same concentrations. The combination of the two selective retinoids (LG100268 and TTNPB) also elicited an additive effect. Therefore, from these experiments it was demonstrated that while either
a retinoid or 1,25(OH)2D3 alone induced
24(OH)ase RNA, the combination of both ligands gave an increased
effect. Additionally, 1,25(OH)2D3 and 9cRA were
approximately equipotent activators of 24(OH)ase in nontransfected CV-1
cells, since each ligand at 108 M gave a
similar induction of RNA levels (9.1- and 9.4-fold, respectively).
We have concluded from the data described herein that 1,25(OH)2D3 and retinoids exert additive effects through RXR heterodimers at the VDREs within the 24(OH)ase promoter. To rule out the possibility that retinoids induce VDR or that vitamin D up-regulates retinoid receptors in these cells, CV-1 cells were treated for 6 h with 10 and 100 nM 9cRA or 1,25(OH)2D3 and 100 nM and 1 µM LG100268 (concentrations that gave effects in both analyses), and receptor levels were quantitated by ligand binding assays. Neither retinoid increased VDR levels, as assayed by specific binding of CV-1 cell extracts to tritiated 1,25(OH)2D3 (data not shown). Additionally, 1,25(OH)2D3 treatment of the cells did not increase RARs or RXRs as assayed by specific binding of the extracts to tritiated 9cRA (data not shown). Therefore, the additive effects of retinoids and 1,25(OH)2D3 via this promoter are not due to ligand-induced up-regulation of the receptor proteins.
Two other vitamin D target genes have also been shown to be regulated by retinoids: osteopontin (34, 35) and osteocalcin (36-38). Osteopontin RNA was induced in rats after a 4-h treatment with tRA regardless of vitamin A or D status and cooperated with 1,25(OH)2D3 to induce increased levels of osteopontin (34). Others have used a heterologous promoter containing two copies of the osteopontin VDRE to show differential effects of retinoids in cotransfection assays (35). Osteocalcin production has been shown to be stimulated by retinoids in primary human osteoblasts, and synergistic induction was observed with tRA and 1,25(OH)2D3 (37), although others have reported down-regulation of osteocalcin by 9cRA in cultured ROS17/2.8 osteosarcoma cells (36). Additionally, the human osteocalcin promoter has been shown to be stimulated by retinoids in cotransactivation assays in osteosarcoma cells through a sequence containing the VDRE (38). RXR ligands may have the ability to regulate a number of vitamin D (and thyroid hormone) target genes through perturbation of the structure of the heterodimer, which may lead to activation or repression. The potential for dual hormone regulation may depend on a number of factors including hormonal status of the organism, cellular receptor complement, promoter context, and the presence of specific receptor-interacting cofactors.
Interestingly, it has been shown that combinations of retinoids and vitamin D compounds have additive or synergistic effects in promoting apoptosis or growth inhibition in breast cancer cells (39), prostate cancer cells (40), and leukemia cells (41) and in growth inhibition and differentiation of leukemia cells (42-44). Therefore, lower concentrations of two ligands together may achieve efficacies that would require increased amounts of either compound alone. Clinically, combination therapy of retinoid and vitamin D analogues may potentially provide a drug treatment regimen that would exhibit a greater therapeutic index than either agent could achieve alone in diseases such as cancer and leukemia.
We thank M. Boehm and L. Zhang for LG100268
and TTNPB, S. White for 9cRA, W. Lamph for pLUCpl, Y. Ohyama for rat
24(OH)ase cDNA, and J. W. Pike for anti-VDR 9A7 antibody.