From the
The relative affinities of the 1
We therefore conclude that IE and ID augment transcriptional
activity of VDR more than 1,25-D
Numerous analogues of 1
A second possible explanation was that the cellular uptake of the
20-epi analogues is more efficient. Analogue IE was shown to have poor
binding affinity for the vitamin D-binding protein (DBP). However, in
culture, the presence or absence of DBP had only a minor effect on the
biological activity of either 1,25-D
Another possibility that has not
been previously tested is that the mode of interaction of the 20-epi
analogues with VDR facilitates its transcriptional activity. To
determine whether the 20-epi analogues augmented VDR action, we tested
early steps in the sequence of events leading to transcriptional
activation of VDR by ligand and compared the efficacy of the analogues
to that of 1,25-D
All transfections were performed by the DEAE-dextran method
(11) , and the cells were then treated for 1 min with 10%
dimethyl sulfoxide. Medium samples for measurements of growth hormone
were collected 2 days after transfection. Growth hormone production
from the reporter gene was measured by a radioimmunoassay as described
by the manufacturer (Nichols Institute, San Juan Capistrano, CA).
To assess receptor occupancy by
the ligand in vivo, monolayers of ROS 17/2.8 or
VDR-transfected COS-1 cells were washed three times with PBS and
incubated for 1 h with ligand in serum-free medium, and then the medium
was discarded. The cells were washed three times in cold PBS, scraped
into 10 ml of PBS, centrifuged, resuspended in KTED, and homogenized.
Aliquots of the homogenates (0.2 ml) were incubated on ice for
3-4 h with 0.2 pmol of [
Fig. 5A shows the complexes
formed after incubation of 1,25-D
To identify qualitative differences in the mode of
interaction of the three ligands with VDR, we compared the
conformational changes induced by the three ligands as reflected in the
pattern of sensitivity to proteases. These assays were performed in
vitro, with
The biological actions of 1,25-D
We chose to investigate the mechanism of
action of the 20-epi analogues because the antiproliferative action of
these compounds is several orders of magnitude higher than that of
1,25-D
In the Introduction we addressed the possibility that
the augmented activity of these analogues is due to their
pharmacokinetic qualities
(8) . While this is a valid
consideration for whole-animal studies or for experiments that require
the continuous presence of the vitamin in serum-enriched culture medium
(such as the antiproliferative assay), we have focused on experimental
systems in which pharmacokinetic qualities of the tested compounds
could be separated from their actual biological activities as agonists
of 1,25-D
To separate the
pharmacological and direct molecular effects of the analogues on VDR,
we tested in vitro the sensitivity of the 20-epi
analogue-induced VDR to proteases and compared it to the protease
sensitivty of the 1,25-D
The earliest events in VDR
activation by the ligand are dimerization with RXR (which was
originally identified as nuclear accessory factor or NAF)
(15) and the subsequent binding to VDRE. Under certain
conditions it is possible to demonstrate ligand-dependent induction of
heterodimerization and binding of VDR to DNA in vitro, for
example, in recombinant receptor expressed in yeast cells and mixed
with RXR-containing cell extracts
(15) . On the other hand, the
VDR-RXR heterodimer expressed in vitro binds strongly to VDRE,
and its binding is not significantly increased by inclusion of ligand
(13) . In our system, VDR and RXR expressed in mammalian cells
had very poor binding activity to osteopontin VDRE in the absence of
ligand. This binding activity was enhanced only by inclusion of ligand
in the culture medium, not in a cell-free system. We therefore
speculate that in mammalian cells a ligand-induced post-translational
modification (possibly phosphorylation) must take place to efficiently
facilitate formation of VDR complexes with RXR or binding of the
VDR
Can the greater biological activities
of the 20-epi analogues be explained only by the pharmacological
qualities and molecular alterations induced by direct interaction with
the receptor? One should not overlook the great discrepancy between the
ED
The RCIs were calculated by plotting the
inverse of the percent maximum binding
[
We thank Dr. J. W. Pike for hVDR, human vitamin D
receptor expression plasmid, and Dr. Pierre Chambon (LGME-U.184) for
the generous gift of the RXR antibodies 1RX-6G12 and 4RX-1D12.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
,25-dihydroxyvitamin
D
(1,25-D
) analogues
20-epi-1
,25-dihydroxyvitamin D
(IE) and
20-epi-22-oxa-24a,26a,27a-tri-homo-1
,25-dihydroxyvitamin D
(ID) to the nuclear vitamin D receptor (VDR) are similar to that
of 1,25-D
, but their antiproliferative action is 1000-fold
greater. We tested whether the greater antiproliferative effect of
these analogues is due to a differential activation of the VDR. In ROS
17/2.8 cells, the effective doses required to produce 50% maximal
stimulation (ED
) of transfected reporter genes driven by
either the osteocalcin or the osteopontin vitamin D-response
elements (VDRE) were 5
10
M,
10
M, and 10
M for 1,25-D
, ID, and IE, respectively.
Similar results were obtained when recombinant human VDR was
cotransfected into CV-1 cells with an osteocalcin VDRE-reporter
plasmid. We found that in vitro the sensitivity of
1,25-D
-induced and analogue-induced receptors to proteases
was different. The ED
for binding to VDRE, as determined
by electrophoretic mobility shift assays, was significantly higher for
1,25-D
-induced than for analogue-induced VDR. The
concentration of retinoid X receptor (RXR) was significantly lower in
1,25-D
-induced than analogue-induced VDR complexes with
VDRE.
does, by producing
conformational changes that enhance dimerization of VDR with RXR. We
suggest that these conformational changes are due to differences in the
contact sites of the 20-epi analogues and 1,25-D
with the
VDR.
,25-dihydroxyvitamin D
(1,25-D
)
(
)
have been
synthesized in the attempt to improve vitamin D therapy of
hyperproliferative conditions and malignancy
(1, 2, 3, 4) . A particularly
interesting group are the 20-epi analogues
(5) . These compounds
have antiproliferative activities 200-7,000-fold greater than
that of 1,25-D
, but their calcemic activity is not
proportionally higher
(5) . Although these compounds may not be
suitable for treatment of cancer because they are very active
immunosuppressants, the mechanism of their facilitated action remains
an interesting issue. Because both the immunosuppressive and
antiproliferative activities of vitamin D are thought to be
transcriptional events mediated by the nuclear vitamin D receptor (VDR)
(6, 7) , the first explanation proposed for the
facilitated action of these compounds was that their affinity to the
VDR was proportionally higher. All of the first generation of 20-epi
analogues were tested for their relative affinity for the chick
intestinal VDR and were found to have affinities similar to that of
1,25-D
(5) . However, another study showed that one
compound in this series, 20-epi-1,25-D
(also known as
MC1288 or analogue IE) had a higher affinity for calf thymus VDR than
1,25-D
did
(8) . Because in both studies binding and
antiproliferative activities were assayed in receptors from different
species, the relevance of the binding data to the augmented action of
these compounds remains unclear and requires further clarification.
or the analogue tested
(8) . A third possible explanation was that the catabolism rate
of the 20-epi analogues was slower than that of 1,25-D
. The
catabolism studies performed with analogue IE showed that it was a good
substrate for 24-hydroxylase but probably a poor substrate for
23-hydroxylase
(8) , suggesting that poor catabolism may
contribute to the facilitated action of this compound and perhaps that
of other analogues in this family.
at each of these steps. We report here
that in vitro, the 20-epi analogues induced a specific
conformational change in the VDR, facilitated dimerization of VDR to
the retinoid X receptor (RXR), and augmented binding to and
transcriptional activation through a vitamin D response element (VDRE)
containing two direct repeats separated by three nucleotides (DR3).
Reagents
Synthetic oligonucleotides were
prepared by the Macromolecular Synthesis and Analysis Facility of M. D.
Anderson Cancer Center. Anti-VDR antibodies were prepared by immunizing
rabbits with a synthetic human VDR peptide conjugated to keyhole limpet
hemocyanin. [-
P]dATP was obtained from ICN
and [
S]methionine and
1,25(OH)
[26,27-
(
)
H]D
from Amersham Corp. A coupled transcription-translation kit was
obtained from Promega. The analogues 20-epi-1
,25-dihydroxyvitamin
D
(MC1288) and
20-epi-22-oxa-24a,26,27a-tri-homo-1
,25-dihydroxyvitamin D
(KH1060) were a generous gift from Dr. L. Binderup (Leo
Pharmaceuticals, Bellerup, Denmark). For convenience, MC1288 was
designated IE and KH1060 was designated ID. The structural formulas of
these compounds are shown in Fig. 1.
Figure 1:
Structural formulas of
1,25-D and the 20-epi analogues.
Cell Culture and Transfections
African green
monkey kidney cell lines (COS-1 and CV-1) were maintained in
Dulbecco's modified Eagle's medium (DMEM). Rat osteosarcoma
ROS 17/2.8 cells were maintained in 50% DMEM and 50% F12. All culture
media were supplemented with 10% fetal bovine serum. Forty-eight h
before transfection, the cells were plated in 35-mm dishes at a density
of 3 10
/dish (ROS 17/2.8 and CV-1) or in 150-mm
dishes at a density of 6
10
/dish (COS-1) in DMEM
and 10% fetal bovine serum. ROS 17/2.8 cells were transfected with 2
µg of plasmid containing either the VDRE from the mouse osteopontin
gene (TGCTCGGGTAGGGTTCACGAGGTTCACTCGAC)
(9) or the VDRE from
the human osteocalcin gene (GGTGACTCACCGGGTGAACGGGGGCATT)
(10) .
These response elements were attached to the thymidine kinase
promoter-growth hormone fusion gene. CV-1 cells were transfected with
the osteocalcin VDRE-reporter fusion gene (4 µg/dish) and the
recombinant human VDR expression vector (2 µg/dish). COS-1 cells
were transfected with 20 µg/dish of recombinant human VDR plasmid.
Preparation of Nuclear Extracts
To test the
ligand-induced DNA binding activity of VDR and to assess the amount of
RXR in the VDRVDRE complexes, nuclear extracts were prepared from
COS-1 cells 48 h after transfection with the human VDR plasmid. Ligands
(1,25-D
, IE, and ID) dissolved in ethanol were added 1 or
24 h before the cells were harvested. The cells were then scraped from
individual dishes into phosphate-buffered saline (PBS), washed twice in
that buffer, and resuspended in 1 ml of buffer A (10 mM HEPES,
pH 7.9, 1.5 mM MgCl
, 10 mM KCl, 0.5
mM dithiothreitol) and incubated on ice for 30 min. After they
had swollen, the cells were homogenized by 5-10 strokes in Dounce
homogenizers and centrifuged for 30 s at 14,000
g. The
supernatants were then discarded, and the nuclear pellets were
resuspended in 50 µl of buffer C (20 mM HEPES, pH 7.9, 400
mM KCl, 1.5 mM MgCl
, 0.2 mM
EDTA, 25% glycerol, 0.5 mM dithiothreitol), set on ice for 30
min, homogenized again, and centrifuged for 30 s at 14,000
g. The nuclear extracts were then collected, frozen in dry ice
immediately, and stored at -80 °C for further analysis.
Electrophoretic Mobility Shift Assays (EMSAs)
A
HindIII fragment containing the mouse osteopontin VDRE was
labeled with P to a specific activity of 1-5
10
counts/min/µg. Each binding reaction included 50
mM KCl, 12 mM HEPES-NaOH, pH 7.9, 1 mM EDTA,
1 mM dithiothreitol, 12% (v/v) glycerol, 0.5 ng of the DNA
probe, 10 µg of nuclear proteins, and 1 µg of poly(dI-dC) as
nonspecific competitor DNA. The binding reactions were performed at
room temperature for 30 min. The complexes were resolved by
electrophoresis through 4% polyacrylamide gels at 4 °C. For
competition experiments and for analysis of the complexes by antibody
binding, the conditions were exactly as described above except that
specific oligonucleotides or the
VDR and
RXR antibodies and
nonspecific DNA were added to the binding reactions before addition of
the probe.
Ligand Binding Assays
To compare the relative
affinity of 1,25-D and the 20-epi analogues to VDR in
vitro, whole-cell extracts from untransfected ROS 17/2.8 cells
were prepared in KTED (10 mM Tris-HCl, pH 7.4, 1.5 mM
EDTA, 0.3 M KCl, and 1 mM dithiothreitol). Briefly,
the cells were scraped into PBS, washed three times, and homogenized
with 10 strokes of a Dounce homogenizer. The homogenates were then
aliquoted into tubes containing 0.2 pmol of
[
H]1,25-D
and increasing
concentrations of nonradioactive ligand. The mixtures were incubated on
ice for 3-4 h, and then free ligand was separated from bound by
hydroxyapatite
(12) . The bound ligand was released from the
hydroxyapatite by ethanol extraction, and the radioactivity was
measured by scintillation counting. The results (see legend to
) were expressed as relative competition index (RCI) by the
method of Wecksler and Norman
(12) , with the RCI for
1,25-D
defined as 100%.
H]1,25-D
with or without 100-fold excess of unlabeled ligand. To assess
the number of unoccupied VDR sites, the free ligand was separated from
the bound by hydroxyapatite as described above.
Ligand-induced Sensitivity to Proteases
Synthetic
human VDR, labeled with [S]methionine (1000
Ci/mmol) was prepared by in vitro coupled
transcription-translation in reticulocyte lysates (Promega) with the
human VDR cDNA inserted into the pGEM4 vector. The receptor was
incubated with 10 nM 1,25-D
or analogues for 10
min at room temperature. Then 0-25 µg/ml trypsin (Calbiochem)
was added, and the mixture was further incubated for 10 min. The
digestion products were analyzed by 12% SDS-polyacrylamide gel
electrophoresis, and the gels were dried and autoradiographed.
Statistical Analysis
The results of the growth
hormone assays were presented as mean ± standard error of the
mean of 3-10 transfections.
Greater Activation of Transcription by 20-epi Analogues
than by 1,25-D
The vitamin D analogues IE and ID
have been shown by Binderup et al.(5) to possess
antiproliferative responses 200-fold and 7,000 fold, respectively,
greater than that of 1,25-D. Because this aspect of vitamin
D action is considered a nuclear receptor-mediated event
(6) ,
we tested whether these augmented effects on cell proliferation can be
reflected in a simplified model to test a single transcriptional
activity of VDR. We used the thymidine kinase promoter-growth hormone
fusion genes containing either the osteocalcin or the osteopontin
VDREs; both have DR3 motifs
(13) . The difference between the
two is that the osteocalcin element also contains an activator protein
1-binding site immediately upstream from the DR3 motif
(14) .
The fusion genes containing these VDREs were transfected into ROS
17/2.8 cells, and growth hormone reporter-protein production was
measured after treatment with increasing concentrations of ligand. The
cells were exposed to the ligands for 1 h in serum-free medium, to
avoid involvement of DBP in the regulation of ligand uptake by the
cells. Fig. 2 A shows that transcription of a reporter
gene containing the osteocalcin VDRE reached 50% of maximal activity
(ED
) at 5
10
M. The
ED
for IE and ID were 10
M
and 10
M, respectively. Similar results
were obtained by transfection of the osteopontin VDRE-containing fusion
gene (Fig. 2 B). We concluded that the 20-epi analogues
increased VDR sensitivity to ligand (as was evident by the shift of the
dose-response curve to the left) but did not increase the levels of
transcription because maximal fold-induction of gene transcription was
similar for all three ligands.
Figure 2:
Transcriptional activity of
1,25-D and 20-epi analogues. ROS 17/2.8 cells were
transfected by the DEAE-dextran method with a thymidine kinase-growth
hormone ( TK/GH) fusion gene containing either the osteocalcin
VDRE ( ocVDRE) ( A) or the osteopontin VDRE
( opVDRE) ( B). C, CV-1 cells were co
transfected with the osteocalcin VDRE and a human VDR expression
vector. Immediately after transfection (for ROS 17/2.8 cells) or 24 h
later (for CV-1 cells), the ligands were added to serum-free culture
medium for 1 h and then removed, the cells were washed twice with PBS,
and DMEM with 10% fetal bovine serum was added. Forty-eight h after
transfection, culture medium was collected and growth hormone levels
were determined by radioimmunoassay. Each point of the dose-response
curve is the average of duplicate transfections. The results shown are
representative of four to six transfection
experiments.
Because later assays of VDR activity
were carried out with recombinant human VDR, we tested whether the
sensitivity of the recombinant human VDR was also augmented by the
20-epi analogues. For that study, the VDR-negative CV-1 cells were
cotransfected with the recombinant human VDR expression vector and the
osteocalcin VDRE-growth hormone fusion gene. Fig. 2 C shows that IE and ID increased the sensitivity of the human VDR in
a manner similar to their effect on the rat VDR in ROS 17/2.8 cells. We
concluded from these experiments that the transcriptional activity of
VDR through a DR3 VDRE motif was augmented by the 20-epi analogues. The
ED for IE transcriptional activity was identical to its
ED
for antiproliferative activity. However, the ED
for transcriptional activity of ID was 100-fold greater than its
ED
for anti-proliferative action. These results suggest
that although the augmented antiproliferative action of the 20-epi
analogues was mediated through enhanced activation of genes with a DR3
motif, an additional unknown mechanism may also be responsible for
their augmented antiproliferative action.
Comparison of Relative Affinities of 1,25-D
The
augmented transcriptional activity of the 20-epi analogues was
reflected by increased receptor sensitivity to the analogues rather
than by an increase in levels of transcription. These results suggest
that either the affinity of the 20-epi analogues for the receptor was
greater than that of 1,25-D and 20-epi Analogues to VDR in Vitro and in Vivo
or that the affinity of the
analogue-induced receptor for other components in the transcriptional
machinery was greater than that of the 1,25-D
-induced VDR.
The original report on these compounds showed that their affinity to
chick intestinal VDR was similar to that of 1,25-D
(5) . However, we could not exclude the possibility that
the binding properties of rat VDR from ROS 17/2.8 cells and those of
the recombinant human VDR are different from those of the chick
receptor. To test that possibility, we performed binding assays with
homogenates from ROS 17/2.8 cells (Fig. 3 A) and from
VDR-transfected COS-1 cells to determine their RCI values
(Fig. 3 B). When we plotted the competition curves for
these compounds, and calculated their RCIs (summarized in
), we found that for the rat VDR the relative affinities of
IE and ID (RCI values of 43 ± 18 and 55 ± 26,
respectively) were lower than that of 1,25-D
(RCI =
100). Similar results were obtained for the human VDR (RCI values of 32
and 34 for IE and ID, respectively).
Figure 3:
Relative affinities of 1,25-D
and the 20-epi analogues to VDR in a cell-free system. Homogenates from
ROS 17/2.8 cells ( A) and human VDR-transfected COS-1 cells
( B) were incubated for 3-4 h at 4 °C with 0.2 pmol
of [
H]1,25-D
and increasing
concentrations of unlabeled 1,25-D
, IE, or ID. The free
ligand was separated from bound by hydoxyapatite, and the amount of
bound ligand was quantified by scintillation counting. The results were
plotted as the reciprocal of binding activity versus the ratio
of the amount of unlabeled competitor and radioactive ligand. The
slopes of the linear plots were calculated to obtain the RCIs. Shown
are representative plots of the two COS-1 and four ROS 17/2.8 binding
assays.
We then considered the
possibilities that the receptor-binding properties of intact cells are
significantly different from the binding properties of cell homogenates
and that the uptake of the 20-epi analogues, even in the absence of
serum, is significantly higher than that of 1,25-D. We
therefore attempted to determine the concentrations of the three
ligands required to reach 50% saturation of VDR in vivo. We
repeated the binding assays by incubating the cells with increasing
concentrations of nonradioactive ligands for 1 h in serum-free medium
and then measured the concentration of unoccupied receptor remaining in
the cell homogenates. Fig. 4shows that in ROS 17/2.8 cells, the
slope of the plots of binding activity of 20-epi analogue-treated cells
was smaller than that of binding activity of 1,25-D
-treated
cells, indicating that either the uptake or the affinity (or both) of
the 20-epi analogues to the VDR in vivo was lower than that of
1,25-D
.
Figure 4:
Relative affinities of 1,25-D
and the 20-epi analogues to VDR in intact cells. ROS 17/2.8 cells were
incubated in serum-free medium for 1 h with increasing concentrations
of either 1,25-D
or the 20-epi analogues. The cells were
then washed twice with cold PBS and homogenized, and the number of
remaining unoccupied binding sites was determined. The results were
plotted as described in the legend to Fig.
3.
Similar results were obtained with the
recombinant human VDR from transfected COS 1 cells (data not shown).
The results of the in vivo and in vitro competition
assays in ROS 17/2.8 cells are summarized in . We concluded
that the augmented transcriptional activity of the 20-epi analogues
under our experimental conditions cannot be attributed to higher
affinity for the VDR or to more efficient delivery into the cells.
Ligand Effect on Binding of VDR to the Osteopontin
VDRE
Because the augmented transcriptional activity of the
20-epi analogues was not due to increased affinity to the VDR or to a
more efficient uptake mechanism, we tested whether the analogues
facilitated binding of the VDR to the VDRE more than did
1,25-D. Two aspects of DNA binding activity were tested:
affinity of the ligand-induced VDR to the osteopontin VDRE and
dose-dependent binding of the ligand-induced VDR to the VDRE. For these
assays VDR was overexpressed in COS-1 cells, exposed to ligand in the
intact cells in serum-free medium for 1 h, and extracted from the
nuclear fraction of the cells. The binding properties of the extracted
VDR to the osteopontin VDRE were tested by EMSA. We used the
osteopontin VDRE and not the osteocalcin VDRE because VDR binding to
the former was more stable and was not accompanied by additional
specific (activator protein 1) and nonspecific binding activities
(14, 15) .
-treated extracts from
human VDR-transfected cells with radiolabeled osteopontin VDRE. We
found that a single complex was formed. The formation of this complex
was blocked by a 50-fold excess of unlabeled osteopontin VDRE, and the
complex contained VDR, as it was partially supershifted by anti-VDR
antibodies. Next, we compared the affinities of
1,25-D
-induced and 20-epi analogue-induced VDR to the
osteopontin VDRE. Receptor preparations from cells treated with
10
M ligand were subjected to EMSA, with
P-labeled osteopontin VDRE as a probe and increasing
concentrations of unlabeled VDRE as a competitor. Fig. 5 B shows that the unlabeled VDRE inhibited the formation of the VDR
complex with the labeled probe in a dose-dependent manner. The
incubation of VDR in vivo with the 20-epi analogues did not
increase its affinity to the VDRE relative to 1,25-D3-induced VDR; 50%
competition was reached at similar concentration of competitor for
1,25-D
-, IE-, and ID-induced VDR.
Figure 5:
Affinity
of ligand-activated VDR for opVDRE. A, nuclear extract from
COS-1 cells transfected with human VDR expression vector and treated
with 10
M 1,25-D
was incubated
without specific competitor ( none) or with a 50-fold excess of
unlabeled specific competitor ( osteopontin VDRE). To test for
presence of VDR in the complex, the nuclear extract was treated with
rabbit anti-human VDR antibodies (
VDR) or with preimmune
serum. The complexes were separated from the free probe by
polyacrylamide gel electrophoresis and visualized by autoradiography.
B, nuclear extracts from COS-1 cells transfected with hVDR and
treated with 10
M 1,25-D
, IE,
or ID were incubated with or without increasing amounts of unlabeled
osteopontin VDRE. The amount of competitor used is shown below the
autoradiograph.
We then tested whether
there were quantitative differences in the amount of VDR binding to
VDRE in 1,25-D-induced and 20-epi analogue-induced
preparations. VDR-transfected cells were treated for 1 h with
increasing concentrations of ligand, and then nuclear extracts were
prepared. The EMSAs were repeated with these receptor preparations and
with osteopontin VDRE as a probe. We found that in the absence of
ligand only a very small amount of complex formed
(Fig. 6 A) and that including ligand induced a
significant increase in VDR binding to DNA. The binding was dose
dependent and reached 50% of maximal value at 10
to
10
M 1,25-D
, at
10
to 10
M ID, and at
10
M IE. Fig. 6 B shows the
relative intensity of the VDR bands from each treatment. In summary, we
found that the analogues facilitated the DNA binding activity of VDR in
a dose-dependent manner.
Figure 6:
Dose-dependent effect of ligand on VDR
binding to osteopontin VDRE. A, nuclear extracts from
untreated and ligand-treated human VDR-transfected COS-1 cells were
incubated with P-labeled osteopontin VDRE and separated by
gel electrophoresis. The molar concentration of each ligand is
indicated above the lanes. B, densitometric scanning of the
VDR
DNA complexes. The results are expressed as percentage of
complex intensity in samples treated with 10
M ligand.
Effect of 20-epi Analogues on Dimerization of VDR with
RXR
A prerequisite for binding of the VDR to a VDRE containing
the DR3 motif is heterodimerization with RXR
(13) . Because we
found that both transcription and DNA binding to these elements were
augmented by the analogues, we hypothesized that these compounds might
also facilitate dimerization of VDR with RXR. To test this hypothesis,
VDR preparations from cells treated with ligand for 1 h in serum-free
medium were incubated with antibodies against RXR or control IgG and
then incubated with the labeled VDRE and subjected to EMSA. The
antibodies used (a generous gift from Dr. P. Chambon) recognize all
three RXR species,(
)
so the assay detected all
possible VDR
RXR complexes, simultaneously. Fig. 7 A shows that the antibodies induced a partial supershift of VDR
complexes, whereas the control IgG did not have any effect on the
complexes. When VDR was pretreated with 10
M ligand, there was no difference in the proportion of
supershifted VDR
RXR complexes for the three ligands. On the other
hand, at ligand concentrations of 10
and
10
M, there was a significant difference
in the proportion of supershifted 1,25-D
-induced and
analogue-induced VDR
RXR complexes. To assess the difference
between receptor preparations quantitatively, we densitometrically
scanned the autoradiograms (Fig. 7 B) and found that the
proportion of VDR
RXR complexes supershifted by the anti-RXR
antibodies was significantly higher in receptor preparations treated
with 10
and 10
M IE and
ID than in those treated with same concentrations of
1,25-D
. These results suggested that there was more RXR
associated with VDR activated by 20-epi analogues than with VDR
activated by 1,25-D
and that the analogues facilitated
heterodimerization. Again, the differences were seen only when VDR was
activated by suboptimal levels of 1,25-D
. We think that
differences were not seen at higher ligand concentrations because the
amount of RXR in the extracts limited heterodimer formation.
Figure 7:
Effect of ligand on VDRRXR complex
formation. A, nuclear extracts from human VDR-transfected
COS-1 cells treated with 10
, 10
,
or 10
M 1,25-D
, IE, or ID were
incubated with [
P]osteopontin VDRE and either
control mouse IgG ( odd-numbered lanes) or with the anti-RXR
antibodies ( even-numbered lanes). The complexes were separated
by gel electrophoresis. The arrows indicate the positions of
the VDR
DNA or the VDR
DNA complexes supershifted by the
antibodies. B. densitometric scanning of the supershifted
VDR
DNA complexes. The results are expressed as percentage of the
intensity of the respective unshifted VDR
band.
Differential Effects of 20-epi Analogues and 1,25-D
The binding and uptake studies did not
reveal a difference in receptor occupancy or affinity to the 20-epi
analogues that correlated with their augmented transcriptional
activity. In fact, the ligand-binding pattern was not correlated with
the dimerization, DNA binding, or transcriptional activities of any of
the compounds tested. We therefore hypothesize that it is the mode of
interaction of the ligands, perhaps their contact sites with VDR, more
than relative affinities of the ligands to the receptor that determines
the efficacy of the activation process that leads to DNA binding and
transcriptional activity. We reasoned that if there are differences in
the mode of interaction they should be reflected by conformational
changes in VDR.
on VDR Conformation
S-labeled synthetic VDR incubated with
either 1,25-D
or the 20-epi analogues, and then treated
with increasing concentrations of trypsin. Fig. 8 A shows
that in the absence of ligand, VDR was rapidly degraded, and only one
short fragment ( fragment d, 22 kDa) was detectable after 10
min of incubation with the protease. Under the same incubation
conditions, treatment of VDR with 1,25-D
changed the
proteolytic pattern of trypsin action: it produced an additional,
slowly migrating fragment ( fragment a, 34 kDa) not seen in the
absence of hormone. Treatment of VDR with ID (Fig. 8 B)
generated an additional fragment ( fragment b, 30 kDa), whereas
the treatment with IE (Fig. 8 B), generated both fragment
b and an additional slow-migrating fragment ( fragment c, 32
kDa) not seen with 1,25-D
-treated VDR. This experiment was
repeated with chymotrypsin, with similar results (data not shown). We
concluded from these experiments that binding of the 20-epi analogues
to VDR induced conformational changes that exposed certain arginine or
lysine residues that are not normally exposed on the
1,25-D
-activated VDR. We speculate that these
conformational modifications facilitate the action of VDR, beginning by
enhancing dimerization with RXR.
Figure 8:
Ligand-induced sensitivity of VDR to
trypsin. In vitro translated VDR labeled with
[S]methionine was incubated with or without
10
M 1,25-D
( A) or the
20-epi analogues IE and ID ( B), before digestion with the
increasing concentrations of trypsin indicated above the lanes. The
digestion products were analyzed by SDS-polyacrylamide gel
electrophoresis and autoradiography. The proteolytic products are
indicated by arrows. The fragments sizes are: a, 34
kDa; b, 32 kDa; c, 30 kDa; d, 22
kDa.
and its
analogues are believed to be mediated through both genomic and
non-genomic signal-transduction pathways
(16, 17, 18, 19) . The extensive search
for analogues of 1,25-D
more suitable for clinical purposes
has yielded a wealth of compounds that can be used as tools for
studying these different aspects of the action of vitamin D
(20, 21) .
. The antiproliferative effect of 1,25-D
is thought to be primarily a genomic action and therefore
believed to be regulated by the nuclear VDR
(6) . The
traditional concept of nuclear receptors activation by a ligand
suggests that activation should be directly proportional to the
ligand's affinity for the receptor. However, the action of the
20-epi analogues presented a paradox: the affinity of these compounds
for VDR is similar or even lower than that of 1,25-D
, but
their antiproliferative action is several orders of magnitude higher
(5) .
action and as ligands for VDR.
-induced VDR. By using a similar
approach for the analysis of ligand interaction with steroid receptors,
it was possible to show differences in the pattern of protease
sensitivity of agonist- and antagonist-treated receptors for
progesterone, estrogen, and androgen
(22, 23, 24) . Our results clearly indicate that
each of the three ligands used in our experiments induced a unique
pattern of sensitivity to trypsin. Therefore, although the apparent
affinity of these compounds to VDR was not higher, it is possible that
the conformational changes they induced facilitated receptor action.
Our interpretation of these results is that the amino acids used by the
20-epi analogues as contact sites on VDR are somewhat different from
the contact sites used by 1,25-D
. Flexibility in ligand
binding requirements that leads to differential activation of receptors
by parental compounds and their analogues has been clearly demonstrated
by deletion analysis of the progesterone receptor
(25) . We are
now in the process of mapping the ligand-binding domain of VDR and
comparing the amino acids required for binding and function of
1,25-D
and of the 20-epi analogues. Our preliminary results
suggest that the binding requirements for these compounds are different
from those of 1,25-D
.
RXR complexes to the VDRE. Because this event was facilitated
by the 20-epi analogues within 1 h of ligand administration in vivo under conditions of similar uptake and receptor saturation by
1,25-D
and the analogues, we are convinced that the
facilitated action of the 20-epi analogues on DNA binding and
dimerization of VDR is not due to their pharmacological properties. We
speculate that the conformational change induced by these compounds
either directly facilitated dimerization of RXR with VDR or perhaps
facilitated a post-translational event that increased the affinity of
ligand-activated VDR for RXR.
for transcription induction of DR3 motif-driven genes
by ID (10
M) and its ED
for
antiproliferation and induction of differentiation (10
to 10
M)
(5) . There are two
possible explanations for this discrepancy. The first is that
ID-induced VDR may be far more transcriptionally active due to the
action of a motif different from DR3 and that genes with such motifs
are important for the regulation of cell proliferation and
differentiation. Examples of diversity in VDREs are the negative VDREs
of the parathyroid hormone and the calcitonin genes. The parathyroid
hormone gene is repressed by weak direct binding of VDR to DNA elements
that are significantly different from DR3
(26) . The calcitonin
gene is down-regulated by a mechanism that does not require direct
interaction of the receptor with DNA
(27) . Another possible
mechanism for the discrepancy in ID ED
is that the 20-epi
analogues, similarly to 1,25-D
(or even more so) may
activate nuclear receptor-independent pathways (the so called
non-genomic responses) that are also important for regulation of cell
proliferation and differentiation. The biochemical and physiological
events induced by 1,25-D
and related compounds that do not
require direct interaction with VDR include activation of calcium
channels
(19) , regulation of the phosphoinositol pathway and
diacylglycerol formation
(18, 28, 29) and
down-regulation of alkaline phosphatase
(30) . Any one of these
events may contribute to regulation of cell growth and differentiation.
Exploring these possibilities is essential for further evaluation of
the mechanism of action of vitamin D analogues.
Table: Relative competition index (RCI) of the
20-epi analogues
H]1,25-D
100 on the ordinate
versus [competitor]/[
H]1,25-D
]
on the abscissa, and dividing the slope of the line for analogue
binding by the slope of the line for 1,25-D
, and
multiplying by 100. By definition, the RCI for 1,25-D
is
100. To measure RCI in culture, increasing concentrations of ligand
were added to cells in serum-free culture medium and incubated for 1 h.
The cells were then washed twice with PBS, scraped off the plates, and
homogenized. The homogenates were then incubated with a constant amount
of [
H]1,25-D
with or without 100-fold
excess of unlabeled 1,25-D
. To measure RCI in
vitro, cell homogenates were incubated with increasing amounts of
ligand and a constant amount of
[
H]1,25-D
. Three experiments were
performed in culture and five in vitro.
, 1
,25-dihydroxyvitamin
D
; VDR, nuclear 1
,25-dihydroxyvitamin D
receptor; IE, 20-epi-1
,25-dihydroxyvitamin D
;
ID, 20-epi-22-oxa-24a,26a,27a-tri-homo-1
,25-dihydroxyvitamin
D
; DBP, vitamin D binding protein; RXR, retinoid X
receptor; VDRE, vitamin D response element; DR
, two direct
repeats separated by three nucleotides; RCI, relative competition
index; EMSA, electrophoretic mobility shift assay; DMEM,
Dulbecco's modified Eagle's medium; PBS, phosphate-buffered
saline.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.