Materials and methods

Compounds

1,25(OH)₂D₃ and its analogs MC903 (^{Carlberg et al, 1994}), GS1500 (^{Mathiasen et al, 1998}), and EB1213 (^{Mørk Hansen et al, 1996}) (for the structure of their side chains see Figure 1) were synthesized in the Department of Chemical Research at LEO Pharmaceutical Products (Ballerup, Denmark). Most characteristic are the cyclopropane ring in MC903 and the aromatic benzene ring in GS1500 and EB1213 combined with an altered stereochemistry at the C20 atom (20-epi). All VDR agonists were dissolved in 2-propanol; further dilutions were made in dimethyl sulfoxide (for in vitro experiments) or in ethanol (for cell culture experiments).

Figure 1.

Structure of 1,25(OH)₂D₃ and its analogs. The structure of the side chain is given.

Full figure and legend (19K)

DNA constructs and in vitro protein translation

The full-length cDNA for human VDR (^{Carlberg et al, 1993}) and human RXR (^{Levin et al, 1992}) were subcloned into the SV40 promoter-driven pSG5 expression vector (Stratagene, Heidelberg, Germany). In vitro translated VDR and RXR proteins were generated by transcribing their respective linearized pSG5-based cDNA expression vector with T₇ RNA polymerase and translating these RNA in vitro using rabbit reticulocyte lysate as recommended by the supplier (Promega, Mannheim, Germany). For mammalian-one-hybrid assays, the DBD of the yeast transcription factor GAL4 (amino acids 1–147) was fused with the cDNA of the human VDR LBD (amino acids 109–427). In reporter gene constructs the luciferase gene was driven either by three copies of the GAL4 binding site or four copies of the DR3-type VDRE of the rat ANF gene promoter (core sequence AGAGGTCATGAAGGACA) (^{Kahlen and Carlberg, 1996}).

Limited protease digestion assay

In vitro translated, [³⁵S]-labeled VDR protein alone or in combination with in vitro translated RXR and 1 ng of unlabeled rat ANF DR3-type VDRE were incubated with graded or saturating concentrations of ligand for 15 min at room temperature in 20 l binding buffer [10 mM HEPES, pH 7.9, 1 mM DTT, 0.2 g per l poly(dI-C) and 5% glycerol]. The buffer was adjusted to 150 mM of monovalent cations by addition of KCl. Trypsin (Promega, final concentration 8.3–16.6 ng per l) was then added and the mixtures were further incubated for 15 min (or indicated times, see Figure 4) at room temperature. The digestion reactions were stopped by adding 25 l protein gel loading buffer (0.25 M Tris, pH 6.8, 20% glycerol, 5% mercaptoethanol, 2% sodium dodecyl sulfate, 0.025% bromophenol blue). The samples were denatured at 85°C for 3 min and electrophoresed through a 15% sodium dodecyl sulfate–polyacrylamide gel. The gels were dried and exposed to a Fuji MP2040S imager screen. The individual protease-sensitive VDR fragments were quantitated on a Fuji FLA2000 reader (Tokyo, Japan) using Image Gauge software.

Figure 4.

Different half-lives of VDR-ligand conformations. Limited protease digestion assays were performed by preincubating in vitro translated [³⁵S]-labeled VDR together with nonlabeled in vitro translated RXR and the rat ANF DR3-type VDRE and saturating concentrations (10 M) of 1,25(OH)₂D₃, its analogs or solvent (as a control). The samples were incubated for indicated times with trypsin and were then electrophoresed through 15% sodium dodecyl sulfate–polyacrylamide gels. The amount of ligand-stabilized VDR conformations (sum of c1_LPD and c3_LPD) in relation to VDR input was quantitated by phosphorimaging. Representative experiments are shown for all four VDR agonists and solvent control. Data points represent the mean of triplicates and the bars indicate SD. The half-lives (t_1/2) were determined from the respective time-course curves.

Full figure and legend (93K)

Gel shift assay

In vitro translated VDR-RXR heterodimers were incubated with graded or saturating concentrations of ligands for 15 min at room temperature in a total volume of 20 l binding buffer. The buffer had been adjusted to 150 mM by addition of KCl. Approximately 1 ng of the [³²P]-labeled rat ANF DR3-type VDRE (50,000 cpm) was added to the protein–ligand mixture and incubation was continued for 20 min. Protein–DNA complexes were resolved through 8% nondenaturing polyacrylamide gels in 0.5  TBE (45 mM Tris, 45 mM boric acid, 1 mM ethylenediamine tetraacetic acid, pH 8.3) and were quantitated on a Fuji FLA2000 reader.

Transient transfections and reporter gene assay

HeLa human cervix carcinoma cells and HaCaT immortalized human keratinocytes were seeded on to six-well plates (10⁵ cells per ml) and grown overnight in phenol red-free Dulbecco's minimal Eagle's medium supplemented with 10% charcoal-treated fetal bovine serum. Liposomes were formed by incubating 1 g of a GAL4 binding site-driven luciferase reporter gene construct and 1 g of an expression vector for a GAL4_DBDVDR_LBD-fusion protein (for mammalian-one-hybrid assays in both cell lines) or 1 g of the rat ANF DR3-type VDRE-driven reporter plasmid and 1 g each of pSG5-based receptor expression vectors for VDR and RXR (for HaCaT cells) with 15–20 g N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP, Roth, Karlsruhe, Germany) for 15 min at room temperature in a total volume of 100 l. After dilution with 900 l phenol red-free Dulbecco's minimal Eagle's medium, the liposomes were added to the cells. Phenol red-free Dulbecco's minimal Eagle's medium supplemented with 30% charcoal-treated fetal bovine serum (500 l) was added 4 h after transfection. At this time, graded concentrations of VDR agonists were also added. The cells were lyzed 16 h after onset of stimulation using the reporter gene lysis buffer (Roche Diagnostics, Mannheim, Germany) for both types of assays and the constant light signal luciferase reporter gene assay was performed as recommended by the supplier (Canberra-Packard, Dreieich, Germany). The luciferase activities were normalized to protein concentration and induction factors were calculated as the ratio of luciferase activity of ligand-stimulated cells to that of solvent controls.

Results

Stabilization of VDR conformation

The interaction of 1,25(OH)₂D₃ and its analogs MC903, GS1500, and EB1213 (for structure of their side chain see Figure 1) with the VDR in solution (Figure 2a) or within VDR-RXR-VDRE complexes (Figure 2b) was analyzed in DNA-independent and DNA-dependent limited protease digestion assays, which were performed with in vitro translated, [³⁵S]-labeled VDR protein alone or in combination with in vitro translated unlabeled RXR and unlabeled rat ANF DR3-type VDRE, respectively. This assay displays a concentration-dependent stabilization of two VDR fragments, c1_LPD (28 kDa) and c3_LPD (23 kDa), that contain major parts of the LBD [from the trypsin cutting site after arginine 173 to either the carboxy-terminus at position 427 (c1_LPD) or to arginine 391 (c3_LPD)] and represent the agonistic and the nonagonistic conformation of the VDR-LBD (^{Herdick et al, 2000a;Herdick and Carlberg, 2000}), whereas a reasonable amount of the VDR fragment c2_LPD is observed only with VDR antagonists (^{Bury et al, 2000;Herdick et al, 2000b}). All four ligands showed the typical profile of VDR agonists, i.e., a stabilization of 60–80% of all VDR molecules in c1_LPD and only 10–20% in c3_LPD (^{Herdick et al, 2000a}). MC903 and EB1213 appeared to be indistinguishable from the natural hormone as they stabilized VDR in solution with an EC₅₀ value of 1.2–1.5 nM, which is approximately five times higher than the concentration needed for a stabilization of the VDR within VDR-RXR-VDRE complexes (EC₅₀ value of 0.2–0.3 nM). Interestingly, GS1500 showed the same sensitivity for the stabilization of the VDR within VDR-RXR-VDRE complexes than the three other VDR agonists (EC₅₀ value of 0.2 nM), but 50 times higher concentrations (EC₅₀ value of 10 nM) were needed to stabilize the VDR in solution.

Figure 2.

Stabilization of VDR conformations by 1,25(OH)₂D₃ and its analogs. Limited protease digestion assays were performed by preincubating in vitro translated [³⁵S]-labeled VDR alone (A) or in combination with nonlabeled in vitro translated RXR and the rat ANF DR3-type VDRE (B) with graded concentrations of 1,25(OH)₂D₃ or its analogs. After digestion with trypsin, samples were electrophoresed through 15% sodium dodecyl sulfate–polyacrylamide gels. The amount of ligand-stabilized VDR conformations 1 (c1_LPD, filled circles) and 3 (c3_LPD, open circles) in relation to VDR input was quantitated by phosphorimaging. Representative experiments are shown for 1,25(OH)₂D₃. Data points represent the mean of triplicates and the bars indicate SD. The EC₅₀ values for the stabilization of c1_LPD were determined from the respective dose–response curves.

Full figure and legend (52K)

Stabilization of VDR-RXR-VDRE complexes

In order to confirm the DNA-dependent sensitivity of the four VDR agonists, ligand-dependent gel shift assays were performed with in vitro translated VDR-RXR heterodimers bound to the rat ANF DR3-type VDRE and graded concentrations of 1,25(OH)₂D₃ and its three analogs (Figure 3). A comparable amount (approximately 20% shifted probe) of concentration-dependent VDR-RXR heterodimer complex formation on the VDRE was observed with all four compounds and provided EC₅₀ values between 0.095 and 0.4 nM. This ligand sensitivity is comparable with that observed in DNA-dependent limited protease digestion assays (Figure 2b). Moreover, this confirms that all three analogs show a sensitivity for the stabilization of VDR-RXR-VDRE complexes that is very close to that of 1,25(OH)₂D₃.

Figure 3.

1,25(OH)₂D₃ and its analogs stabilize VDR-RXR heterodimer complex formation on DNA. Gel shift experiments were performed with in vitro translated VDR-RXR heterodimers, which were preincubated at room temperature with graded concentrations of 1,25(OH)₂D₃ or its analogs and the [³²P]-labeled rat ANF DR3-type VDRE. Protein–DNA complexes were separated from free probe through 8% nondenaturing polyacrylamide gels. The amount of VDR-RXR-VDRE complexes in relation to free probe was quantitated by phosphorimaging. A representative experiment is shown for 1,25(OH)₂D₃. Data points represent the mean of triplicates and the bars indicate SD. The EC₅₀ values for VDR-RXR-VDRE complex formation were determined from the respective dose–response curves.

Full figure and legend (79K)

The kinetics of VDR-ligand dissociation within VDR-RXR-VDRE complexes was analyzed by DNA-dependent limited protease digestion assays, which were performed with in vitro translated VDR-RXR heterodimers bound to the rat ANF DR3-type VDRE and saturating concentrations of 1,25(OH)₂D₃ and the three analogs and solvent as a control (Figure 4). The incubation time with trypsin varied between 15 min and 24 h. It is important to note that trypsin was found to be still active even after 24 h of incubation (data not shown). The amount of ligand-stabilized VDR was quantitated and plotted over time, which allowed a determination of the half-life (t_1/2) of the VDR–ligand complex. Interestingly, the three analogs showed clearly different t_1/2 values, which were found to be lower (MC903, t_1/2 = 438 min) and higher (GS1500, t_1/2 = 1175 min and EB1213, t_1/2 = 2717 min) than that of the natural hormone (t_1/2 = 660 min). If only the stabilization of c1_LPD is determined, the respective t_1/2 values are 472, 260, 1260, and 3375 min for 1,25(OH)₂D₃, MC903, GS1500, and EB1213, respectively, i.e., no differences in the ranking of the VDR ligands.

Functional activity of VDR agonists in HeLa and HaCaT cells

In order to test the functionality of 1,25(OH)₂D₃ and the three selected analogs, classical mammalian-one-hybrid assays were performed in HeLa human cervix carcinoma cells that were transiently transfected with an expression vector for a fusion protein containing the DBD of the yeast transcription factor GAL4 and the LBD of the VDR together with a reporter gene construct containing a GAL4 binding site-driven luciferase gene (Figure 5). The stimulation of the cells with graded ligand concentrations provided a 25–35-fold induction of reporter gene activity with EC₅₀ values of 1.0 nM for 1,25(OH)₂D₃, 0.1 nM for MC903, 0.11 nM for GS1500, and 0.14 nM for EB1213. This indicates that the in vitro sensitivity of the three analogs for the stabilization of VDR-RXR-VDRE complexes (Figure 2 and Figure 3) translates well to their sensitivity in HeLa cells. In contrast, the natural hormone appears to be approximately 10 times less sensitive in living cells than in vitro.

Figure 5.

Agonistic effects of 1,25(OH)₂D₃ and its analogs in vivo. Luciferase reporter gene assays were performed with extracts from HeLa cells that were transiently transfected with a reporter gene construct-driven by three copies of the GAL4 binding site and an expression vector for a GAL4_DBDVDR_LBD fusion protein (as schematically depicted above). The cells were treated for 16 h with graded concentrations of 1,25(OH)₂D₃ or its analogs. Stimulation of luciferase activity was calculated in comparison with solvent-induced controls. Data points represent the mean of triplicates and the bars indicate SD. The EC₅₀ values for stimulation of VDR-driven gene activity were determined from the respective dose–response curves.

Full figure and legend (32K)

The functional analysis of 1,25(OH)₂D₃ and its analogs was extended to HaCaT immortalized human keratinocytes as a representative 1,25(OH)₂D₃ target tissue. Mammalian-one-hybrid assays (Figure 6a) as well as traditional reporter gene assays (Figure 6b), that used overexpressed VDR and RXR proteins and a DR3-type VDRE-driven luciferase gene, were performed in this cell line. Interestingly, the mammalian-one-hybrid assay (Figure 6a) provided for all four ligands more than 100-fold induction of gene activity, whereas the traditional assay (Figure 6b) only showed an inducibility of 25-fold. Both types of reporter gene assays provided for GS1500 and EB1213 EC₅₀ values of 0.2–0.31 nM, i.e., values that are very comparable with that obtained in HeLa cells (Figure 5). This demonstrates that mammalian-one-hybrid assays are as sensitive as traditional reporter gene assays. Moreover, the EC₅₀ values that were obtained in both assays for 1,25(OH)₂D₃ (1.1 and 2.8 nM) are in accordance with the results from HeLa cells (1.0 nM, see Figure 5). In contrast, in HaCaT cells the sensitivity of MC903 for activation of gene activity (EC₅₀ values of 1.0 and 2.1 nM) was found to be 10–21-fold lower than in HeLa cells (Figure 5).

Figure 6.

Agonistic effects of 1,25(OH)₂D₃ and its analogs in HaCaT cells. Luciferase reporter gene assays were performed with extracts from HaCaT cells that were transiently transfected with a reporter gene construct-driven by three copies of the GAL4 binding site and an expression vector for a GAL4_DBDVDR_LBD fusion protein (A) or with a luciferase reporter gene construct driven by four copies of the rat ANF DR3-type VDRE together with the expression vectors for VDR and RXR (B) as schematically depicted above the respective figures. The cells were treated for 16 h with graded concentrations of 1,25(OH)₂D₃ or its analogs. Stimulation of luciferase activity was calculated in comparison with solvent-induced controls. Data points represent the mean of triplicates and the bars indicate SD. The EC₅₀ values for stimulation of VDR-driven gene activity were determined from the respective dose–response curves.

Full figure and legend (38K)

Discussion

The nuclear receptor superfamily contains a series of transcription factors that are of high impact because they can be specifically regulated in their activity by small lipophilic compounds of natural or synthetic origin. The protein–DNA complex of nuclear receptor and its specific response element can be considered as a molecular switch for those genes that contain such a response element in their promoter region. The VDR appears to be an ideal member of the nuclear receptor superfamily, as apart from hypercalcemia no significant side-effects of its specific natural ligand, 1,25(OH)₂D₃, are known. This makes prevention of hypercalcemia a primary target for the development of therapeutically important VDR agonists. In this study, the molecular action of the natural hormone was compared with that of three analogs that had been identified by biologic screenings to be low calcemic. Interestingly, on the level of the activation of RXR- and DNA-complexed VDR, i.e., on the level of the molecular switch, the sensitivity of all three analogs (EC₅₀ values of approximately 0.2 nM) was found to be not significantly different to that of 1,25(OH)₂D₃ (Table I). This identity was observed by two different methods, DNA-dependent limited protease digestion assays and ligand-dependent gel shift assays that appear to be very accurate tools for an in vitro evaluation of nuclear receptor–DNA–ligand interactions. Similar results have been obtained recently with other potent (but higher calcemic) 1,25(OH)₂D₃ analogs, such as 20-epi-1,25(OH)₂D₃, 20-methyl-1,25(OH)₂D₃ and an analog with two side chains, referred to as Gemini (^{Herdick et al, 2000a}). In that report, in addition to the above-mentioned methods, supershifts with coactivator proteins and gel shift clipping assays were used, but in all cases the EC₅₀ value for the activation of the VDR by any of these ligands was found to be approximately 0.1 nM (^{Herdick et al, 2000a}). This indicates that there appears to be a threshold of VDR activation of 0.1–0.2 nM that even optimized synthetic VDR agonists may not be able to exceed. This means that there is probably no synthetic VDR agonists having an affinity for the VDR that is significantly higher than that of the natural hormone. This observation is supported by traditional ligand binding assays (^{Binderup et al, 1994;Bouillon et al, 1995;van den Bemd et al, 1995}).

Table I - Comparison of the four tested VDR ligands.

Full table

Interestingly, the 1,25(OH)₂D₃ analogs GS1500 and EB1213 showed the same EC₅₀ values in the functional assays in HeLa and HaCaT cells as in the in vitro assays (approximately 0.2 nM). This indicates that the in vitro assays represent well the ligand sensitivity of the VDR in living cells and in turn suggests that both analogs are not significantly metabolized or absorbed by cellular or serum proteins. This conclusion appears to hold true for MC903 in HeLa cells, but not in HaCaT cells, where the EC₅₀ value was found to be 22-fold higher. The reason for this difference is yet unknown and apparently not related to a fast metabolism of MC903 in HaCaT cells, in which the analog was shown to be rather stable (Løgsted Nielsen and Kissmeyer, unpublished results). In contrast, 1,25(OH)₂D₃ showed higher EC₅₀ values in the functional assays in HeLa and HaCaT cells compared with the in vitro assays. The functional assays are performed in the presence of serum and thereby also vitamin D binding protein. Therefore, it is likely that the binding of 1,25(OH)₂D₃ to vitamin D binding protein has suppressed the free entry 1,25(OH)₂D₃ into the cells and thereby increased the EC₅₀ value (^{Vanham et al, 1988}). As most 1,25(OH)₂D₃ analogs have a reduced affinity for vitamin D binding protein compared with the natural hormone (^{Kissmeyer et al, 1995}) the same difference in activity between functional and in vitro assays is, as observed, not expected.

On first glance, the functionality of MC903, GS1500, and EB1213 in vitro and in HeLa cells appears to be identical (Table I); however, in proliferation assays in HaCaT cells GS1500 and EB1213 are known to be more potent than MC903 and 1,25(OH)₂D₃ (^{Mørk Hansen et al, 1996}). This may be due to important differences in stabilizing either the VDR in solution or VDR-RXR-VDRE complexes over time. Compared with the three other tested VDR agonists, GS1500 showed a clear preference for DNA-bound VDR-RXR complexes (Table I). A selectivity for DNA-bound VDR-RXR heterodimers means that DNA-independent actions of the VDR, such as the inhibition of IL-2 gene expression via the prevention of DNA binding of the transcription factor NF-AT (^{Alroy et al, 1995}), will not be stimulated (or only at clearly higher concentrations) by the respective ligand. Moreover, GS1500 stabilized DNA-bound VDR-RXR complexes nearly twice as long as 1,25(OH)₂D₃, whereas MC903 kept the complex stable only for a shorter time period than the natural hormone; however, EB1213 appears to be most potent as it stabilizes VDR-RXR-VDRE complexes more than four times longer than 1,25(OH)₂D₃. An increased ligand-dependent stabilization of VDR-RXR-VDRE complexes would then result in longer-lasting effects of the respective ligand on gene activation.

In conclusion, the molecular evaluation of the analogs MC903, GS1500, and EB1213 in comparison with the natural hormone has indicated that they are all potent VDR agonists that show a very similar sensitivity in stabilizing the molecular switches of nuclear 1,25(OH)₂D₃ signaling, i.e., DNA-bound VDR-RXR heterodimers. Individual properties of the compounds, however, could also be identified, of which the skin cell-specific reduced sensitivity of MC903, the DNA selectivity of GS1500 and the long-lasting stabilization of VDR-RXR-VDRE complexes by EB1213 are the most remarkable.

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Acknowledgments

This work was supported by the Sonderforschungsbereich 503, project A6, the Medical faculty of the Heinrich-Heine-Universität, DFG-grant Ca229/1, the Fonds der Chemischen Industrie and the Wilhelm Sander Foundation (all to C.C.).