Department of Medicine Stanford University School of Medicine Stanford, California 94305
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Direct measurement of the VDR-RXR heterodimerization in the presence of vitamin D analogs in intact cells has not been reported. In this study, we used the yeast two-hybrid system, developed by Fields and Song (28), to evaluate the specific interaction between the VDR and the RXR induced by 1,25-(OH)2D3 and six vitamin D analogs. This system permits a quantitative and rapid estimation of the strength of intracellular protein-protein interaction (29). We have applied this technique as a novel approach to study the role of 1,25-(OH)2D3 and analogs in promoting VDR-RXR heterodimerization. It is our hypothesis that the selective and differential potencies of the various analogs arise, in part, from differences in their ability to induce the heterodimerization of VDR with RXR. To ascertain whether the data obtained from the yeast two-hybrid system on heterodimerization would be useful in predicting analog potency, we evaluated 1,25-(OH)2D3 and the analogs for their ability to augment VDR action in the sequence of events leading to transcriptional activation of a target gene. We assayed 1) the ligand-binding affinity for VDR; 2) the DNA-binding activity of liganded VDR-RXR heterodimer to the VDRE derived from the human osteocalcin gene; 3) the induction of transcription of a VDRE-reporter gene and 4) the growth inhibition of human prostate cancer cells. Our findings suggest that the level of transcriptional activation induced by 1,25-(OH)2D3 and its analogs correlates well with the strength of VDR-RXR heterodimerization, as measured by the yeast two-hybrid system. Although our data indicate that heterodimerization is generally better than ligand binding or DNA binding as a predictor of potency, the potency of all analogs is not fully revealed by measurement of heterodimerization activity. The exceptions indicate that the outcome of ligand-dependent transactivation is more complex than what the assay of a single molecular event in the hormone action pathway discloses. Our data suggest that the ability to induce heterodimerization, although important, is only one determinant of ligand potency.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Use of the Yeast Two-Hybrid System to Study Heterodimerization
The actions of 1,25-(OH)2D3 and analogs
are dependent on binding to VDR. It is known that the ligand-inducible
VDR-dependent transactivation requires RXR, a partner for
heterodimerization of VDR (21). To test whether the strength of VDR-RXR
heterodimerization is dependent on the structure of the ligand being
bound to VDR, we used the yeast two-hybrid system to detect VDR-RXR
interactions in intact cells. This was accomplished by employing a
diploid yeast cell that expresses both VDR and RXR as GAL4 fusion
proteins and determining the activity of a GAL4-dependent
ß-galactosidase reporter gene. A diagrammatic representation of the
system is shown in Fig. 2A. The transcription of the
lac Z gene was dependent on the juxtaposition of the GAL4
DNA-binding domain (DBD) and the GAL4 activation domain (AD) at the
GAL4- binding sites attached to the CYC1 promoter. The only way the DBD
and the AD of GAL4 can be juxtaposed in this system is for VDR and RXR
to interact. The expression of the HIS 3 gene is similarly dependent on
GAL4 since it is driven by a GAL1 promoter.
|
To prove that the interaction between VDR and RXR fusion proteins was
necessary for ß-galactosidase activity, the diploid strain was
constructed to contain various control plasmids and fusion proteins.
These were tested for their growth on supplemented minimal medium
lacking tryptophan, leucine, and with or without histidine. Only the
yeast diploids expressing the VDR and RXR GAL4 fusion proteins were
able to grow on medium that lacked histidine but contained
3-aminotriazole. The growth of the different diploids is shown in Fig. 2B. The results show that some specific interaction between VDR and RXR
occurs in the absence of any added ligand.
When all the diploids were cultured in complete synthetic medium
lacking only tryptophan and leucine, the levels of ß-galactosidase
activity were highest in the diploid containing the VDR and RXR GAL4
fusion proteins (Fig. 2C). This level was 5-fold higher than the basal
levels obtained for other combinations of fusion proteins used as
controls in this experiment. The control plasmids expressed fusion
proteins for lamin C or the TATA-binding protein (TBP) or they were the
vectors pAS2 and pGAD424 without any inserts.
Diploids containing the plasmids pAS2-VDR and pGAD-RXR showed
maximal ß-galactosidase activity, demonstrating a strong interaction
between VDR and RXR
. Interestingly, diploids carrying pAS2-RXR
alone appeared to express a relatively high level of ß-galactosidase,
with a level twice that of background, regardless of which fusion
protein was being expressed from the pGAD424 vector. It is possible
that GAL4-DBD-RXR
fusion protein alone can interact with the yeast
general transcription machinery and activate the reporter genes to some
degree.
Differential Ability of
1,25-(OH)2D3
and
1,25-(OH)2D3-Analogs
to Enhance VDR-RXR Heterodimerization
We next determined whether the level of ß-galactosidase activity
changed due to addition of various doses of
1,25-(OH)2D3 in the diploid yeast cells
expressing VDR and RXR GAL4 fusions. As shown in Fig. 3, the addition of 1,25-(OH)2D3 to the yeast
culture medium stimulated ß-galactosidase activity over the basal
level in the absence of any ligand in a dose-dependent manner, and the
maximal activity seen at 100 nM
1,25-(OH)2D3 was approximately 4-fold higher
than the basal level. These results indicate that
1,25-(OH)2D3 binding enhances the affinity of
the specific VDR-RXR interaction. When the RXR ligand,
9-cis-retinoic acid, was added to the medium there was no
change in the basal level of ß-galactosidase activity (data not
shown). When 1,25-(OH)2D3-analogs were added to
the medium in graded concentrations from 1 to 1000 nM, the
ß-galactosidase activity was induced in an analog-specific manner. As
shown in Fig. 4
, the order of potency of the
1,25-(OH)2D3-analogs to induce
ß-galactosidase activity was: KH-1060 >
1,25-(OH)2D3 > EB-1089 > ED-71 >
MC-903 > Ro245531 > Ro242287. Thus, the
1,25-(OH)2D3 analog KH-1060 was the most potent
in stimulating ß-galactosidase activity and Ro242287 was the
weakest. Data are also expressed as percent of
1,25-(OH)2D3 activity in Table 1
.
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The actions of 1,25-(OH)2D3 can be broadly divided into two categories: calcemic activity and antiproliferative activity. Synthetic 1,25-(OH)2D3-analogs have been found to have a reduced calcemic action while maintaining or exhibiting enhanced antiproliferative actions, and this selectivity of response is the major reason for interest in these molecules. The basis for their differential effects appears to be multifactorial and probably has several distinct mechanisms. In vivo, an altered affinity of an analog for the vitamin D-binding protein (DBP) in the serum appears to be important, since DBP determines the level of free hormone available to target tissues (33). Once at the target cell, the permeability of the cell membrane to the analog as well as other pharmacokinetic parameters might contribute to differential effects. Also different rates of metabolic conversion of their active to inactive forms in various tissues no doubt plays a roles in their unique patterns of activity (34).
In addition to these in vivo factors, it is clear that the
analogs differ from 1,25-(OH)2D3 in their
intracellular mechanism of action. We (32) and others (17) have shown
that high-affinity binding of the ligand to VDR is necessary for
VDR-induced function, but differences in the binding affinities among
analogs do not sufficiently explain differences in their potencies. One
may have expected that the VDR binding affinity would determine the
potency of various analogs in assays of transactivation and growth
inhibition. However, the affinities of the different ligands for the
receptor did not correlate well with the potency of the ligands to
cause transcriptional activation (Fig. 8A), and growth inhibition
(Table 1
).
|
Since the assays of VDR binding and DNA binding are somewhat lacking in their ability to predict potency or explain selective actions of analogs, our interest has been directed at the VDR-RXR interaction. Binding of 1,25-(OH)2D3 or its analogs to VDR induces conformational changes in the VDR (27), and the differences in the steric constraints induced might differentially alter the ability of VDR to be phosphorylated, to interact with RXR or other proteins, or might change the half-life of the protein. Thus, in specific target cells with different species or concentrations of interacting proteins, the VDR-mediated calcemic activity might be reduced and the relative antiproliferative potency enhanced by different 1,25-(OH)2D3 analogs. To assess the extent of conformational changes in VDR induced by analog binding, other investigators have used unique approaches. Peleg et al. (27) employed a protease sensitivity assay to demonstrate that vitamin D analogs with 20-epi orientation conferred distinct sensitivities to protease digestion and greater responsiveness to a VDRE-regulated reporter gene than 1,25-(OH)2D3. Cheskis et al. (18) showed that surface plasmon resonance determined kinetic parameters of VDR-RXR interaction in the presence and absence of analogs.
Although these in vitro studies indeed provide insights into the analog-induced conformational changes of the VDR protein, a variety of intracellular or in vivo conditions may also be important in understanding the functional changes of VDR in vivo. For these reasons, we chose to use the yeast two-hybrid system as an intracellular system to measure the in vivo effects of vitamin D analogs on the VDR-RXR heterodimerization and to compare these intracellular effects of ligands with their abilities to transactivate a target gene in transient cotransfection assays or to inhibit cellular proliferation. The yeast two-hybrid method is a sensitive assay to determine the strength of protein-protein interaction (28), and our data demonstrate that this method can also be used to investigate how protein-protein interactions are regulated by specific effectors, such as ligands of receptor proteins. It has been reported that the strength of protein-protein interaction, as predicted by the ß-galactosidase assay in the two-hybrid system, generally correlates with that determined in vitro by directly assaying the association of proteins in solution or by using the dissociation constant (Kd) for binding to consensus DNA motifs to extrapolate an approximate minimum affinity (29).
Our study of the interaction of VDR-RXR using the yeast two-hybrid
system has revealed several interesting findings. First, it should be
noted that a VDR-RXR interaction is easily detectable in the absence of
1,25-(OH)2D3, as shown in Fig. 2. This result
indicates that a basal level of ligand-independent heterodimerization
is present (35). However, we did not detect any association between the
VDR and the TBP or the formation of RXR-RXR homodimers in the absence
of ligand. As shown in Fig. 3
, 1
, 25-(OH)2D3
enhanced the VDR-RXR interaction in a dose-dependent manner. It is
possible that ligand binding alters the conformation of VDR so that the
ligand-bound receptor exposes distinct interfaces that enhance RXR
interaction. Interestingly, addition of 9-cis-retinoic acid,
the RXR ligand, to culture medium of the same yeast cells did not
affect the VDR-RXR interaction (data not shown). This observation
indicates that the two components in the VDR-RXR complex have different
ligand-binding capacity. We are currently investigating the molecular
mechanism for this difference.
We have analyzed how well the ability of an analog to influence the
strength of VDR-RXR heterodimerization may predict its potency as
measured by VDRE-CAT induction (Figs. 4 and 7
) or growth inhibition of
prostate cancer cells (Table 1
). We found that the strength of
interaction between VDR and RXR induced by analogs correlated well with
their ability to transactivate a target gene in the case of
1,25-(OH)2D3 and compounds with side-chain
modifications such as KH-1060, Ro245531, EB-1089, MC-903, and
Ro242287, as shown in Fig. 8B
. However, analog ED-71 enhanced the
VDR-RXR interaction more than most of compounds (except KH-1060 and
1,25-(OH)2D3) (Fig. 4
), but in the
transactivation assay, ED-71 has no effect at a concentration of 1 or
10 nM (Fig. 7
). These results suggest that ED-71 induced a
conformation of the VDR that was suitable for the RXR dimerization, but
was not sufficient for transactivation. ED-71 was also relatively
inactive in promoting the binding of VDR-RXR dimer to VDRE in EMSA
(Fig. 6
). This analog was the exception demonstrating that
heterodimerization activity does not always predict potency. Among the
panel of analogs examined, ED-71 is unique in that it has a
modification in the A ring. From the structural aspect, the A ring of
1,25-(OH)2D3 is dynamically equilibrated
between the
-form and the ß-form. The 2ß-(3-hydroxypropoxy)
substitution may disturb the equilibrium of the chair-chair conformers
interconversion of the A ring and make a new hydrogen bond with
1
-hydroxyl group in the A ring of ED-71 or the ligand-binding pocket
of the VDR. Thus, by lacking the configurational flexibility of the A
ring, the conformational changes in the VDR induced by ED-71 were
presumably different from those induced by
1,25-(OH)2D3 or other analogs with side-chain
modifications.
Figure 8B shows a regression plot comparing
heterodimerization potency with transactivation potency. The
relationship between the two activities is excellent, with a
correlation coefficient of 0.815, P < 0.0001
(r2 = 0.663). (If ED-71 is omitted, the r2
value increases to 0.744). The r2 value of 0.663
(coefficient of determination) indicates that 66.3% of the dependent
variables variation (transactivation activity) is explained by the
independent variable (heterodimerization). The same relationship
between VDR binding and transactivation has an r2 value of
0.304 (Fig. 8A
), which is substantially less than for
heterodimerization. The correlation between VDR binding and
heterodimerization has an r2 value of 0.649. In other
words, the variation in analog-VDR binding activity presents
approximately 65% of the variation in analog-induced VDR-RXR
heterodimerization. The variation in heterodimerization contributes to
about 66% of the variation in analog-induced transactivation. But the
variation in VDR binding only explains approximately 31% of the
variation in transactivation (0.65 x 0.66) (Fig. 8C
).
The conventional reporter gene assay for steroid ligand/receptor studies in mammalian cells can be complicated by the unavoidable background of endogenous receptors and cofactors. Cell type-specific factors that interact with receptors may alter the action of ligands on receptor function. Therefore, in studying specific steps in a receptors function, the yeast system is helpful because: 1) it lacks intracellular receptors and 2) human steroid receptors function in yeast cells. In fact, many steroid-responsive transcription units have been successfully reconstituted in Saccharomyces cerevisiae expressing recombinant mammalian steroid receptors (35, 36, 37).
The transactivational capability of steroid-receptor complexes is not only determined by the cellular context but also by the promoter context of the reporter gene (38, 39). Although yeast transactivation systems are useful for steroid/receptor studies, they may be complicated by the types of promoters chosen and by other differences between yeast and mammalian cells. One potential drawback is that the transactivation activity of steroid receptors in yeast cells is dependent upon how efficiently the recombinant mammalian receptors interact with the yeast general transcriptional machinery. The interactions among these heterologous proteins may not always be sensitive enough to reflect mammalian effects of steroids or receptors. This is exemplified by the recent finding that VDR/RXR-dependent transcriptional activity in yeast is constitutive in nature and does not appear to be inducible by 1,25-(OH)2D3 (35).
In contrast, the yeast two-hybrid assay is based on the reconstitution of a functional yeast transcriptional activator GAL4, and the consequent activation of reporter genes (HIS 3 and Lac Z) is under the control of a GAL4-responsive promoter (28). Heterologous proteins are not involved in this yeast transcription unit. The assay is sensitive enough that it is well suited for detecting weak or transient interactions between two proteins of interest. Our findings that the VDR-RXR heterodimerization is inducible by 1,25-(OH)2D3 and its analogs in yeast indicate that the system can be used to determine the effect of ligand on receptor function (heterodimerization) regardless of the cellular and promoter context. Moreover, this approach allows one to isolate the heterodimerization strength from other factors affecting potency such as binding to DBP or target organ metabolism.
In summary, we have shown that 1,25-(OH)2D3-analogs differentially enhanced VDR-RXR heterodimerization in vivo. The ligand-induced heterodimerization of VDR and RXR, as measured in yeast cells, correlates well with the level of transactivation of target genes in mammalian cells. The yeast two-hybrid system is thus a supplemental measure that is convenient and rapid and yields insight into the critical step of heterodimerization providing specific information on analog ability to influence protein-protein interactions. This assay may therefore be useful in the evaluation of new analogs and in designing structural changes in future analogs based on the ability of the compounds to induce this critical step in vitamin D action.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aprotinin, pepstatin, and soybean trypsin inhibitor were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Liquid tissue culture media were purchased from Mediatech (Herndon, VA). FCS, penicillin, streptomycin, and lipofectamine were obtained from GIBCO/BRL (Grand Island, NY). Partially purified VDR was purchased from Panvera Corporation (Madison, WI). The monoclonal anti-chicken VDR antibody, 9A7, was the gift of Dr. J. W. Pike (Ligand Pharmaceuticals, San Diego, CA). The anti-mouse RXR antibody, 4RX-1D12, was the gift of Dr. P. Chambon (Strasbourg, France). Restriction enzymes, T4 polynucleotide kinase, and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA). Oligonucleotides were synthesized by Operon Technologies (Alameda, CA). The plasmids and yeast strains comprising the two-hybrid yeast system, as well as extensive protocols, were obtained from Clontech (Palo Alto, CA). Solid yeast media were purchased from Difco (Detroit, MI) and Bio101 (San Diego, CA). Various other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).
Plasmids and Plasmid Constructs
The following plasmids and plasmid constructs were used in this
study:
pEUK-C1-VDR.
The human VDR cDNA sequence (obtained from Dr. J. W Pike, Ligand Pharmaceuticals) was isolated by EcoRI digestion and ligated into the EcoRI site of pBluescript SK. Plasmid DNA was purified, digested with XhoI/BamHI, and inserted into the expression vector pEUK-C1 (obtained from Clontech, Palo Alto, CA) in the appropriate orientation to allow overexpression of VDR in mammalian cells.
pOC(VDRE)2-CAT.
The plasmid pOC(VDRE)2-CAT, carrying the human OC-VDRE
sequences in duplicate upstream of the thymidine kinase promoter and
the CAT gene, was obtained from Dr. L. Freedman (Memorial
Sloan-Kettering Cancer Center, New York, NY).
pSVgal.
The plasmid pSVgal (obtained from Promega, Madison, WI) carried the
ß-galactosidase gene linked to the SV40 promoter.
pAS2-VDR.
The plasmid pAS2-VDR was constructed by inserting the entire human VDR
coding sequence between the BamHI and SalI sites
of the plasmid pAS2 (obtained from Clontech) to produce an in-frame
fusion gene between the DBD of the GAL4 gene and the VDR cDNA gene. The
insert was the product of a PCR in which a plasmid carrying VDR cDNA
was used as a template, and the primers were
5'-TCCGGGATCCGTATGGAGGCAATGGCG-3' and
5'-GAGGTCGACTAGTCAG-GAGATCTCATT-3'. Both of the potential VDR
initiation codons are present in this construction. The plasmid pAS2
carries a selectable TRP1 gene.
pGAD424-RXR.
The plasmid pGAD424-RXR was constructed by inserting the entire
human RXR
coding sequence between the BamHI and
SalI sites of the plasmid pGAD424 (obtained from Clontech)
to produce an in-frame fusion gene between the AD of the GAL4 gene and
the RXR
cDNA gene. The insert was the product of a PCR in which a
plasmid carrying RXR
cDNA (obtained from Dr. R. A. Evans, Salk
Institute, San Diego, CA) was used as a template, and the primers were
5'-CCGGGGATCCGTGCCATGGA-CACCAAACAT-3' and
5'-CCGCTCGAGGGCCTAAGTCATTTGGTG-3'. The plasmid pGAD424 carries a
selectable LEU2 gene.
pGAD424-TBP.
The plasmid pGAD424-TBP was constructed by inserting the entire human
TBP coding sequence between the BamHI and SalI
sites of the plasmid pGAD424 to produce an in-frame fusion gene between
the AD of the GAL4 gene and the TBP cDNA gene. The insert was the
product of a PCR in which a plasmid carrying human TBP cDNA (obtained
from Dr. R. Kornberg, Stanford University, Palo Alto, CA) was used as a
template and the primers were 5'-CCGAATTCATGGATCAGAACAGCCTG-3' and
5'-GGAGATCTTACGTCGTCTTCCTGAATCC-3'.
pG5-RXR.
The plasmid pG5-RXR was constructed by inserting the human RXR
coding sequence between the BglII and SalI sites
of the plasmid pG5 (obtained from Dr. R. S. Fuller, Stanford
University) downstream of the GAPDH promoter. The insert was the
product of a PCR in which a plasmid carrying RXR
was used as a
template and the primers were 5'-GAAGATCTAGACATGGACACCAAACAT-3' and
5'-CCGCTCGAGGGCC-TAAGTCATTTGGTG-3'. The plasmid pG5 harbors a
selectable URA3 gene.
pAS2-RXR.
pAS2-RXR was constructed using the insert used in making
pGAD424-RXR
.
pLaminC.
pLaminC was obtained from Clontech (Palo Alto, CA).
DNA manipulations and DNA fragment amplifications were performed according to standard protocols (40).
Mammalian Cell Culture
The monkey kidney fibroblast cell line, Cos-7 (obtained from
American Type Culture Collection, Rockville, MD), was grown in DMEM
containing one g/liter glucose and supplemented with 10% FCS and the
antibiotics penicillin and streptomycin. LNCaP cells were cultured in
RPMI-1640 supplemented with 5% FCS and antibiotics. These cell lines
were grown at 37 C in an atmosphere of 5% CO2.
Cell Proliferation Assay
For cell proliferation assay, LNCaP cells were seeded at an
initial density of 50,000 cells per well in a six-well plate. After
overnight culture, the cells were exposed to 10 nM
1,25-(OH)2D3 or different analogs.
1,25-(OH)2D3 or analogs were added with media
replenishment every other day. At the end of the 6-day period, cellular
proliferation was assessed by determination of attained DNA mass using
the method of Burton (41).
Expression of VDR in Cos-7 and Ligand Competition Assay
Cos-7 cell monolayers were grown to 80% confluence in 100-mm
tissue culture dishes. Cells in each dish were transfected with 2 µg
pEUK-C1-VDR and lipofectamine as recommended by the manufacturer.
Forty-eight hours after transfection, the cells were collected, rinsed
with PBS, resuspended in KTEDM buffer (300 mM KCl, 10
mM Tris-HCl, 1.5 mM EDTA, 1 mM
dithiothreitol, and 10 mM sodium molybdate), containing
soybean trypsin inhibitor (10 µg/ml), leupeptin (1 µg/ml),
pepstatin (2 µg/ml), and aprotinin (1 µg/ml), and disrupted by
sonication at 4 C. The disrupted cells were centrifuged at 210,000
x g for 35 min at 4 C and the supernatant retained. The
protein concentration of the supernatant was determined by the method
of Bradford (42). Typically, an aliquot of 200 µl of supernatant,
containing 100200 µg protein, was incubated with
[3H]-1,25-(OH)2D3 at 1
nM and increasing concentrations of
1,25-(OH)2D3 analogs in a range of 1100
nM final concentration for 4 h at 4 C. Bound and free
[3H]1,25-(OH)2D3 were separated
using hydroxylapatite (43). Nonspecific binding was determined by
measuring binding in the presence of a 250-fold excess of radioinert
1,25-(OH)2D3. The concentration of the
radioinert 1,25-(OH)2D3 stock solution was
confirmed by measuring its absorbance at 265 nm and calculating its
concentration using a molar extinction coefficient of 18,200 for
1,25-(OH)2D3 at this wavelength.
The Yeast Two-Hybrid System
The Saccharomyces cerevisiae strains CG-1945 (MATa,
ura352, his3200, lys2801, trp1901, ade2101, leu23,
leu2112, gal4542, gal80538, LYS2::GAL1-HIS3,
cyhr2, URA3::(GAL4
17-mers)3-CYC1-lacZ) and Y187 (MAT, ura352, his3200,
trp1901, ade2101, leu23, leu2112, gal4
, gal80
, met,
URA3::GAL1-lacZ) were used. Yeast strains were transformed
with plasmid DNA using the method of Hill et al. (44) or
using the EZ Yeast Transformation kit (ZYMO Research, Orange, CA). The
yeast strains were grown in YPD medium (2% peptone, 1% yeast extract,
2% glucose) before transformation. Transformants were selected on
plates containing complete synthetic medium which consisted of 0.17%
yeast nitrogen base (without amino acids and ammonium sulfate), 0.5%
ammonium sulfate, 2% glucose, 2% agar, and amino acid, adenine, and
uracil supplements according to Sherman et al. (45). One or
more amino acids were omitted from the medium depending on which
plasmid was being selected.
The strain CG-1945 was transformed with pAS2-VDR DNA and transformants
were selected on plates containing complete synthetic medium but
lacking tryptophan. Transformants were maintained on the same medium.
The strain Y187 was transformed with pGAD424-RXR DNA and
transformants selected on plates containing complete synthetic medium
lacking leucine. A diploid strain was obtained by mating CG-1945
carrying pAS2-VDR with Y187 carrying pGAD424-RXR
on YPD plates and
selecting for diploid cells on minimal medium lacking supplements
except for adenine and uracil. Diploid cells selected in this manner
expressed both the GAL4 fusion proteins of VDR and RXR
. When diploid
cells were grown on solid medium without histidine, 3-aminotriazole was
routinely added at a concentration of 5 mM to suppress the
low basal level of HIS 3 expression according to the
manufacturers protocol PT10281 (Clontech, Palo Alto, CA).
ß-Galactosidase Assays
For ß-galactosidase assays, at least three independent diploid
colonies were grown overnight at 30 C in complete synthetic medium but
lacking tryptophan and leucine. The culture was diluted 1:20 into fresh
medium, and 1,25-(OH)2D3 or its analogs were
added at concentrations of 1, 10, 100, and 1000 nM. After a
further 16 h growth at 30 C, to an A600 of about
0.51.0, ß-galactosidase assays were performed. Briefly, cell
pellets from 1.5-ml cultures were permeabilized with three cycles of
freeze/thaw and incubated for 1 h at 30 C with
o-nitrophenyl-ß-D-galactopyranoside (ONPG) as
described (29). A420 of the incubations was normalized to
A600 of cell culture. One unit of ß-galactosidase is
defined as the amount of enzyme that hydrolyzes 1 µmol ONPG to
o-nitrophenol and D-galactose per min at 30
C.
EMSA
The EMSAs were performed following the protocol of Cheskis
et al. (18) with modifications. A yeast extract was prepared
from cells of the Saccharomyces cerevisiae strain CB023
(MATa pep4:: HIS3
prb1::hisG
prc1::hisG ura3
leu2 trp1ade2 Gal+
cir0) carrying the plasmid pG5-RXR
, which expressed the
human RXR
. The cells were grown in complete minimal medium lacking
uracil and harvested and disrupted in a "Bead Beater" in KTEDM
buffer. Protein concentration was determined using the Bradford assay
(37). Aliquots were frozen at -80 C and used as needed. A
double-stranded human OC-VDRE DNA fragment with the sequence
5'-TTGGTGACTCACCGGGTGAACGGGGGCATT-3' was end-labeled using
[
-32P]ATP and T4 polynucleotide kinase, according to
the suppliers recommendations (New England Biolabs, Beverly, MA).
In a typical assay, 5 ng human VDR and 10 µg yeast extract containing
the human RXR were incubated for 30 min at ambient temperature in a
binding buffer (20 mM Tris-HCl, pH 7.9, 1 mM
EDTA, 100 mM KCl, 10% glycerol, 0.1% NP-40, 1
mM dithiothreitol, 50 mg/ml poly
deoxyinosinic-deoxycytidylic acid) in the presence of
1,25-(OH)2D3,
1,25-(OH)2D3-analog, or vehicle (ethanol) in a
final volume of 30 µl. Thirty femtomoles (2550 x
103 dpm) of VDRE DNA were added to the binding mixture, and
the mixture was incubated for a further 20 min at ambient temperature.
Twenty microliters of the binding mixture was electrophoresed on a 6%
polyacrylamide gel using 0.5 x TBE buffer (45 mM
Tris-borate, 1 mM EDTA) at 85 V. The gel was dried on
filter paper and exposed to x-ray film. In some experiments, the
monoclonal anti-VDR antibody, 9A7, or anti-RXR antibody, 4RX-1D12,
diluted 1:40, was added before the addition of the VDRE DNA. Some
assays contained excess unlabeled VDRE DNA to assess nonspecific
protein-DNA interactions.
Transactivation Assay
Cos-7 cells were seeded at 3 x 105 cells per
dish in 60-mm tissue culture dishes (Corning, NY) in DMEM containing
10% FBS and antibiotics. Plasmid DNA was incubated with appropriate
amounts of lipofectamine for 30 min at room temperature to form
liposomes. The DNA-lipid mixture was added to the cells in serum-free
medium after 18 h of growth. Each transfection contained 1 µg
pOC(VDRE)2-CAT DNA, 0.5 µg pEUK-C1-VDR DNA, and 0.5 µg
pSVgal DNA. The control plasmid pSVgal was used to monitor transfection
efficiency. After 16 h of incubation, 3 ml DMEM containing 10%
FBS were added to each dish along with
1,25-(OH)2D3 or
1,25-(OH)2D3 analogs (1100 nM),
as appropriate. Cells were harvested after a further 32 h of
incubation with the ligands at 37 C. Cell lysates were prepared using
the Reporter Lysis Buffer (Promega, Madison, WI) according to the
manufacturers instructions. The ß-galactosidase activity was
measured by adding 100 µl cell lysate to 100 µl of a
double-strength buffer (120 mM
Na2HPO4, 80 mM
NaH2PO4, 2 mM MgCl2,
100 mM ß-mercaptoethanol, 1.33 mg/ml ONPG). The mixture
was incubated for 3060 min at 37 C, and the reaction was stopped by
the addition of 0.5 ml 1 M Na2CO3.
The level of ß-galactosidase activity was determined by measuring
A420. The level of CAT expression was determined by using
the CAT-ELISA kit (Boehringer Mannheim Biochemical, Indianapolis, IN)
and the results expressed as picograms of CAT per mU ß-galactosidase.
One unit of ß-galactosidase hydrolyzes 1 µmol ONPG to
o-nitrophenol and galactose per min at pH 7.5 at 37 C.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
This work was supported by NIH Grant DK-42482 and a grant from American Institute for Cancer Research. X.Y. Zhao was supported by NIH Grant 5T32DK-072120, and C. Gross was supported by NIH Grant 1K08DK-024590.
Received for publication July 7, 1996. Revision received October 30, 1996. Accepted for publication November 26, 1996.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|