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-Hydroxyvitamin D3- 26,23S-Lactone Antagonizes the Nuclear Vitamin D Receptor by Mediating a Unique Noncovalent Conformational Change
Department of Biochemistry (C.M.B., J.E.B., A.W.N.) University
of California-Riverside Riverside, California 92521
Department of Bone and Calcium Metabolism (S.I.) Teijin
Institute for Biomedical Research Tokyo 191-8512, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-Dihydroxyvitamin
D3-26,23-lactone (TEI-9647; MK) has been
reported to antagonize the 1,25-dihydroxyvitamin
D3 nuclear receptor (VDR)- mediated increase in
transcriptional activity. Using a transient transfection system
incorporating the osteocalcin VDRE (vitamin D response element) in
Cos-1 cells, we found that 20 nM MK antagonizes
VDR-mediated transcription by 50% when driven by 1
nM
1,25(OH)2D3. Four
analogs of
1,25(OH)2D3, also at
1 nM, were antagonized 25 to 39% by 20
nM MK. However, analogs with 16-ene/23-yne or
20-epi modifications, which have a significantly lower agonist
ED50 for the VDR than
1,25(OH)2D3, were
antagonized by 20 nM MK only at 100
pM or 10 pM,
respectively. One possible mechanism for antagonism is that the
25-dehydro alkene of MK might covalently bind the ligand-binding site
of the VDR rendering it inactive. Utilization of a ligand exchange
assay, however, demonstrated that MK bound to VDR is freely exchanged
with 1,25(OH)2D3 in
vitro. These data support the apparent correlation between VDR
transcriptional activation by agonists and the effective range of MK
antagonism by competition. Furthermore, protease sensitivity analysis
of MK bound to VDR indicates the presence of a unique conformational
change in the VDR ligand-binding domain, showing a novel doublet of VDR
fragments centered at 34 kDa, whereas
1,25(OH)2D3 as a
ligand produces only a single 34-kDa fragment. In comparison, the
natural metabolite 1,25dihydroxyvitamin
D3-26,23-lactone yields only the 30-kDa
fragment that is produced by all ligands to varying degrees.
Collectively, these results support that MK is a potent partial
antagonist of the VDR for
1,25(OH)2D3 and its
analogs when in appropriate excess of the agonist. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,25(OH)2-vitamin D3
[1,25(OH)2D3] is known
to have numerous physiological functions (1). These functions may be
manifested either by the classical genomic mechanism (2, 3) or by the
initiation of a complex signal transduction cascade that has been
termed a nongenomic rapid response (4). In the genomic response,
1,25(OH)2D3 binds to the
ligand-binding domain of the nuclear vitamin D receptor protein (VDR)
which heterodimerizes with retinoid X receptor (RXR) and
recruits transcriptional coactivators such as SRC-1 (5) and the DRIP
complex (6).
Among the many biological functions of
1,25(OH)2D3, the most
studied are its role in calcium homeostasis and cellular
differentiation. Both
1,25(OH)2D3 and many
analogs have been shown to mediate inhibition of the growth of various
cancers; however, the utility of the hormone as a chemotherapeutic
agent is reduced by its calcemic action, which is harmful above
physiological concentrations (7, 8). Currently, many researchers are
striving to produce analogs of
1,25(OH)2D3 that are
able to discriminate between actions for cellular differentiation and
calcium absorption/mobilization (2).
In the search for ligands that discriminate between desirable and undesirable functionality, occasionally some have been found to be antagonists, such as raloxifene for the estrogen receptor (9) and RU-486 for the progesterone receptor (10). In the case of the estrogen system, some of these antagonists have been shown to confer only partial antagonism and show useful agonist properties in other cell types or metabolic pathways (raloxifene and tamoxifen for example).
Recently, an analog of the natural metabolite
1,25(OH)2D3-26,23S-lactone
has been discovered to function as an antagonist to the VDR-mediated
actions of
1,25(OH)2D3. This
antagonistic analog,
25-dehydro-1-OH-D3-26,23S-lactone (see
Fig. 1
) [TEI-9647 or MK], has
been shown to block
1,25(OH)2D3-induced
cellular differentiation in HL-60 but not in NB4 cells (11). In cells
overexpressing a transiently transfected VDR, a 10-fold excess of
analog MK in combination with
1,25(OH)2D3 results in a
50% reduction in transactivation of a reporter plasmid driven by the
24-hydroxylase promoter. The same concentration ratio also was shown to
reduce
1,25(OH)2D3-induced
p21WAF1,CIP1 expression (12) and modulate the
protein-protein interaction between the VDR/RXR heterodimers and
between VDR and SRC-1 without modifying the nuclear localization or DNA
binding properties of the VDR (13).
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-OH-D3-26,23S-lactone is not
known. One possibility is that the reactive C-25/C-26 primary alkene
may bind covalently within the hydrophobic pocket of the ligand-binding
domain of the VDR to block the transcriptional action of the receptor.
In this communication we extend the action of the antagonistic analog
to the osteocalcin vitamin D response element (VDRE) and indicate that
it functions not by covalent modification of the VDR but rather by
displacing the far more agonistic
1,25(OH)2D3 in a
concentration-dependent manner. The antagonistic action occurs only
over a limited concentration range, however, that is dependent on the
activity of the agonist. Furthermore, evidence for a unique
conformational change in the ligand-binding domain of the VDR upon
antagonist binding is also shown. Together, these results support a
mechanism by which MK, when in appropriate excess, acts as a potent
partial antagonist for
1,25(OH)2D3 and its
analogs. |
RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,25(OH)2D3-OH-D3-26,23S-lactone (MK
TEI-9647; see Fig. 1
), has previously been shown to reduce
transcriptional activation of
1,25(OH)2D3 for the
25(OH)D-24-hydroxylase gene by 50% in transiently expressed Cos-7
cells (12, 13). Figure 2 shows the
reporter activity as fold-activation relative to the vehicle control
over a range of 0.1 to 100 nM
1,25(OH)2D3 with and
without analog MK in 10-fold excess. Here we show that a 10-fold excess
of MK over 10 nM
1,25(OH)2D3 results in a
50% reduction of transcriptional activity in transiently transfected
Cos-1 cells using a promoter containing the osteocalcin VDRE linked to
the human GH reporter. Unlike the earlier report (14), we found that
treatment with MK alone results in a significant 2.5-fold agonist
activity over the vehicle control for the osteocalcin reporter. For the
purpose of approximating physiological concentrations and assuring that
the transactivation achieved by
1,25(OH)2D3 is not
attenuated by nearing the maximal activation of the system, we selected
1 nM
1,25(OH)2D3 to be used
for further experiments. Thus, in this experiment a 10-fold excess of
MK over 1,25(OH)2D3 is a
significant inhibitor of the transactivation of the osteocalcin
promoter.
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,25(OH)2D3, the
transfected cells were treated with 1 nM
1,25(OH)2D3 and MK
ranging from 0.1 nM and 1000 nM (Fig. 3). In this experiment, transactivation
of the osteocalcin reporter by 1 nM
1,25(OH)2D3 is
significantly antagonized by 30% at 1 nM MK and 36% at 10
nM MK (Fig. 3). Although, the maximal antagonism of 52%
occurs at a 100-fold excess (100 nM) of MK, only an 80%
confidence level can be attributed to the combination being different
from MK alone. It is evident (Fig. 3) that antagonism by MK increases
with the concentration of MK although the agonist activity attributed
to MK rises also. The agonist activity of MK and the antagonistic
action on 1,25(OH)2D3
transactivation are equivalent at 1000 nM, but the maximum
antagonism occurs at 100 nM. The inability of MK to
antagonize 1,25(OH)2D3
at a level greater than 60% is the result of MK being both an
antagonist and a weak agonist. Therefore, the antagonistic resultant of
the MK-1,25(OH)2D3
interaction is a combination of MK agonism and antagonism for
1,25(OH)2D3. To focus on
the antagonistic effect in the absence of a significant agonist effect,
we chose an MK concentration of 20 nM. At this level, MK is
only mildly agonistic (0.2–0.8 fold activation over background; data
not shown) and produces a 40% antagonism of transactivation as
compared with the same level of
1,25(OH)2D3 alone (Fig. 3).
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,25(OH)2D3 where it
acts to generate a receptor conformation that is not competent with
regard to transactivation. A second hypothesis is that the C-25, C-26
primary alkene adjacent to a carbonyl group of the lactone serves as a
nucleophile that may form a 1,4-nucleophilic addition to either
histidine 305 or histidine 397 of the VDR; these residues have been
shown to hydrogen bond with the 25-hydroxyl of
1,25(OH)2D3 (14). In
this case, the VDR would be covalently bound at the ligand-binding site
by a relatively inactive ligand. A hypothetical mechanism for the
covalent addition is shown in Fig. 4A.
The neutral nitrogen of a histidine could donate its lone pair of
electrons to form a bond with C-27 of MK. This bond would
simultaneously shift the electron density to the C-25/C-26 bond to form
a more highly substituted alkene and forming a oxygen anion of the
carbonyl group that could be stabilized by the second local histidine
residue, if it was positively charged. Decay of the oxygen anion would
regenerate the carbonyl causing electron density in the adjacent alkene
to be transferred to a bond between C-25 and hydrogen from the charged
nitrogen. The hydrogen-nitrogen bond energy would be returned to
regenerate the lone pair on nitrogen.
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,25(OH)2D3 as a control
with VDR present in vitamin D-deficient chick duodenal mucosal
chromatin. After a 4-h incubation at 0 C, the analog was washed away
and TPCK
(L-1-tosylamido-2phenylethyl-chloromethyl-ketone) was
added to covalently bind in the ligand binding site of any unoccupied
VDR (15). Once the unreacted TPCK was washed away, the VDR is incubated
at 37 C in the presence of excess
[3H]1,25(OH)2D3.
The radioactive ligand can then exchange with the sites previously
protected by a noncovalently bound ligand. Figure 4B shows exchanged
radioactive ligand bound as percent reoccupancy. As expected, all of
the unoccupied ligand binding domains (LBDs) are irreversibly occupied
by TPCK in the vehicle control and, as a result, none of the VDR is
available for reoccupancy. At the low concentration of both
1,25(OH)2D3 and MK, the
VDR ligand binding site is only partially occupied, allowing TPCK to
covalently bind only to a portion of the receptors resulting in partial
reoccupancy. The high concentration of
1,25(OH)2D3 is able to
protect nearly all of the receptors (better than 96%), which would
indicate that the VDR is nearly saturated. For the high concentration
of analog MK, 89% of the VDR was occupied and was later fully
exchanged for the tritium-labeled ligand. This would indicate that
analog MK does not bind irreversibly to the VDR. Therefore, the theory
that antagonism of MK functions by displacing the more agonistic
1,25(OH)2D3 is our
working hypothesis.
Antagonistic Action of MK on
1,25(OH)2D3
Analogs
If antagonism results from the ability of MK to interfere with the
interaction of stronger agonists for the VDR, then MK should be able to
antagonize other VDR agonists in a manner that is proportional to their
agonistic activities. To test this theory, four ligands were chosen on
the basis of their relative competitive index (RCI) and cell
differentiation ratio values (2, 16, 17). The chemical structure, the
RCI, and the ratio of ED50 for cell
differentiation in HL-60 or U-937 cells vs.
1,25(OH)2D3 (CDR) of
these analogs is shown in the bottom row of Fig. 1. Each of
these analogs was added both in the presence and absence of 20
nM analog MK to transiently transfected Cos-1
cells overexpressing the VDR and a plasmid vector containing the
osteocalcin VDRE reporter construct (18). The transcriptional activity
of each of these analogs as compared with
1,25(OH)2D3 is shown in
Fig. 5. It is evident that the
transcriptional activity is not directly related to either RCI or the
cell differentiation ratio values. For instance, both the RCI and cell
differentiation ratio of AW
[1-F-25-(OH)-16-ene-23-yne-D3 ]are much lower
than EV
[22-(m-(dimethylhydroxymethyl)phenyl)-23,24,25,26,27-pentanor-1-OH-D3],
yet AW induces transactivation by 34% more. However, antagonism by MK
is directly proportional to the transactivational activity for each of
these analogs including
1,25(OH)2D3. Antagonism
ranges from 44% for analog AW to 30% for both analogs Q and HQ.
Another apparent trend is that as the agonist activity weakens
(transcriptional activity is reduced), the antagonist action of analog
MK diminishes. This trend can also be explained by the stronger agonist
displacement hypothesis. When weaker agonists are used, the
transcriptional activation of the agonist is less distinguishable from
the poorly agonistic analog MK.
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,25(OH)2D3 will require
an increased molar excess of the antagonist to induce antagonism. The
20-epi analog IE (19, 20) and the 16-ene,23-yne analog V (7, 21, 22),
which have increased biological effectiveness when present as ligands
for the VDR relative to
1,25(OH)2D3, were used
to test whether this were true. To test this hypothesis, dose-response
curves of both analogs IE and V from 0.001 to 10 nM were
used in the transfected Cos-1 cell system with and without the addition
of 20 nM MK. Reporter activity was assayed at 30 h
after dosing, and the results are reported as fold activation over
vehicle control (Fig. 6). Analog IE was
antagonized by MK only in the 0.01–0.1 nM range. At 0.001
nM, IE does not show any activation of transcription. At
0.01 nM, IE shows a 2-fold activation that is antagonized
70% by the addition of 20 nM MK. This difference is
significant at better than 99% confidence interval. MK antagonism
toward analog IE retreats rapidly at higher IE concentrations as
indicated by the 18% antagonism of 0.1 nM IE. Analog V at
0.1 nM (Fig. 6B) is antagonized 42% in the presence of 20
nM MK with a difference of better than a 95% confidence.
Analog V at 0.01 nM shows only a small amount of activity
(<1.5-fold of basal expression), which is less than the agonist
response of MK in the control (1.6-fold).
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,25(OH)2D3. To address
this hypothesis, the VDR was subjected to limited proteolysis by
trypsin in the presence of a saturating concentration of ligand.
Alterations in the conformation of the protein, as reflected by
different trypsin-mediated cleavage sites, can then be detected on
SDS-PAGE as the fragmentation pattern changes. This occurs by exposing
or shielding lysine and arginine residues that are differentially
protected from proteolysis by steric hindrance. In the case of the VDR,
the binding of
1,25(OH)2D3 produces
protease-resistant fragments at 34 kDa and 28 kDa (18, 19). The 34-kDa
and 28-kDa fragments have been shown by N-terminal sequencing to be
cleaved at the C terminus of arginine 173; thus, they contain the
C-terminal ligand-binding domain (23). Using this method we have
observed the formation of a unique conformation that occurs upon
binding of the MK antagonist to the VDR. This fragment is
characterized by a doublet of bands at 34 kDa (specifically 34.6 kDa
and 33.3 kDa mass apparent; see Fig. 7)
and a fragment at 30 kDa. This 30-kDa band is also present in a
very small amount in the
1,25(OH)2D3
fragmentation pattern and is seen as the predominant band for the
natural metabolite lactone BS,
1,25R(OH)2-D3-26,23S-lactone.
The 30-kDa fragment, as is reported here, is thought to be the same
fragment as has been previously referred to as the 28-kDa fragment
(23).
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-OH-D3-26,23S-lactone (MK) can
function as an antagonist of the VDR. In this report we have studied
the molecular basis of MK’s antagonistic properties with respect to
its possible covalent attachment to the VDR-LBD and as to whether the
presence of MK in the VDR-LBD alters the conformation of the protein as
assessed by protease sensitivity analysis.
A model to describe the possible covalent addition of MK to the
VDR is presented in Fig. 4A. However, our exchange assay has indicated
that no significant covalent binding of MK to the VDR takes place
within 4 h at 0 C (Fig. 4B). Thus, we conclude that the MK
interaction with VDR is freely reversible.
It is obvious that there is a small but significant agonist activity
that can be attributed to MK. At 1000 nM, MK alone shows
39% of the activity induced by 1 nM
1,25(OH)2D3 alone on the
osteocalcin VDRE (Fig. 3). Agonist activity induced by a combination of
both 1000 nM MK and 1 nM
1,25(OH)2D3 falls within
the standard error of the agonist activity of
1,25(OH)2D3. As a
partial antagonist, the antagonism of the MK and
1,25(OH)2D3 is actually
the product of two components, the subtractive antagonistic effect of
MK on the 1,25(OH)2D3
agonist and the additive effect of MK and
1,25(OH)2D3 agonism.
When the ligand being antagonized is comparatively much more active
than MK, as is the case of V (21, 22) and IE (19, 20) (Fig. 6), the
equilibrium of interaction with the VDR is shifted away from MK because
of its relatively poorer affinity so that the antagonistic activity of
MK is again reduced. These analogs require a 200- to 2,000-fold excess
of MK, respectively, as compared with only a 20-fold excess shown for
1,25(OH)2D3. This would
imply that when using this system, the effective range of MK antagonism
is only useful for agonists with a level of transactivation in the
range of
1,25(OH)2D3.
We have shown that as the VDR interacts with progressively weaker
agonists, the antagonism by MK is correspondingly weaker (Fig. 5). When
the ligand being antagonized by MK is comparatively less active, the
agonist activity of MK is more significant because the subtractive
effect is reduced. Therefore, the antagonism of MK results from the
ability to displace ligands that have more agonist activity in a
concentration-dependent manner.
The ability of a ligand to induce transactivation of the VDR can be
described as a combination of affinity, kinetics, and effectiveness at
producing an optimal protein conformation. Using the protease
sensitivity assay, we could observe changes in the protein
conformation of the ligand-binding domain. With
1,25(OH)2D3 as the
ligand, a single 34-kDa trypsin fragment is produced. However, ligands
that transactivate poorly or not at all, such as
1-OH-D3 or 25-OH-D3,
predominantly produce only a 30-kDa fragment (data not shown).
20-Epi-1,25(OH)2D3,
which is 500–2,000 times more potent than
1,25(OH)2D3 in
transactivation of the VDR (19), generates not only the 34-kDa fragment
of 1,25(OH)2D3 and a
minor amount of the 30-kDa fragment but also a novel 32 kDa fragment
(18, 23). Antagonist MK, however, consistently produces a unique
pattern of three fragments with nearly equal intensity at 34.6, 33.3,
and 30 kDa (Fig. 7). The 30-kDa fragment is exactly the same size as
that seen very faintly when
1,25(OH)2D3 is the
ligand, and predominantly when the natural metabolite lactone BS is the
ligand. However, both the 34.6- and 33.3-kDa fragments are somewhat
different than the fragmentation pattern of any previously tested
analog.
Previous researchers determined by N-terminal sequencing that both the 34-kDa and 30-kDa (previously cited as 28 kDa) bands are C-terminal fragments from cleavage at arginine-173 (23). The 34-kDa band contains a 19 residue portion of the hinge region and the entire LBD. Having the same N terminus as the 34-kDa band, the 30-kDa band must result from further trypsinization near the C terminus. An increase in the intensity of this fragment could be explained as the failure of the ligand to coordinate the active closed conformation (26, 27, 28) of helices 10–12, leaving them more susceptible to proteolytic cleavage. We speculate that the increase in the 30-kDa band protease sensitivity is indicative of more time spent in a transcriptionally inactive state.
The MK-induced doublet at 34 kDa, however, is less easily explained. It
could possibly be formed as a consequence of altered VDR
phosphorylation in the transcription/translation reticulocyte system
used to produce the [35S]-VDR employed for the
protease sensitivity assay. More likely, however, VDR binding of MK may
result from the exposure of alternate trypsin sites at either end of
the 34-kDa fragment produced by cleavage at Arg174 (23), which is
characteristic of
1,25(OH)2D3 and other
agonistic ligands. A candidate cleavage point located two amino acids
before the beginning of helix 12, Lys413, is 1.5 kDa smaller than the
Arg174 cleavage product as determined from the primary sequence (29).
This value is almost exactly the difference in molecular mass between
the two fragments reported here. The example of an altered positioning
of helix 12 resulting from antagonist binding into the estrogen
receptor has been previously reported (9, 30). This further supports
the idea that the doublet of fragments centered at 34 kDa upon MK
binding results from proteolysis of a differentially exposed trypsin
site near helix 12 of the VDR.
This publication is the first report of MK antagonism toward the
osteocalcin VDRE and in Cos-1 cells. For this reason, it is difficult
to ascertain whether the differences between results seen for this
system and those reported are the result of using a different
promoter, the cell line, or a yet-unidentified factor.
Transcriptional antagonism of the reporter plasmid containing the SV40
promoter having two human 25-OH-D3 24-hydroxlase
gene VDREs has been previously shown in three different cell lines
using 10 nM
1,25(OH)2D3 and 100
nM MK concentrations. In Cos-7 cells, MK shows
approximately 60% antagonism compared with
1,25(OH)2D3 induction
alone. MK agonist activity in these cells is only about 3% of the
activation by
1,25(OH)2D3 (24). In
HeLa cells, MK has a similar antagonism of approximately 45% but twice
the MK agonist activity (
8% of 10 nM 1,
25(OH)2D3 alone). Saos-2
cells show a much larger antagonistic response of around 85% with less
than 3% MK agonist activity (13). In the present study, the Cos-1
cells display a 54% antagonism but have a larger MK agonist activity
that is 11% of the
1,25(OH)2D3-induced
activity at these same concentrations.
It has been suggested that the larger antagonistic response to MK in
Saos-2 cells may be due to a possible
1,25(OH)2D3-specific
functionality resulting from the bone-derived osteosarcoma cells,
whereas, kidney (Cos-1 and Cos-7)- or cervical tissue-derived (HeLa)
cells might not be as responsive due to an absence of such
functionality (13). A reason for the larger agonist response of MK in
the Cos-1/osteocalcin system, which is nearly 3 times that of the
morphologically similar Cos-7 cells, may be due to the use of the
osteocalcin reporter, but no data are presented to support such a
hypothesis.
Previous to MK, no antagonists of the genomic action of
1,25(OH)2D3 on the VDR
had been reported, although,
1ß,25(OH)2D3 has been
shown to antagonize the nongenomic or rapid actions of
1,25(OH)2D3 to stimulate
transcaltachia or to open chloride channels in ROS 17/2.8 cells (31, 32). However, antagonists or partial antagonists of other steroid
hormones already have been shown to be useful pharmaceuticals. The
estrogen system has a number of clinically relevant antagonists such as
raloxifene, tamoxifen, and ICI182,780 (9). Another prominent
pharmaceutical antagonist of both progesterone and glucocorticoid is
mifepristone (RU486) (10).
In conclusion, we have shown that the antagonism of MK is not due to a covalent binding to the VDR but, rather, the freely reversible interaction with a ligand that is partial antagonist and agonist. For the osteocalcin VDRE in Cos-1 cells, this antagonism has been shown to occur against all ligands tested, but the effective range of antagonism is dependent on the transactivation activity of the agonist relative to MK. Also, we have shown that when the VDR-LBD is occupied by MK there results a unique conformational change in the protein which mediates the antagonist properties of the analog.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,25(OH)2D3,
its analogs, or a combination with a final ethanol concentration of
0.1%. At 30 h after analog treatment, the cell medium was
harvested to measure GH reporter by RIA (Nichols Institute Diagnostics, San Juan Capistrano, CA). The data is presented as
fold activation relative to ethanol control. All experiments were
carried out on triplicate samples and the data are expressed as the
mean ± SEM.
Exchange Assay
Duodenal mucosa from 3-week-old vitamin D-deficient White
Leghorn cockerels (Hyline International, Lakeview CA), fed from hatch
on a standard rachitogenic diet (33), was homogenized in 10
mM Tris, 1.5 mM EDTA, 1 mM
dithiothreitol, pH 7.4 (TED) to make a 10% homogenate. The crude
chromatin fraction was prepared by our standard procedure (34)
involving three washes of the nuclear fraction in TED with 0.5% Triton
X-100 (Sigma), wash and resuspension in TED. The chromatin
fraction was divided into five aliquots and treated with either ethanol
(vehicle control, 5 µl/ml) or
1,25(OH)2D3 at 0.5 or 2
nM (final concentration) or MK at 5 or 200 nM.
Ligands were incubated with the chromatin prep for 4 h on ice,
followed by a wash and resuspension in TED. For the measurement of
unoccupied receptor, a standard binding assay was set up using receptor
from each treatment group binding to
[3H]-1,25(OH)2D3
(100 Ci/mmol, Amersham Pharmacia Biotech, Arlington
Heights, IL) in the presence or absence of a 200-fold excess of
1,25(OH)2D3. The
proportion of occupied receptor in each sample was measured using our
exchange assay (35). Briefly, aliquots of the chromatin receptor
preparation were incubated with TPCK (Sigma) for 30 min on
ice to covalently block unoccupied receptor binding sites. Next,
aliquots of the TPCK-treated chromatin receptor were incubated with
[3H]-1,25(OH)2D3
in the presence or absence of
1,25(OH)2D3 at 37 C for
30 min, followed by hydroxyapatite (Bio-Rad Laboratories, Inc. Hercules CA) separation of bound and free ligand (on ice)
and liquid scintillation counting. Data are presented as percent
reoccupancy, which is the percent normalized average of the
disintegrations per minute (DPM) from triplicate samples divided
by duplicate background samples containing a large excess of unlabeled
1,25(OH)2D3. The data
from two separate experiments are shown as the mean ±
SEM.
Protease Sensitivity Assay
The sensitivity of ligand-bound receptor to limited proteolysis
was determined using our protease sensitivity assay (18).
[35S]-labeled VDR was prepared using the TNT
coupled transcription and translation system (Promega Corp., Madison, WI) from the pGEM-4 plasmid containing an insert
for wild-type VDR cDNA and [35S]methionine
(1,000 Ci/mmol, Amersham Pharmacia Biotech). Aliquots (5
µl) of the [35S]-VDR were incubated for 20
min at room temperature with ligand (10 µM), followed by
treatment with 15 µg trypsin/ml (Sigma) for 20 min at
room temperature. Samples were run on 12% SDS-PAGE. The radioactive
bands showing the [35S]-VDR proteolytic
fragments were visualized by autoradiography.
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FOOTNOTES |
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Received for publication May 9, 2000. Revision received August 3, 2000. Accepted for publication August 7, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,25(OH)2-vitamin
D3: A case study of transcaltachia (the rapid
hormonal stimulation of intestinal calcium transport). In: Feldman D,
Glorieux FH, Pike JW (eds) Vitamin D. Academic Press, San Diego, CA, pp
233–256
,25-Dihydroxy-16-ene-23-yne-vitamin
D3 prolongs survival time of leukemic mice. Proc
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analogs antagonize differentiation of human leukemia cells (HL-60
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