Analysis of Vitamin D Analog-Induced Heterodimerization of Vitamin D Receptor with Retinoid X Receptor Using the Yeast Two-Hybrid System

Xiao-Yan Zhao, T. Ross Eccleshall, Aruna V. Krishnan, Coleman Gross and David Feldman

Department of Medicine Stanford University School of Medicine Stanford, California 94305


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Several synthetic analogs of 1{alpha},25-dihydroxyvitamin D3 [1,25-(OH)2D3] are potent inducers of cellular differentiation and inhibitors of cell growth, yet they are less calcemic than 1,25-(OH)2D3 itself. The mechanisms by which these vitamin D analogs elicit a different profile of cellular activities than 1,25-(OH)2D3 are not fully understood. We propose that the analogs bind to the vitamin D receptor (VDR) to produce a conformational change that is more or less constrained than that induced by 1,25-(OH)2D3. This conformational change determines the extent of the VDR-retinoid X receptor (RXR) heterodimerization which, in turn, determines the interaction with other factors that specify the selectivity and magnitude of gene transactivation. We used the yeast two-hybrid system to evaluate a series of six vitamin D analogs for their ability to induce VDR-RXR heterodimerization. The VDR-RXR interaction was elicited by the analogs in a concentration-dependent manner. To evaluate how this activity compared with other known steps in 1,25-(OH)2D3 action, we also measured the ability of the same six analogs to bind to VDR, to enhance the binding of VDR-RXR to DNA, to transactivate a vitamin D-response element-reporter construct, and to inhibit proliferation in mammalian cells. Our results indicate that, for most analogs, the level of transcriptional activation correlates well with the strength of VDR-RXR heterodimerization in intact cells. We conclude that the yeast two-hybrid system provides a useful means to investigate heterodimerization potency and that this property contributes significantly to the overall pattern of analog activity. The yeast two-hybrid system, being an intact cell assay and easy to perform, may be a useful supplement to the conventional assays employed to screen vitamin D analogs.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The hormone 1{alpha},25-dihydroxyvitamin D3 [1,25-(OH)2D3] is well known for its role in maintaining calcium homeostasis in the body (1, 2, 3). But recent findings indicate that 1,25-(OH)D3 has a much wider scope of activity that includes inhibition of cellular proliferation, promotion of differentiation, and immunosuppression (4, 5, 6). These observations have led to a consideration of using 1,25-(OH)2D3 for numerous new therapeutic applications such as treatment of cancer, psoriasis, and autoimmune diseases. The use of 1,25-(OH)2D3 for these emerging clinical applications is limited by the dose-dependent induction of hypercalcemia when it is given at the substantial doses required for these actions. To reduce this hypercalcemic potential, a large number of synthetic vitamin D analogs have been developed, and many of these carry side-chain substitutions (7, 8, 9, 10, 11, 12, 13). At present, several analogs appear to be therapeutically useful, exhibiting significant antiproliferative, prodifferentiation, and immunosuppressive activity while being less calcemic than 1,25-(OH)2D3 in vivo (7, 8, 9, 10, 11, 12, 13). In this study, we have investigated several analogs (Fig. 1Go) because they have demonstrated therapeutic utility, while exhibiting a reduced tendency to cause hypercalcemia. Except for Ro24–2287, the analogs are relatively potent compared with 1,25-(OH)2D3. Ro24–2287 binds poorly to the vitamin D receptor (VDR) and was included as a negative control. The mechanisms underlying the dissociation of the antiproliferative and differentiation activities from the calcemic potential of these analogs are under intense investigation (14, 15, 16, 17, 18).



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Figure 1. Chemical Structure of 1,25-(OH)2D3 and Analog Side-Chain Modifications

X = OH except Ro24–2287 where X = H.

 
Most of the biological effects of 1,25-(OH)2D3 and its analogs are mediated by the VDR, which is a member of the nuclear receptor superfamily (19, 20). The VDR must heterodimerize with the retinoid X receptor (RXR) (21, 22, 23) to activate transcription. Dimerization is dependent on sites within the ligand-binding domain of the VDR that contains a series of nine heptad repeats (24, 25). The VDR-RXR heterodimer binds to specific DNA sequences known as vitamin D response elements (VDREs) in the promoter region of target genes and subsequently results in the transactivation of these genes (26). The transcriptional activation of a target gene is a ligand-dependent event, although the exact molecular role of the ligand in this process is not totally clear. Some in vitro studies have indicated that the ligand may induce a conformational change in the VDR structure that modulates the ability of VDR to dimerize, be phosphorylated, bind to VDREs, or interact with coactivators and repressors involved in target gene transactivation (18, 27).

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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Vitamin D Analogs
The chemical structures of the 1,25-(OH)2D3 analogs used in this study are shown in Fig. 1Go. ED-71 (1{alpha}, 25-dihydroxy-2ß-3-hydroxypropoxy-vitamin D3) has an A ring modification. The analogs with modification of the side chain only are MC-903, 1{alpha},24S-dihydroxy-22-ene-25,26,27-cyclopropylvitamin D3 (introduction of a double bond at C22, a hydroxyl group at C24, and cyclopropyl modification of the side chain); EB-1089, 1{alpha},25-dihydroxy-22,24-diene-24,26,27-trihomovitamin D3 (introduction of two double bonds at C22, C24 and three additional methyl groups); and KH-1060, 1{alpha},25-dihydroxy-20-epi 22-ene-24, 26, 27-trihomovitamin D3 (20-epi, substitution of oxygen atom for the methylene group at C22, three additional methyl groups). Ro24–5531, 1{alpha},25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol, and Ro24–2287, 25-hydroxy-16,23-diene-cholecalciferol, have modifications of both the D ring (double bond at C16) and the side chain. In Ro24–2287, there is a deletion of the 1-{alpha} hydroxyl group while in Ro24–5531 there is the introduction of a double bond at C23 and the addition of six fluoro groups.

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. 2AGo. 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.



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Figure 2. Specific Interactions of VDR with RXR{alpha} in the Yeast Two-Hybrid System

A, The two-hybrid system: The GAL 4 DBD-VDR fusion protein and the GAL 4 AD-RXR{alpha} fusion protein are illustrated. The specific interactions between VDR and RXR{alpha} lead to GAL4-dependent transcription of both the reporter genes, lac Z and HIS 3. B, Growth patterns of various yeast diploids on the histidine-containing media (+histidine) or on the histidine-deficient media (-histidine). Diploid strains were obtained by mating CG-1945 carrying pAS2-VDR, pAS2-RXR, pLaminC, or pAS2 (from top to bottom) with Y187 carrying pGAD-RXR, pGAD-TBP, or pGAD (from left to right) individually, as described in Materials and Methods. C, ß-Galactosidase expression in various yeast diploids as determined in liquid cultures. ß-Galactosidase activity was assayed as described in Materials and Methods and values are given as ß-gal Units.

 
The diploid strain was derived from two haploid strains, one of which expressed the VDR-GAL4 DBD fusion protein, and the other expressed the RXR{alpha}-GAL4 AD fusion protein. The strain CG-1945 carrying the plasmid pAS2-VDR was mated with the strain Y187 carrying the plasmid pGAD-RXR{alpha} on solid medium, and diploid cells were selected on minimal medium containing adenine and uracil and without 3-aminotriazole.

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. 2BGo. 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. 2CGo). 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{alpha} showed maximal ß-galactosidase activity, demonstrating a strong interaction between VDR and RXR{alpha}. Interestingly, diploids carrying pAS2-RXR{alpha} 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{alpha} 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. 3Go, 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. 4Go, 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 > Ro24–5531 > Ro24–2287. Thus, the 1,25-(OH)2D3 analog KH-1060 was the most potent in stimulating ß-galactosidase activity and Ro24–2287 was the weakest. Data are also expressed as percent of 1,25-(OH)2D3 activity in Table 1Go.



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Figure 3. Dose-Response Effect of 1,25-(OH)2D3 on the VDR-RXR{alpha} Interaction in the Yeast Two-Hybrid System

A diploid yeast cell expressing both the GAL4 fusion proteins of VDR and RXR{alpha} was inoculated into minimal yeast culture medium. Aliquots of the overnight culture were incubated with vehicle (ethanol) or increasing concentrations of 1,25-(OH)2D3 for 16 h at 30 C. Yeast cells were collected and subjected to ß-galactosidase assay, as described in Materials and Methods. The ß-galactosidase units were calculated and shown as means from three experiments with SEM. Data were evaluated by ANOVA and tested for significance using Scheffe’s F test. P < 0.05 was considered significant. The P value for difference between 0 and 1 nM is less than 0.005.

 


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Figure 4. Dose-Response Effect of 1,25-(OH)2D3-Analogs on the VDR-RXR{alpha} Interaction in the Yeast Two-Hybrid System

A diploid yeast cell expressing both GAL4 fusion proteins of VDR and RXR{alpha} was inoculated into minimal yeast culture medium. Aliquots of the overnight culture were incubated with increasing concentrations of 1,25-(OH)2D3 or its analogs for 16 h at 30 C. Yeast cells were collected and subjected to ß-galactosidase assay as described in Materials and Methods. The ß-galactosidase units were calculated and shown as means from three experiments with SEM.

 

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Table 1. Effect of 1,25-(OH)2D3 and Analogs on Receptor Binding, VDR-RXR Heterodimerization, DNA Binding, Transactivation, and Growth Inhibition

 
Ability of 1,25-(OH)2D3 and 1,25-(OH)2D3-Analogs to Compete with [3H] 1,25-(OH)2D3 for VDR Binding
We next attempted to compare the heterodimerization potency of each analog to its ability to bind to the VDR. To compare the relative binding affinities of 1,25-(OH)2D3 and 1,25-(OH)2D3-analogs for VDR, ligand-binding competition experiments were performed with [3H]1,25-(OH)2D3 and unlabeled analogs as competitors. Cell extracts containing VDR were obtained from Cos-7 cells transiently transfected with the expression vector pEUKC1 carrying the VDR cDNA (30). In this system, the binding capacity (Nmax) of the extract was approximately 300 fmol [3H]1,25-(OH)2D3 bound per mg protein as determined by equilibrium binding analysis (data not shown). The nonspecific binding in this assay was less than 20% of total binding. As shown in Fig. 5Go, 1Go, 25-(OH)2D3 had the highest affinity of the seven compounds tested, and it competed for 50% of the binding sites at a concentration of 1.8 nM. Ro24–2287 had the lowest affinity, exhibiting only 30% competition at a concentration of 100 nM. MC-903, ED-71, EB-1089, KH-1060, and Ro24–5531 were 1.7-, 1.9-, 2.5-, 2.5-, and 6-fold less potent than 1,25-(OH)2D3, respectively. The relative VDR-binding activities of all analogs are summarized in Table 1Go.



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Figure 5. Competition Binding Analysis of VDR by Specific [3H]1,25-(OH)2D3 Binding

Cos-7 cells were transfected with a VDR expression vector, as described in Materials and Methods. Soluble extracts were prepared in KTEDM buffer at 4 C and incubated for 4 h in the presence of 1 nM [3H]1,25-(OH)2D3 with or without a 250-fold excess of radioinert hormone. Analog concentrations were at 1-, 10- and 100-fold excess of [3H]-1,25-(OH)2D3. Values shown are means from two experiments performed in duplicate. The data are represented as percent of specific binding in the absence of any competing steroid. A level of 100% represents 300 fmol [3H]1,25-(OH)2D3 bound per mg protein.

 
Ability of 1,25-(OH)2D3 and 1,25-(OH)2D3-Analogs to Enhance the Binding of VDR-RXR Heterodimers to the VDRE in Electrophoretic Mobility Shift Assays (EMSA)
The data that analogs modulate the strength of interaction between VDR and RXR suggests that analogs may also affect the DNA-binding activity of the VDR-RXR dimer. EMSA was used to study the interaction of VDR-RXR with the VDRE sequence from the human osteocalcin (OC) gene (31). As shown in Fig. 6AGo, under the conditions of the experiment in the absence of ligand, the VDR and the RXR{alpha} weakly bound to the labeled OC-VDRE probe (lane 1). This binding resulted in a retarded band that migrated slower than the free probe. The band containing the VDR-RXR{alpha}-VDRE complex was absent when the incubation mixture contained a 200-fold excess of unlabeled VDRE probe (lane 16) or in the presence of the anti-VDR antibody 9A7 (lane 17). The same complex was supershifted in the presence of the anti-RXR antibody 4RX-1D12 (lane 18). Addition of 1,25-(OH)2D3 or its analogs to the reaction mixture generally enhanced the DNA-binding activity of VDR-RXR{alpha} complex (lanes 2–15). 1,25-(OH)2D3 stimulated the protein-DNA interaction at a concentration of 10 and 100 nM (lanes 2–3), and KH-1060 was more potent than 1,25-(OH)2D3 (lanes 8–9). The gel shift pattern was minimally affected by Ro24–2287 (lanes 14, 15). Using densitometry to quantitate the VDR-RXR-VDRE bands (Fig. 6BGo), the ranking of analogs in their ability to enhance the VDR-RXR{alpha}-VDRE interaction at a concentration of 10 nM is as follows: KH-1060 > EB-1089 > 1,25D = MC-903 > Ro24–5531 > ED-71> Ro24–2287. Their potencies to enhance VDR-RXR binding to the VDRE are also expressed as percent of 1,25-(OH)2D3 activity, as shown in Table 1Go.



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Figure 6. Dose-Response Effect of 1,25-(OH)2D3-Analogs on the VDR-RXR Interaction with OC-VDRE in EMSA

A, EMSA were carried out as described in Materials and Methods using the OC-VDRE. 1,25-(OH)2D3 and analogs were tested at 10 and 100 nM. Lane 1, no ligand; lanes 2–15, 1,25-(OH)2D3 and analogs at concentrations 10 and 100 nM; lane 16, 10 nM 1,25-(OH)2D3 + 200-fold excess unlabeled VDRE; lane 17, 10 nM 1,25-(OH)2D3 + anti-VDR antibody, 9A7; lane 18, 10 nM 1,25-(OH)2D3 + anti-RXR antibody. B, Absorbance of each protein-DNA complex band was quantified by densitometry.

 
Ability of 1,25-(OH)2D3 and Its Analogs to Transactivate the pOC(VDRE)2-Chloramphenicol Acetyl Transferase (CAT) Reporter Gene
We next determined whether the ability of 1,25-(OH)2D3-analogs to enhance the VDR-RXR interaction would correlate with their ability to stimulate transactivation of a target gene. Cos-7 cells were transiently transfected with the VDR expression vector as well as a reporter gene construct, pOC(VDRE)2-CAT, which has a bacterial CAT reporter gene driven by a thymidine kinase promoter with two copies of the VDRE derived from the human OC gene. The transfected cells were treated with various 1,25-(OH)2D3-analogs at concentrations of 1, 10, and 100 nM. As shown in Fig. 7Go, all of the analogs tested enhanced CAT production at concentrations of 1 and 10 nM except for Ro24–2287 and ED-71. However, ED-71 at a concentration of 100 nM stimulated CAT to the level observed with EB-1089, MC-903, and Ro24–5531 at the same concentration. KH-1060 induced more CAT production than 1,25-(OH)2D3 at every dose tested. The profile of transactivation activity relative to 1,25-(OH)2D3, also shown in Table 1Go, is KH-1060 > 1,25-(OH)2D3 > MC-903 > Ro24–5531 = EB-1089 > ED-71 > Ro24–2287.



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Figure 7. Dose-Response Effect of 1,25-(OH)2D3-Analogs on the Transactivation of pOC(VDRE)2-CAT

Cos-7 cells were transiently transfected with a VDR expression vector pEUK-c1-VDR, a reporter plasmid pOC(VDRE)2CAT, and pSV2ßgal. Transfected cells were treated with 1,25-(OH)2D3 or analogs at various concentrations as indicated for at least 32 h and harvested 48 h post-transfection. The amount of CAT expression was determined by CAT-ELISA assay, and transfection efficiency was normalized by ß-galactosidase activity. Values are shown as a ratio of CAT protein produced to ß-galactosidase activity and presented as a percent of control activity determined in the absence of any ligand (100%). Values are the means of three separate transfection experiments with SEM.

 
Ability of 1,25-(OH)2D3 and Its Analogs to Inhibit the Growth of LNCaP Cells
To compare the potency of 1,25-(OH)2D3-analogs with 1,25-(OH)2D3 in a cell proliferation assay, we used the LNCaP human prostate cancer cell line as a model system (32). LNCaP cells were treated with 1,25-(OH)2D3 or various analogs at a concentration of 10 nM in serum-containing medium for 6 days. Fresh ligand was added every other day. At the end of the incubation, cells were harvested and the attained DNA mass was determined as an index of cellular proliferation. The rank order of growth-inhibitory potency is as follows: KH-1060 > EB-1089 > Ro24–5531 > MC-903 > 1,25-(OH)2D3 > ED-71 > Ro24–2287. The antiproliferative potencies, as a percent of 1,25-(OH)2D3 activity, are shown in Table 1Go.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study we explored the intracellular potencies of 1,25-(OH)2D3 and six 1,25-(OH)2D3-analogs in terms of some of the essential steps in the hormone action pathway. We hypothesized that by studying 1,25-(OH)2D3-analog action directly at the level of ligand-induced heterodimerization, insight might be gained into the mechanism by which the 1,25-(OH)2D3-analogs differed in functional potency in regard to transactivation of target genes or inhibition of cell growth. We examined two other molecular events important in the 1,25-(OH)2D3 action pathway, i.e. hormone binding to VDR and VDR-RXR binding to DNA. Our goal was to compare the potencies of the analogs derived from these assays with that obtained from a study of the induction of heterodimerization as assessed by the yeast two-hybrid system. Two functional responses were studied to assess potency, specifically transactivation of an OC reporter gene construct and the inhibition of LNCaP cell growth. At each step, we compared the potency of each analog with 1,25-(OH)2D3. The six 1,25-(OH)2D3-analogs were selected for study because of their potent antiproliferative activities (11, 12, 32), and in the case of Ro24–2287, because of its low binding affinity for VDR (32).

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. 8AGo), and growth inhibition (Table 1Go).



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Figure 8. Relationship of Vitamin D Analog-Induced Transactivation to Ligand Binding and Heterodimerization

A, Statistical analysis of correlation of ligand-VDR binding activity with the degree of ligand-induced VDR-dependent transactivation. All data in Figs. 5Go and 7Go at concentrations of 1, 10, and 100 nM were subjected to a correlation/covariance analysis and plotted as a simple regression curve. R, correlation coefficient, is 0.551, P = 0.0085, R2 = 0.303. B, Statistical analysis of correlation of ligand-induced VDR-RXR heterodimerization with the degree of transactivation. All data in Figs. 4Go and 7Go at concentrations of 1, 10, and 100 nM were subjected to a correlation/covariance analysis and plotted as a simple regression curve. R, correlation coefficient, is 0.815, P < 0.0001, R2 = 0.663. C. R2 values for regression plots derived from data in three assays: ligand binding, heterodimerization, and transactivation. All data in Figs. 4Go, 5Go, and 7Go at concentrations of 1, 10, and 100 nM were subjected to correlation/covariance and regression analyses. R, correlation coefficient; R2, determination coefficient.

 
The effect of 1,25-(OH)2D3-analogs on the DNA-binding activity of the VDR-RXR dimer has been investigated previously (16, 17, 18). We found that 1,25-(OH)2D3 and the analogs enhanced DNA-binding activity of the VDR-RXR dimer in a dose-dependent manner (Fig. 6Go). However, in our hands, the assay was only qualitatively useful.

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. 2Go. 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. 3Go, 1Go, 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. 4Go and 7Go) or growth inhibition of prostate cancer cells (Table 1Go). 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, Ro24–5531, EB-1089, MC-903, and Ro24–2287, as shown in Fig. 8BGo. However, analog ED-71 enhanced the VDR-RXR interaction more than most of compounds (except KH-1060 and 1,25-(OH)2D3) (Fig. 4Go), but in the transactivation assay, ED-71 has no effect at a concentration of 1 or 10 nM (Fig. 7Go). 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. 6Go). 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 {alpha}-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{alpha}-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 8BGo 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 variable’s 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. 8AGo), 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. 8CGo).

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 receptor’s 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
[3H]-1,25-(OH)2D3, with a specific activity of 170 Ci/mmol, and [{gamma}-32P]ATP, with a specific activity of 3000 Ci/mmol, were purchased from Amersham (Arlington Heights, IL). Nonradioactive 1,25-(OH)2D3 and the 1,25-(OH)2D3-analogs, Ro24–2287 and Ro24–5531, were the gifts of Dr. M. Uskokovic (Hoffmann-La Roche Co., Nutley, NJ). The 1,25-(OH)2D3-analogs MC-903, KH-1060, and EB-1089 were the gifts of Dr. L. Binderup (Leo Pharmaceutical Products, Ballerup, Denmark). The 1,25-(OH)2D3 analog ED-71 was the gift of Dr. Y. Nishii (Chugai Pharmaceuticals, Tokyo, Japan).

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{alpha}.
The plasmid pGAD424-RXR{alpha} was constructed by inserting the entire human RXR{alpha} 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{alpha} cDNA gene. The insert was the product of a PCR in which a plasmid carrying RXR{alpha} 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{alpha}.
The plasmid pG5-RXR{alpha} was constructed by inserting the human RXR{alpha} 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{alpha} 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{alpha}.
pAS2-RXR{alpha} was constructed using the insert used in making pGAD424-RXR{alpha}.

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 100–200 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 1–100 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, ura3–52, his3–200, lys2–801, trp1–901, ade2–101, leu2–3, leu2–112, gal4–542, gal80–538, LYS2::GAL1-HIS3, cyhr2, URA3::(GAL4 17-mers)3-CYC1-lacZ) and Y187 (MAT{alpha}, ura3–52, his3–200, trp1–901, ade2–101, leu2–3, leu2–112, gal4{Delta}, gal80{Delta}, 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{alpha} 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{alpha} 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{alpha}. 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 manufacturer’s protocol PT1028–1 (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 0.5–1.0, 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{Delta}::hisG ura3 leu2 trp1ade2 Gal+ cir0) carrying the plasmid pG5-RXR{alpha}, which expressed the human RXR{alpha}. 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 [{gamma}-32P]ATP and T4 polynucleotide kinase, according to the supplier’s recommendations (New England Biolabs, Beverly, MA).

In a typical assay, 5 ng human VDR and 10 µg yeast extract containing the human RXR{alpha} 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 (25–50 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 (1–100 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 manufacturer’s 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 30–60 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
 
We thank Dr. M. Uskokovic, Hoffmann-La Roche Co. (Nutley, NJ), for kindly providing us with the Ro24–5531, Ro24–2287, and 1,25-(OH)2D3; Dr. L. Binderup, Leo Pharmaceutical Products (Ballerup, Denmark), for MC-903, EB-1089, and KH-1060; Dr. Y. Nishii, Chugai Pharmaceutical (Tokyo, Japan), for ED-71; Dr. L. Freedman, Memorial Sloan-Kettering Cancer Center (New York, NY), for pOC(VDRE)2CAT; Dr. R. Kornberg, Stanford University (Palo Alto, CA), for human TBP cDNA, Dr. J. W. Pike, Ligand Pharmaceutical (San Diego, CA), for the human VDR cDNA and anti-VDR monoclonal antibody 9A7; and Dr. P. Chambon (Strasbourg, France) for anti-RXR monoclonal antibody 4RX-1D12. 1.


    FOOTNOTES
 
Address requests for reprints to: David Feldman, M.D., Division of Endocrinology, Gerontology and Metabolism, Stanford University Medical Center, Room S005, Stanford, California 94305-5103.

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-0721–20, and C. Gross was supported by NIH Grant 1K08DK-02459–0.

Received for publication July 7, 1996. Revision received October 30, 1996. Accepted for publication November 26, 1996.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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