©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Distinct Conformational Changes Induced by 20-epi Analogues of 1,25-Dihydroxyvitamin D Are Associated with Enhanced Activation of the Vitamin D Receptor (*)

Sara Peleg , Mani Sastry , Elaine D. Collins (1), June E. Bishop (1), Anthony W. Norman (1)(§)

From the (1) Department of Medical Specialties, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030 and the Department of Biochemistry, University of California, Riverside, California 92521

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The relative affinities of the 1,25-dihydroxyvitamin D (1,25-D) analogues 20-epi-1,25-dihydroxyvitamin D (IE) and 20-epi-22-oxa-24a,26a,27a-tri-homo-1,25-dihydroxyvitamin D (ID) to the nuclear vitamin D receptor (VDR) are similar to that of 1,25-D, but their antiproliferative action is 1000-fold greater. We tested whether the greater antiproliferative effect of these analogues is due to a differential activation of the VDR. In ROS 17/2.8 cells, the effective doses required to produce 50% maximal stimulation (ED) of transfected reporter genes driven by either the osteocalcin or the osteopontin vitamin D-response elements (VDRE) were 5 10 M, 10 M, and 10 M for 1,25-D, ID, and IE, respectively. Similar results were obtained when recombinant human VDR was cotransfected into CV-1 cells with an osteocalcin VDRE-reporter plasmid. We found that in vitro the sensitivity of 1,25-D-induced and analogue-induced receptors to proteases was different. The ED for binding to VDRE, as determined by electrophoretic mobility shift assays, was significantly higher for 1,25-D-induced than for analogue-induced VDR. The concentration of retinoid X receptor (RXR) was significantly lower in 1,25-D-induced than analogue-induced VDR complexes with VDRE.

We therefore conclude that IE and ID augment transcriptional activity of VDR more than 1,25-D does, by producing conformational changes that enhance dimerization of VDR with RXR. We suggest that these conformational changes are due to differences in the contact sites of the 20-epi analogues and 1,25-D with the VDR.


INTRODUCTION

Numerous analogues of 1,25-dihydroxyvitamin D (1,25-D)() have been synthesized in the attempt to improve vitamin D therapy of hyperproliferative conditions and malignancy (1, 2, 3, 4) . A particularly interesting group are the 20-epi analogues (5) . These compounds have antiproliferative activities 200-7,000-fold greater than that of 1,25-D, but their calcemic activity is not proportionally higher (5) . Although these compounds may not be suitable for treatment of cancer because they are very active immunosuppressants, the mechanism of their facilitated action remains an interesting issue. Because both the immunosuppressive and antiproliferative activities of vitamin D are thought to be transcriptional events mediated by the nuclear vitamin D receptor (VDR) (6, 7) , the first explanation proposed for the facilitated action of these compounds was that their affinity to the VDR was proportionally higher. All of the first generation of 20-epi analogues were tested for their relative affinity for the chick intestinal VDR and were found to have affinities similar to that of 1,25-D(5) . However, another study showed that one compound in this series, 20-epi-1,25-D (also known as MC1288 or analogue IE) had a higher affinity for calf thymus VDR than 1,25-D did (8) . Because in both studies binding and antiproliferative activities were assayed in receptors from different species, the relevance of the binding data to the augmented action of these compounds remains unclear and requires further clarification.

A second possible explanation was that the cellular uptake of the 20-epi analogues is more efficient. Analogue IE was shown to have poor binding affinity for the vitamin D-binding protein (DBP). However, in culture, the presence or absence of DBP had only a minor effect on the biological activity of either 1,25-D or the analogue tested (8) . A third possible explanation was that the catabolism rate of the 20-epi analogues was slower than that of 1,25-D. The catabolism studies performed with analogue IE showed that it was a good substrate for 24-hydroxylase but probably a poor substrate for 23-hydroxylase (8) , suggesting that poor catabolism may contribute to the facilitated action of this compound and perhaps that of other analogues in this family.

Another possibility that has not been previously tested is that the mode of interaction of the 20-epi analogues with VDR facilitates its transcriptional activity. To determine whether the 20-epi analogues augmented VDR action, we tested early steps in the sequence of events leading to transcriptional activation of VDR by ligand and compared the efficacy of the analogues to that of 1,25-D at each of these steps. We report here that in vitro, the 20-epi analogues induced a specific conformational change in the VDR, facilitated dimerization of VDR to the retinoid X receptor (RXR), and augmented binding to and transcriptional activation through a vitamin D response element (VDRE) containing two direct repeats separated by three nucleotides (DR3).


EXPERIMENTAL PROCEDURES

Reagents

Synthetic oligonucleotides were prepared by the Macromolecular Synthesis and Analysis Facility of M. D. Anderson Cancer Center. Anti-VDR antibodies were prepared by immunizing rabbits with a synthetic human VDR peptide conjugated to keyhole limpet hemocyanin. [-P]dATP was obtained from ICN and [S]methionine and 1,25(OH)[26,27-() H]D from Amersham Corp. A coupled transcription-translation kit was obtained from Promega. The analogues 20-epi-1,25-dihydroxyvitamin D (MC1288) and 20-epi-22-oxa-24a,26,27a-tri-homo-1,25-dihydroxyvitamin D (KH1060) were a generous gift from Dr. L. Binderup (Leo Pharmaceuticals, Bellerup, Denmark). For convenience, MC1288 was designated IE and KH1060 was designated ID. The structural formulas of these compounds are shown in Fig. 1.


Figure 1: Structural formulas of 1,25-D and the 20-epi analogues.



Cell Culture and Transfections

African green monkey kidney cell lines (COS-1 and CV-1) were maintained in Dulbecco's modified Eagle's medium (DMEM). Rat osteosarcoma ROS 17/2.8 cells were maintained in 50% DMEM and 50% F12. All culture media were supplemented with 10% fetal bovine serum. Forty-eight h before transfection, the cells were plated in 35-mm dishes at a density of 3 10/dish (ROS 17/2.8 and CV-1) or in 150-mm dishes at a density of 6 10/dish (COS-1) in DMEM and 10% fetal bovine serum. ROS 17/2.8 cells were transfected with 2 µg of plasmid containing either the VDRE from the mouse osteopontin gene (TGCTCGGGTAGGGTTCACGAGGTTCACTCGAC) (9) or the VDRE from the human osteocalcin gene (GGTGACTCACCGGGTGAACGGGGGCATT) (10) . These response elements were attached to the thymidine kinase promoter-growth hormone fusion gene. CV-1 cells were transfected with the osteocalcin VDRE-reporter fusion gene (4 µg/dish) and the recombinant human VDR expression vector (2 µg/dish). COS-1 cells were transfected with 20 µg/dish of recombinant human VDR plasmid.

All transfections were performed by the DEAE-dextran method (11) , and the cells were then treated for 1 min with 10% dimethyl sulfoxide. Medium samples for measurements of growth hormone were collected 2 days after transfection. Growth hormone production from the reporter gene was measured by a radioimmunoassay as described by the manufacturer (Nichols Institute, San Juan Capistrano, CA).

Preparation of Nuclear Extracts

To test the ligand-induced DNA binding activity of VDR and to assess the amount of RXR in the VDRVDRE complexes, nuclear extracts were prepared from COS-1 cells 48 h after transfection with the human VDR plasmid. Ligands (1,25-D, IE, and ID) dissolved in ethanol were added 1 or 24 h before the cells were harvested. The cells were then scraped from individual dishes into phosphate-buffered saline (PBS), washed twice in that buffer, and resuspended in 1 ml of buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl, 10 mM KCl, 0.5 mM dithiothreitol) and incubated on ice for 30 min. After they had swollen, the cells were homogenized by 5-10 strokes in Dounce homogenizers and centrifuged for 30 s at 14,000 g. The supernatants were then discarded, and the nuclear pellets were resuspended in 50 µl of buffer C (20 mM HEPES, pH 7.9, 400 mM KCl, 1.5 mM MgCl, 0.2 mM EDTA, 25% glycerol, 0.5 mM dithiothreitol), set on ice for 30 min, homogenized again, and centrifuged for 30 s at 14,000 g. The nuclear extracts were then collected, frozen in dry ice immediately, and stored at -80 °C for further analysis.

Electrophoretic Mobility Shift Assays (EMSAs)

A HindIII fragment containing the mouse osteopontin VDRE was labeled with P to a specific activity of 1-5 10 counts/min/µg. Each binding reaction included 50 mM KCl, 12 mM HEPES-NaOH, pH 7.9, 1 mM EDTA, 1 mM dithiothreitol, 12% (v/v) glycerol, 0.5 ng of the DNA probe, 10 µg of nuclear proteins, and 1 µg of poly(dI-dC) as nonspecific competitor DNA. The binding reactions were performed at room temperature for 30 min. The complexes were resolved by electrophoresis through 4% polyacrylamide gels at 4 °C. For competition experiments and for analysis of the complexes by antibody binding, the conditions were exactly as described above except that specific oligonucleotides or the VDR and RXR antibodies and nonspecific DNA were added to the binding reactions before addition of the probe.

Ligand Binding Assays

To compare the relative affinity of 1,25-D and the 20-epi analogues to VDR in vitro, whole-cell extracts from untransfected ROS 17/2.8 cells were prepared in KTED (10 mM Tris-HCl, pH 7.4, 1.5 mM EDTA, 0.3 M KCl, and 1 mM dithiothreitol). Briefly, the cells were scraped into PBS, washed three times, and homogenized with 10 strokes of a Dounce homogenizer. The homogenates were then aliquoted into tubes containing 0.2 pmol of [H]1,25-D and increasing concentrations of nonradioactive ligand. The mixtures were incubated on ice for 3-4 h, and then free ligand was separated from bound by hydroxyapatite (12) . The bound ligand was released from the hydroxyapatite by ethanol extraction, and the radioactivity was measured by scintillation counting. The results (see legend to ) were expressed as relative competition index (RCI) by the method of Wecksler and Norman (12) , with the RCI for 1,25-D defined as 100%.

To assess receptor occupancy by the ligand in vivo, monolayers of ROS 17/2.8 or VDR-transfected COS-1 cells were washed three times with PBS and incubated for 1 h with ligand in serum-free medium, and then the medium was discarded. The cells were washed three times in cold PBS, scraped into 10 ml of PBS, centrifuged, resuspended in KTED, and homogenized. Aliquots of the homogenates (0.2 ml) were incubated on ice for 3-4 h with 0.2 pmol of [H]1,25-D with or without 100-fold excess of unlabeled ligand. To assess the number of unoccupied VDR sites, the free ligand was separated from the bound by hydroxyapatite as described above.

Ligand-induced Sensitivity to Proteases

Synthetic human VDR, labeled with [S]methionine (1000 Ci/mmol) was prepared by in vitro coupled transcription-translation in reticulocyte lysates (Promega) with the human VDR cDNA inserted into the pGEM4 vector. The receptor was incubated with 10 nM 1,25-D or analogues for 10 min at room temperature. Then 0-25 µg/ml trypsin (Calbiochem) was added, and the mixture was further incubated for 10 min. The digestion products were analyzed by 12% SDS-polyacrylamide gel electrophoresis, and the gels were dried and autoradiographed.

Statistical Analysis

The results of the growth hormone assays were presented as mean ± standard error of the mean of 3-10 transfections.


RESULTS

Greater Activation of Transcription by 20-epi Analogues than by 1,25-D

The vitamin D analogues IE and ID have been shown by Binderup et al.(5) to possess antiproliferative responses 200-fold and 7,000 fold, respectively, greater than that of 1,25-D. Because this aspect of vitamin D action is considered a nuclear receptor-mediated event (6) , we tested whether these augmented effects on cell proliferation can be reflected in a simplified model to test a single transcriptional activity of VDR. We used the thymidine kinase promoter-growth hormone fusion genes containing either the osteocalcin or the osteopontin VDREs; both have DR3 motifs (13) . The difference between the two is that the osteocalcin element also contains an activator protein 1-binding site immediately upstream from the DR3 motif (14) . The fusion genes containing these VDREs were transfected into ROS 17/2.8 cells, and growth hormone reporter-protein production was measured after treatment with increasing concentrations of ligand. The cells were exposed to the ligands for 1 h in serum-free medium, to avoid involvement of DBP in the regulation of ligand uptake by the cells. Fig. 2 A shows that transcription of a reporter gene containing the osteocalcin VDRE reached 50% of maximal activity (ED) at 5 10 M. The ED for IE and ID were 10 M and 10 M, respectively. Similar results were obtained by transfection of the osteopontin VDRE-containing fusion gene (Fig. 2 B). We concluded that the 20-epi analogues increased VDR sensitivity to ligand (as was evident by the shift of the dose-response curve to the left) but did not increase the levels of transcription because maximal fold-induction of gene transcription was similar for all three ligands.


Figure 2: Transcriptional activity of 1,25-D and 20-epi analogues. ROS 17/2.8 cells were transfected by the DEAE-dextran method with a thymidine kinase-growth hormone ( TK/GH) fusion gene containing either the osteocalcin VDRE ( ocVDRE) ( A) or the osteopontin VDRE ( opVDRE) ( B). C, CV-1 cells were co transfected with the osteocalcin VDRE and a human VDR expression vector. Immediately after transfection (for ROS 17/2.8 cells) or 24 h later (for CV-1 cells), the ligands were added to serum-free culture medium for 1 h and then removed, the cells were washed twice with PBS, and DMEM with 10% fetal bovine serum was added. Forty-eight h after transfection, culture medium was collected and growth hormone levels were determined by radioimmunoassay. Each point of the dose-response curve is the average of duplicate transfections. The results shown are representative of four to six transfection experiments.



Because later assays of VDR activity were carried out with recombinant human VDR, we tested whether the sensitivity of the recombinant human VDR was also augmented by the 20-epi analogues. For that study, the VDR-negative CV-1 cells were cotransfected with the recombinant human VDR expression vector and the osteocalcin VDRE-growth hormone fusion gene. Fig. 2 C shows that IE and ID increased the sensitivity of the human VDR in a manner similar to their effect on the rat VDR in ROS 17/2.8 cells. We concluded from these experiments that the transcriptional activity of VDR through a DR3 VDRE motif was augmented by the 20-epi analogues. The ED for IE transcriptional activity was identical to its ED for antiproliferative activity. However, the ED for transcriptional activity of ID was 100-fold greater than its ED for anti-proliferative action. These results suggest that although the augmented antiproliferative action of the 20-epi analogues was mediated through enhanced activation of genes with a DR3 motif, an additional unknown mechanism may also be responsible for their augmented antiproliferative action.

Comparison of Relative Affinities of 1,25-D and 20-epi Analogues to VDR in Vitro and in Vivo

The augmented transcriptional activity of the 20-epi analogues was reflected by increased receptor sensitivity to the analogues rather than by an increase in levels of transcription. These results suggest that either the affinity of the 20-epi analogues for the receptor was greater than that of 1,25-D or that the affinity of the analogue-induced receptor for other components in the transcriptional machinery was greater than that of the 1,25-D-induced VDR. The original report on these compounds showed that their affinity to chick intestinal VDR was similar to that of 1,25-D(5) . However, we could not exclude the possibility that the binding properties of rat VDR from ROS 17/2.8 cells and those of the recombinant human VDR are different from those of the chick receptor. To test that possibility, we performed binding assays with homogenates from ROS 17/2.8 cells (Fig. 3 A) and from VDR-transfected COS-1 cells to determine their RCI values (Fig. 3 B). When we plotted the competition curves for these compounds, and calculated their RCIs (summarized in ), we found that for the rat VDR the relative affinities of IE and ID (RCI values of 43 ± 18 and 55 ± 26, respectively) were lower than that of 1,25-D (RCI = 100). Similar results were obtained for the human VDR (RCI values of 32 and 34 for IE and ID, respectively).


Figure 3: Relative affinities of 1,25-D and the 20-epi analogues to VDR in a cell-free system. Homogenates from ROS 17/2.8 cells ( A) and human VDR-transfected COS-1 cells ( B) were incubated for 3-4 h at 4 °C with 0.2 pmol of [H]1,25-D and increasing concentrations of unlabeled 1,25-D, IE, or ID. The free ligand was separated from bound by hydoxyapatite, and the amount of bound ligand was quantified by scintillation counting. The results were plotted as the reciprocal of binding activity versus the ratio of the amount of unlabeled competitor and radioactive ligand. The slopes of the linear plots were calculated to obtain the RCIs. Shown are representative plots of the two COS-1 and four ROS 17/2.8 binding assays.



We then considered the possibilities that the receptor-binding properties of intact cells are significantly different from the binding properties of cell homogenates and that the uptake of the 20-epi analogues, even in the absence of serum, is significantly higher than that of 1,25-D. We therefore attempted to determine the concentrations of the three ligands required to reach 50% saturation of VDR in vivo. We repeated the binding assays by incubating the cells with increasing concentrations of nonradioactive ligands for 1 h in serum-free medium and then measured the concentration of unoccupied receptor remaining in the cell homogenates. Fig. 4shows that in ROS 17/2.8 cells, the slope of the plots of binding activity of 20-epi analogue-treated cells was smaller than that of binding activity of 1,25-D-treated cells, indicating that either the uptake or the affinity (or both) of the 20-epi analogues to the VDR in vivo was lower than that of 1,25-D.


Figure 4: Relative affinities of 1,25-D and the 20-epi analogues to VDR in intact cells. ROS 17/2.8 cells were incubated in serum-free medium for 1 h with increasing concentrations of either 1,25-D or the 20-epi analogues. The cells were then washed twice with cold PBS and homogenized, and the number of remaining unoccupied binding sites was determined. The results were plotted as described in the legend to Fig. 3.



Similar results were obtained with the recombinant human VDR from transfected COS 1 cells (data not shown). The results of the in vivo and in vitro competition assays in ROS 17/2.8 cells are summarized in . We concluded that the augmented transcriptional activity of the 20-epi analogues under our experimental conditions cannot be attributed to higher affinity for the VDR or to more efficient delivery into the cells.

Ligand Effect on Binding of VDR to the Osteopontin VDRE

Because the augmented transcriptional activity of the 20-epi analogues was not due to increased affinity to the VDR or to a more efficient uptake mechanism, we tested whether the analogues facilitated binding of the VDR to the VDRE more than did 1,25-D. Two aspects of DNA binding activity were tested: affinity of the ligand-induced VDR to the osteopontin VDRE and dose-dependent binding of the ligand-induced VDR to the VDRE. For these assays VDR was overexpressed in COS-1 cells, exposed to ligand in the intact cells in serum-free medium for 1 h, and extracted from the nuclear fraction of the cells. The binding properties of the extracted VDR to the osteopontin VDRE were tested by EMSA. We used the osteopontin VDRE and not the osteocalcin VDRE because VDR binding to the former was more stable and was not accompanied by additional specific (activator protein 1) and nonspecific binding activities (14, 15) .

Fig. 5A shows the complexes formed after incubation of 1,25-D-treated extracts from human VDR-transfected cells with radiolabeled osteopontin VDRE. We found that a single complex was formed. The formation of this complex was blocked by a 50-fold excess of unlabeled osteopontin VDRE, and the complex contained VDR, as it was partially supershifted by anti-VDR antibodies. Next, we compared the affinities of 1,25-D-induced and 20-epi analogue-induced VDR to the osteopontin VDRE. Receptor preparations from cells treated with 10 M ligand were subjected to EMSA, with P-labeled osteopontin VDRE as a probe and increasing concentrations of unlabeled VDRE as a competitor. Fig. 5 B shows that the unlabeled VDRE inhibited the formation of the VDR complex with the labeled probe in a dose-dependent manner. The incubation of VDR in vivo with the 20-epi analogues did not increase its affinity to the VDRE relative to 1,25-D3-induced VDR; 50% competition was reached at similar concentration of competitor for 1,25-D-, IE-, and ID-induced VDR.


Figure 5: Affinity of ligand-activated VDR for opVDRE. A, nuclear extract from COS-1 cells transfected with human VDR expression vector and treated with 10 M 1,25-D was incubated without specific competitor ( none) or with a 50-fold excess of unlabeled specific competitor ( osteopontin VDRE). To test for presence of VDR in the complex, the nuclear extract was treated with rabbit anti-human VDR antibodies ( VDR) or with preimmune serum. The complexes were separated from the free probe by polyacrylamide gel electrophoresis and visualized by autoradiography. B, nuclear extracts from COS-1 cells transfected with hVDR and treated with 10 M 1,25-D, IE, or ID were incubated with or without increasing amounts of unlabeled osteopontin VDRE. The amount of competitor used is shown below the autoradiograph.



We then tested whether there were quantitative differences in the amount of VDR binding to VDRE in 1,25-D-induced and 20-epi analogue-induced preparations. VDR-transfected cells were treated for 1 h with increasing concentrations of ligand, and then nuclear extracts were prepared. The EMSAs were repeated with these receptor preparations and with osteopontin VDRE as a probe. We found that in the absence of ligand only a very small amount of complex formed (Fig. 6 A) and that including ligand induced a significant increase in VDR binding to DNA. The binding was dose dependent and reached 50% of maximal value at 10 to 10 M 1,25-D, at 10 to 10 M ID, and at 10 M IE. Fig. 6 B shows the relative intensity of the VDR bands from each treatment. In summary, we found that the analogues facilitated the DNA binding activity of VDR in a dose-dependent manner.


Figure 6: Dose-dependent effect of ligand on VDR binding to osteopontin VDRE. A, nuclear extracts from untreated and ligand-treated human VDR-transfected COS-1 cells were incubated with P-labeled osteopontin VDRE and separated by gel electrophoresis. The molar concentration of each ligand is indicated above the lanes. B, densitometric scanning of the VDRDNA complexes. The results are expressed as percentage of complex intensity in samples treated with 10 M ligand.



Effect of 20-epi Analogues on Dimerization of VDR with RXR

A prerequisite for binding of the VDR to a VDRE containing the DR3 motif is heterodimerization with RXR (13) . Because we found that both transcription and DNA binding to these elements were augmented by the analogues, we hypothesized that these compounds might also facilitate dimerization of VDR with RXR. To test this hypothesis, VDR preparations from cells treated with ligand for 1 h in serum-free medium were incubated with antibodies against RXR or control IgG and then incubated with the labeled VDRE and subjected to EMSA. The antibodies used (a generous gift from Dr. P. Chambon) recognize all three RXR species,() so the assay detected all possible VDRRXR complexes, simultaneously. Fig. 7 A shows that the antibodies induced a partial supershift of VDR complexes, whereas the control IgG did not have any effect on the complexes. When VDR was pretreated with 10 M ligand, there was no difference in the proportion of supershifted VDRRXR complexes for the three ligands. On the other hand, at ligand concentrations of 10 and 10 M, there was a significant difference in the proportion of supershifted 1,25-D-induced and analogue-induced VDRRXR complexes. To assess the difference between receptor preparations quantitatively, we densitometrically scanned the autoradiograms (Fig. 7 B) and found that the proportion of VDRRXR complexes supershifted by the anti-RXR antibodies was significantly higher in receptor preparations treated with 10 and 10 M IE and ID than in those treated with same concentrations of 1,25-D. These results suggested that there was more RXR associated with VDR activated by 20-epi analogues than with VDR activated by 1,25-D and that the analogues facilitated heterodimerization. Again, the differences were seen only when VDR was activated by suboptimal levels of 1,25-D. We think that differences were not seen at higher ligand concentrations because the amount of RXR in the extracts limited heterodimer formation.


Figure 7: Effect of ligand on VDRRXR complex formation. A, nuclear extracts from human VDR-transfected COS-1 cells treated with 10, 10, or 10 M 1,25-D, IE, or ID were incubated with [P]osteopontin VDRE and either control mouse IgG ( odd-numbered lanes) or with the anti-RXR antibodies ( even-numbered lanes). The complexes were separated by gel electrophoresis. The arrows indicate the positions of the VDRDNA or the VDR DNA complexes supershifted by the antibodies. B. densitometric scanning of the supershifted VDRDNA complexes. The results are expressed as percentage of the intensity of the respective unshifted VDR band.



Differential Effects of 20-epi Analogues and 1,25-D on VDR Conformation

The binding and uptake studies did not reveal a difference in receptor occupancy or affinity to the 20-epi analogues that correlated with their augmented transcriptional activity. In fact, the ligand-binding pattern was not correlated with the dimerization, DNA binding, or transcriptional activities of any of the compounds tested. We therefore hypothesize that it is the mode of interaction of the ligands, perhaps their contact sites with VDR, more than relative affinities of the ligands to the receptor that determines the efficacy of the activation process that leads to DNA binding and transcriptional activity. We reasoned that if there are differences in the mode of interaction they should be reflected by conformational changes in VDR.

To identify qualitative differences in the mode of interaction of the three ligands with VDR, we compared the conformational changes induced by the three ligands as reflected in the pattern of sensitivity to proteases. These assays were performed in vitro, with S-labeled synthetic VDR incubated with either 1,25-D or the 20-epi analogues, and then treated with increasing concentrations of trypsin. Fig. 8 A shows that in the absence of ligand, VDR was rapidly degraded, and only one short fragment ( fragment d, 22 kDa) was detectable after 10 min of incubation with the protease. Under the same incubation conditions, treatment of VDR with 1,25-D changed the proteolytic pattern of trypsin action: it produced an additional, slowly migrating fragment ( fragment a, 34 kDa) not seen in the absence of hormone. Treatment of VDR with ID (Fig. 8 B) generated an additional fragment ( fragment b, 30 kDa), whereas the treatment with IE (Fig. 8 B), generated both fragment b and an additional slow-migrating fragment ( fragment c, 32 kDa) not seen with 1,25-D-treated VDR. This experiment was repeated with chymotrypsin, with similar results (data not shown). We concluded from these experiments that binding of the 20-epi analogues to VDR induced conformational changes that exposed certain arginine or lysine residues that are not normally exposed on the 1,25-D-activated VDR. We speculate that these conformational modifications facilitate the action of VDR, beginning by enhancing dimerization with RXR.


Figure 8: Ligand-induced sensitivity of VDR to trypsin. In vitro translated VDR labeled with [S]methionine was incubated with or without 10 M 1,25-D ( A) or the 20-epi analogues IE and ID ( B), before digestion with the increasing concentrations of trypsin indicated above the lanes. The digestion products were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The proteolytic products are indicated by arrows. The fragments sizes are: a, 34 kDa; b, 32 kDa; c, 30 kDa; d, 22 kDa.




DISCUSSION

The biological actions of 1,25-D and its analogues are believed to be mediated through both genomic and non-genomic signal-transduction pathways (16, 17, 18, 19) . The extensive search for analogues of 1,25-D more suitable for clinical purposes has yielded a wealth of compounds that can be used as tools for studying these different aspects of the action of vitamin D (20, 21) .

We chose to investigate the mechanism of action of the 20-epi analogues because the antiproliferative action of these compounds is several orders of magnitude higher than that of 1,25-D. The antiproliferative effect of 1,25-D is thought to be primarily a genomic action and therefore believed to be regulated by the nuclear VDR (6) . The traditional concept of nuclear receptors activation by a ligand suggests that activation should be directly proportional to the ligand's affinity for the receptor. However, the action of the 20-epi analogues presented a paradox: the affinity of these compounds for VDR is similar or even lower than that of 1,25-D, but their antiproliferative action is several orders of magnitude higher (5) .

In the Introduction we addressed the possibility that the augmented activity of these analogues is due to their pharmacokinetic qualities (8) . While this is a valid consideration for whole-animal studies or for experiments that require the continuous presence of the vitamin in serum-enriched culture medium (such as the antiproliferative assay), we have focused on experimental systems in which pharmacokinetic qualities of the tested compounds could be separated from their actual biological activities as agonists of 1,25-D action and as ligands for VDR.

To separate the pharmacological and direct molecular effects of the analogues on VDR, we tested in vitro the sensitivity of the 20-epi analogue-induced VDR to proteases and compared it to the protease sensitivty of the 1,25-D-induced VDR. By using a similar approach for the analysis of ligand interaction with steroid receptors, it was possible to show differences in the pattern of protease sensitivity of agonist- and antagonist-treated receptors for progesterone, estrogen, and androgen (22, 23, 24) . Our results clearly indicate that each of the three ligands used in our experiments induced a unique pattern of sensitivity to trypsin. Therefore, although the apparent affinity of these compounds to VDR was not higher, it is possible that the conformational changes they induced facilitated receptor action. Our interpretation of these results is that the amino acids used by the 20-epi analogues as contact sites on VDR are somewhat different from the contact sites used by 1,25-D. Flexibility in ligand binding requirements that leads to differential activation of receptors by parental compounds and their analogues has been clearly demonstrated by deletion analysis of the progesterone receptor (25) . We are now in the process of mapping the ligand-binding domain of VDR and comparing the amino acids required for binding and function of 1,25-D and of the 20-epi analogues. Our preliminary results suggest that the binding requirements for these compounds are different from those of 1,25-D.

The earliest events in VDR activation by the ligand are dimerization with RXR (which was originally identified as nuclear accessory factor or NAF) (15) and the subsequent binding to VDRE. Under certain conditions it is possible to demonstrate ligand-dependent induction of heterodimerization and binding of VDR to DNA in vitro, for example, in recombinant receptor expressed in yeast cells and mixed with RXR-containing cell extracts (15) . On the other hand, the VDR-RXR heterodimer expressed in vitro binds strongly to VDRE, and its binding is not significantly increased by inclusion of ligand (13) . In our system, VDR and RXR expressed in mammalian cells had very poor binding activity to osteopontin VDRE in the absence of ligand. This binding activity was enhanced only by inclusion of ligand in the culture medium, not in a cell-free system. We therefore speculate that in mammalian cells a ligand-induced post-translational modification (possibly phosphorylation) must take place to efficiently facilitate formation of VDR complexes with RXR or binding of the VDRRXR complexes to the VDRE. Because this event was facilitated by the 20-epi analogues within 1 h of ligand administration in vivo under conditions of similar uptake and receptor saturation by 1,25-D and the analogues, we are convinced that the facilitated action of the 20-epi analogues on DNA binding and dimerization of VDR is not due to their pharmacological properties. We speculate that the conformational change induced by these compounds either directly facilitated dimerization of RXR with VDR or perhaps facilitated a post-translational event that increased the affinity of ligand-activated VDR for RXR.

Can the greater biological activities of the 20-epi analogues be explained only by the pharmacological qualities and molecular alterations induced by direct interaction with the receptor? One should not overlook the great discrepancy between the ED for transcription induction of DR3 motif-driven genes by ID (10 M) and its ED for antiproliferation and induction of differentiation (10 to 10 M) (5) . There are two possible explanations for this discrepancy. The first is that ID-induced VDR may be far more transcriptionally active due to the action of a motif different from DR3 and that genes with such motifs are important for the regulation of cell proliferation and differentiation. Examples of diversity in VDREs are the negative VDREs of the parathyroid hormone and the calcitonin genes. The parathyroid hormone gene is repressed by weak direct binding of VDR to DNA elements that are significantly different from DR3 (26) . The calcitonin gene is down-regulated by a mechanism that does not require direct interaction of the receptor with DNA (27) . Another possible mechanism for the discrepancy in ID ED is that the 20-epi analogues, similarly to 1,25-D (or even more so) may activate nuclear receptor-independent pathways (the so called non-genomic responses) that are also important for regulation of cell proliferation and differentiation. The biochemical and physiological events induced by 1,25-D and related compounds that do not require direct interaction with VDR include activation of calcium channels (19) , regulation of the phosphoinositol pathway and diacylglycerol formation (18, 28, 29) and down-regulation of alkaline phosphatase (30) . Any one of these events may contribute to regulation of cell growth and differentiation. Exploring these possibilities is essential for further evaluation of the mechanism of action of vitamin D analogues.

  
Table: Relative competition index (RCI) of the 20-epi analogues

The RCIs were calculated by plotting the inverse of the percent maximum binding [H]1,25-D 100 on the ordinate versus [competitor]/[H]1,25-D] on the abscissa, and dividing the slope of the line for analogue binding by the slope of the line for 1,25-D, and multiplying by 100. By definition, the RCI for 1,25-D is 100. To measure RCI in culture, increasing concentrations of ligand were added to cells in serum-free culture medium and incubated for 1 h. The cells were then washed twice with PBS, scraped off the plates, and homogenized. The homogenates were then incubated with a constant amount of [H]1,25-D with or without 100-fold excess of unlabeled 1,25-D. To measure RCI in vitro, cell homogenates were incubated with increasing amounts of ligand and a constant amount of [H]1,25-D. Three experiments were performed in culture and five in vitro.



FOOTNOTES

*
This study was supported by ARP Grant 15078 from the Texas Board of Higher Education (to S. P.), by National Institutes of Health Grant DK-09012 (to A. W. N.), and by Research Grant CA-16672 awarded by the National Cancer Institute, Department of Health and Human Services to the M. D. Anderson Cancer Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, The University of California, Riverside, CA 92521. Tel.: 909-787-4777; Fax: 909-787-4784.

The abbreviations used are: 1,25-D, 1,25-dihydroxyvitamin D; VDR, nuclear 1,25-dihydroxyvitamin D receptor; IE, 20-epi-1,25-dihydroxyvitamin D; ID, 20-epi-22-oxa-24a,26a,27a-tri-homo-1,25-dihydroxyvitamin D; DBP, vitamin D binding protein; RXR, retinoid X receptor; VDRE, vitamin D response element; DR, two direct repeats separated by three nucleotides; RCI, relative competition index; EMSA, electrophoretic mobility shift assay; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline.

P. Chambon, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. J. W. Pike for hVDR, human vitamin D receptor expression plasmid, and Dr. Pierre Chambon (LGME-U.184) for the generous gift of the RXR antibodies 1RX-6G12 and 4RX-1D12.


REFERENCES
  1. Koeffler, H. P., Amatruda, T., Ikekawa, N., Kobayashi, Y., and DeLuca, H. F. (1984) Cancer Res. 44, 5624-5628 [Abstract]
  2. Zhou, J.-Y., Norman, A. W., Akashi, M., Chen, D-L., Uskokovic, M. R., Aurrecoechea, J. M., Dauben, W. G., Okamura, W. H., and Koeffler, H. P. (1991) Blood 78, 75-82 [Abstract]
  3. Zhou, J.-Y., Norman, A. W., Lubbert, M., Collins, E. D., Uskokovic, M. R., and Koeffler, H. P. (1989) Blood 74, 82-93 [Abstract]
  4. Bouillon, R., Okamura, W. H., and Norman, A. W. (1995) Endocrine Rev. 16, 200-257 [Medline] [Order article via Infotrieve]
  5. Binderup, L., Latini, S., Binderup, E., Bretting, C., Calverley, M., and Hansen, K. (1991) Biochem. Pharmacol. 42, 1565-1575
  6. Haussler, M. R. (1986) Annu. Rev. Nutr. 6, 527-562 [CrossRef][Medline] [Order article via Infotrieve]
  7. Amento, E. P. (1987) Steroids 49, 55-72 [CrossRef][Medline] [Order article via Infotrieve]
  8. Dilworth, F. J., Calverley, M. J., Makin, H. L. J,. and Jones, G. (1994) Biochem. Pharmacol. 47, 987-993 [CrossRef][Medline] [Order article via Infotrieve]
  9. Noda, M., Vogel, R. L., Craig, A. M., Prahl, J., DeLuca, H. F., and Denhardt, D. T. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9995-9999 [Abstract]
  10. Ozono, K., Liao, S. A., Kerner, S. A., Scott, R. A., and Pike, J. W. (1990) J. Biol. Chem. 265, 21881-21888 [Abstract/Free Full Text]
  11. Peleg, S., Cote, G. J., Abruzzese, R. V., and Gagel, R. F. (1989) Henry Ford Hosp. Med. J. 37, 194-197 [Medline] [Order article via Infotrieve]
  12. Wecksler, W. R., and Norman, A. W. (1980) Methods Enzymol. 67, 488-497 [Medline] [Order article via Infotrieve]
  13. Yu, V. C., Delsert, C., Andersen, B., Holloway, J. M., Devary, O. V., Naar, A. M., Kim, S. Y., Boudin, J.-M., Glass, C. K., and Rosenfeld, M. G. (1991) Cell 67, 1251-1266 [Medline] [Order article via Infotrieve]
  14. Scule, R., Umensono, K., Mangelsdorf, D. J., Bolado, J., Pike, J. W., and Evans, R. M. (1990) Cell 61, 497-504 [Medline] [Order article via Infotrieve]
  15. Sone, T., Kerner, S., and Pike, J. W. (1991) J. Biol. Chem. 266, 23296-23305 [Abstract/Free Full Text]
  16. Pike, J. W. (1991) Annu. Rev. Nutr. 11, 189-216 [CrossRef][Medline] [Order article via Infotrieve]
  17. Nemere, I., Yoshimoto, Y., and Norman, A. W. (1984) Endocrinology 115, 1467-1483
  18. Lieberherr, M., Grosse, B., Duchambon, P., and Drueke, T. (1989) J. Biol. Chem. 264, 20403-20406 [Abstract/Free Full Text]
  19. Caffrey, J. M., and Farach-Carson, M. C. (1989) J. Biol. Chem. 264, 20265-20274 [Abstract/Free Full Text]
  20. Farach-Carson, M. C., Sergeev, I., and Norman, A. W. (1991) Endocrinology 129, 1878-1884
  21. Norman, A. W., Okamura, W. H., Farach-Carson, M. C., Allewaert, K., Branisteanu, D., Nemere, I., Muralidharan, K. R., and Bouillon, R. (1993) J. Biol. Chem. 268, 13811-13819 [Abstract/Free Full Text]
  22. Allan, G. F., Leng, X., Tsai, S. Y., Weigel, N. L., Edwards, D. P., Tsai, M.-J., and O'Malley, B. W. (1992) J. Biol. Chem. 267, 19513-19520 [Abstract/Free Full Text]
  23. Beekman, J. M., Alan, G. F., Tsai, S. Y., Tsai, M.-J., and O'Malley, B. W. (1993) Mol. Endocrinol. 7, 1266-1274 [Abstract]
  24. Kuil, C. W., and Mulder, E. (1994) Mol. Cell. Endocrinol. 102, R1-R5 [Medline] [Order article via Infotrieve]
  25. Vegeto, E., Allan, G. F., Schrader, W. T., Tsai, M.-J., McDonell, D. P., and O'Malley, B. W. (1992) Cell 69, 703-713 [Medline] [Order article via Infotrieve]
  26. Demay, M. B., Kiernan, M. S., DeLuca, H. F., and Kronenberg, H. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8097-8101 [Abstract]
  27. Peleg, S., Abruzzese, R. V., Cooper, C. W., and Gagel, R. F. (1993) Mol. Endocrinol. 7, 999-1008 [Abstract]
  28. Simboli-Campbell, M., Franko, D. J., and Welsh, J. E. (1992) Cell Signal. 4, 99-109 [Medline] [Order article via Infotrieve]
  29. Simboli-Campbell, M., Gagnon, A. M., Franks, D. J., and Welsh, J. E. (1993) J. Biol. Chem. 269, 3257-3264 [Abstract/Free Full Text]
  30. Schwartz, Z., Brooks, R., Swain, L., Del Toro, F., Norman, A. W., and Boyan, B. (1992) Endocrinology 130, 2495-2504 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.