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Molecular Endocrinology, doi:10.1210/me.2003-0072
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Molecular Endocrinology 17 (10): 2028-2038
Copyright © 2003 by The Endocrine Society

Corepressor Excess Shifts the Two-Side Chain Vitamin D Analog Gemini from an Agonist to an Inverse Agonist of the Vitamin D Receptor

Manuel Macias Gonzalez, Petra Samenfeld, Mikael Peräkylä and Carsten Carlberg

Departments of Biochemistry (M.M.G., P.S., C.C.) and Chemistry (M.P.), University of Kuopio, FIN-70211 Kuopio, Finland

Address all correspondence and requests for reprints to: Professor Carsten Carlberg, Department of Biochemistry, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. E-mail: carlberg{at}messi.uku.fi.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS AND DISCUSSION
 Conclusion
 MATERIALS AND METHODS
 REFERENCES
 
The vitamin D receptor (VDR) is an endocrine nuclear receptor that binds with high affinity its natural ligand 1{alpha},25-dihydroxyvitamin D3. Gemini is a 1{alpha},25-dihydroxyvitamin D3 analog with two identical side chains that, despite its significantly increased volume, binds to the VDR and can function as a potent agonist. This study demonstrates that, at excess corepressor (CoR) levels, Gemini shifts from an agonist to an inverse agonist that actively recruits CoR proteins to the VDR and mediates superrepression. Under these conditions Gemini stabilizes the VDR into a silent conformation, in which helix 12 of the ligand-binding domain is repositioned and thus unable to contribute to coactivator interaction. Amino acid F422 has been described as the lock of helix 12 and seems to be the most critical VDR residue in the inverse agonistic action of Gemini. Molecular dynamics simulations of the Gemini-VDR complex support these observation by indicating that the second side chain of Gemini induces tension to the receptor structure that can be released by a shift of helix 12. Taken together, Gemini is the first described (conditional) inverse agonist to an endocrine nuclear receptor and may function as a sensor for the cell-specific coactivator/CoR ratio.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS AND DISCUSSION
 Conclusion
 MATERIALS AND METHODS
 REFERENCES
 
THE NUCLEAR RECEPTOR (NR) for the biologically active form of vitamin D, 1{alpha},25-dihydroxyvitamin D3 [1{alpha},25(OH)2D3], the vitamin D receptor (VDR), is one of the 11 endocrine members of the NR superfamily, i.e. it binds its ligand with high affinity [dissociation constant (Kd) value of 1 nM or lower (1)]. 1{alpha},25(OH)2D3 plays an important role in calcium resorption and bone formation (2) and was also shown to act as a regulator of cellular proliferation, differentiation, and apoptosis (3). Like all NRs, VDR has a highly conserved DNA-binding domain and a less conserved ligand-binding domain (LBD). The LBD is formed by 12 {alpha}-helices, and its overall architecture is similar for all NRs (4). VDR acts preferentially as a heterodimer with the retinoid X receptor (RXR) on specific DNA sequences in promoter regions of 1{alpha},25(OH)2D3 target genes, referred to as 1{alpha},25(OH)2D3 response elements (VDREs) (5). The VDR-RXR-VDRE complex is the core of DNA-dependent 1{alpha},25(OH)2D3 signaling (6), and the stabilization of the agonistic conformation of the LBD of the VDR via the repositioning of the most carboxy-terminal {alpha}-helix (helix 12) is the crucial step in this signaling process. In detail, this is achieved by a hydrogen bond between the C25-hydroxyl group of 1{alpha},25(OH)2D3 and H397 of the receptor (7) and is supported by an additional, less important, hydrogen bond with H305 (8). In the presence of agonist, H397 is able to form van der Waals contacts with F422 of helix 12. This keeps helix 12 in a position that is optimal for the charge clamp between E420 (helix 12) and K246 (helix 3), which allows the LXXLL NR interaction domain of coactivator (CoA) proteins into a hydrophobic cleft on the surface of the LBD (9). CoA proteins, in turn, contact and recruit other components of the basal transcriptional machinery, the result of which is enhanced transcription of 1{alpha},25(OH)2D3 target genes (10).

Nearly all NRs are known to interact with CoA proteins, but it is unclear, whether all form complexes with corepressor (CoR) proteins, such as NR corepressor (NCoR), Alien, and others (11). Classical CoRs act as a specific bridge between transcription factors and histone deacetylases (HDACs), which are enzymes that locally close chromatin (12). It is thought that NRs interact with CoRs only in the absence of ligand, i.e. in their apo-form. Agonistic ligand binding to the LBD of the VDR initiates the dissociation of CoRs. VDR has been shown to interact with the CoR proteins NCoR, Alien, and Pit-1 (13, 14). This provides VDR with intrinsic repressing activity in the absence of ligand. It is thought that nonliganded VDR-RXR heterodimers actively repress 1{alpha},25(OH)2D3 target genes by binding to their respective VDREs.

Humans have 37 orphan NR superfamily members and, for some of them in past years, low-affinity ligands have been identified (Kd value of 1 µM or higher) (15). One of these adopted orphan NRs is the constitutive androstane receptor (CAR), which is an unusual member of the family, because it interacts in the absence of ligand with CoAs and displays constitutive activity (16). Furthermore, the natural CAR ligand androstanol acts as an inverse agonist by decreasing CoA interaction (17) and increasing CAR-CoR complex formation (13). This opens the question of whether inverse agonists exist only for constitutively active NRs or whether inverse agonists can be developed for other ligand-activated members of the NR superfamily, such as VDR.

More than 2000 analogs of 1{alpha},25(OH)2D3 have been synthesized in an effort to improve the potency and specificity of the physiological effects of vitamin D (18). The large majority of these analogs have been modified at their side chain, which in most cases increases their metabolic stability and the half-life of the VDR-ligand complex (19, 20). Superagonists carry only minor modifications compared with the natural hormone and stabilize the same agonistic VDR conformation via the H397-F422 interaction (21). An exception is Gemini, which is the first 1{alpha},25(OH)2D3 analog that carries two side chains (22, 23). This analog has approximately 25% higher volume than the natural hormone. Gemini was shown to act as a potent agonist, and in the presence of CoAs, RXR, and DNA the ligand is able to bind the VDR in its agonistic (active) conformation (22, 24). Molecular dynamics (MD) simulations of the Gemini-VDR complex demonstrated that one of the two side chains of Gemini has the same location as in the natural hormone, whereas for the second side chain two approximately equal positions were identified (25). Receptor mutagenesis, CoA interaction studies in vitro, and functional assays in living cells confirmed that Gemini uses both possible positions. However, Gemini has also been shown to stabilize a silent (nonagonistic) conformation of the VDR, in which the receptor favors CoR interaction to complex formation with CoAs (24, 26). This study attempts to answer the question, whether Gemini can also act as an inverse agonist to the VDR. In functional assays it could be demonstrated that CoR excess shifts Gemini from an agonist to an inverse agonist. Amino acid F422 was shown to have a critical role in this process as confirmed by in vitro interaction studies with NCoR and conformational analysis by limited protease digestion. MD simulations indicated that the second side chain of Gemini creates tension within the LBD of VDR, which in excess of CoR proteins can be released by shifting helix 12 into an inverse agonistic position. Gemini therefore seems to be a conditional inverse agonist of the VDR.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
Conclusion
 MATERIALS AND METHODS
 REFERENCES
 
The VDRE of the human osteocalcin gene promoter is composed of three NR binding sites forming direct repeats of six and three spacing nucleotides (27). In addition, it is overlaid by a binding site for the transcription factor AP-1 (28) (see Fig. 1Go for sequence and schematic structure). Binding of AP-1 provides the VDRE with a basal activity in the MCF-7 human breast cancer model cell line that is significantly higher than that of other known VDREs (29). Therefore, this VDRE is suited for monitoring studies in which both activating and repressing activities of the VDR could be measured (14). Treatment with the HDAC inhibitor trichostatin A (TSA) allows monitoring the derepressed state of the VDRE, i.e. its basal activity at maximally deacetylated chromatin.



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  		Fig. 1. Agonistic and Inverse Agonistic Action of Gemini

Luciferase reporter gene assays were performed with extracts from MCF-7 cells that were transiently transfected with a reporter gene construct driven by one copy of the complex VDRE of the human osteocalcin gene promoter (A–C) or four copies of the DR3-type VDRE of the rat ANF gene promoter (D) and the indicated expression vectors for wild-type or mutant VDR and RXR and full-length NCoR proteins. The cells were treated for 16 h with ethanol (0.1%), TSA, 1{alpha},25(OH)2D3, or Gemini (each 100 nM). Relative luciferase activity was calculated in comparison to solvent-induced controls and normalized to the basal activity of VDRwt-RXRwt heterodimers. Columns represent the mean of at least three experiments and the tips of the bars indicate SD. Statistical analysis was performed by two-tailed, paired Student’s t test, and P values were calculated in reference to basal activity of VDRwt-RXRwt heterodimers (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

 
In this study, reporter gene assays were performed from extracts of MCF-7 cells being transiently transfected with a luciferase reporter gene construct under the control of one copy of the human osteocalcin VDRE fused with the thymidine kinase (tk) promoter (Fig. 1Go, A–C). The activity level of the derepressed state of the VDRE appeared to be independent from overexpression of VDR, RXR, or NCoR in the cells and therefore served as an internal reference (compare lanes 2, 5, 8, 11, 14, 17, 20, and 23 in Fig. 1AGo and lanes 2 and 4 in Fig. 1CGo). The first series of assays (Fig. 1AGo) studied the influence of the overexpressing of VDR, RXR, and NCoR on VDRE-driven reporter gene activity. In reference to the derepressed state (lane 2), endogenous NRs repressed the basal activity by 26% (lane 1). A stimulation with 1{alpha},25(OH)2D3 resulted in a 2.3-fold increase (lane 3) of this basal activity. Overexpression of NCoR effected further 8% reduction of the basal activity (34%, lane 4) and slightly attenuated activation by 1{alpha},25(OH)2D3 (2.1-fold, lane 6). The overexpression of wild-type VDR (VDRwt) had no effect on the ligand-activated state of the VDRE (lanes 9 and 12), but reduced the basal activity by 58% (lane 7). Interestingly, the cotransfection of NCoR enhanced repression to 24% of reference basal activity (lane 10). Overexpressed wild-type RXR (RXRwt) alone did not affect the ligand-stimulated state of the VDRE (lanes 15 and 18), but nearly completely abolished repression of the basal activity (lanes 13 and 16) while having no effect on the ligand-stimulated state of the VDRE (lanes 15 and 18). The combined overexpression of VDRwt and RXRwt did not influence the ligand-stimulated state (lanes 21 and 24), but resulted in an approximately 2-fold repression of basal activity (lanes 19 and 22). Interestingly, when both VDRwt and RXRwt are overexpressed, the cotransfection of NCoR induced no superrepression (compare lanes 22 and 10). In summary, these data indicate that there is minor repression of the model VDRE via endogenously expressed VDR that can be increased by overexpression of VDRwt. In contrast, RXRwt alone has no repressive effects but blocks the repression by endogenous and overexpressed VDR. Interestingly, NCoR induces superrepression only when VDR exceeds the amount of RXR.

In the next series of reporter gene assays (Fig. 1BGo) the impact of helix 12 of both RXR and VDR on the activity of the osteocalcin VDRE was studied. When both VDRwt and RXRwt are overexpressed simultaneously, 1{alpha},25(OH)2D3 and Gemini provided a 4- and 4.6-fold induction of basal activity, respectively (compare lanes 2 and 3 with lane 1). The overexpression of NCoR did not affect significantly the basal activity (lane 4) or the induction by the natural ligand (4.5-fold, lane 5), but clearly reduced the inducibility by Gemini (2.7-fold, lane 6). The deletion of helix 12 of VDR (VDR{Delta}H12) in the presence of RXRwt did not affect the basal activity (lane 7), but blunted the induction by 1{alpha},25(OH)2D3 (1.2-fold, lane 8) and reduced the stimulation by Gemini (1.8-fold, lane 9). The overexpression of NCoR slightly reduced the basal activity (lane 10), rendered the natural hormone ineffective (1.1-fold induction, lane 11), and turned Gemini from an agonist (lane 9) to an inverse agonist (2.0-fold reduction, lane 12). The deletion of helix 12 of RXR (RXR{Delta}H12) in the presence of VDRwt reduced the basal activity of the VDRE (56% of reference, lane 13) and therefore increased the observed inducibility by 1{alpha},25(OH)2D3 (6.1-fold, lane 14) and by Gemini (7.0-fold, lane 15). The overexpression of NCoR led to superrepression of the basal activity (24% of reference, lane 16), kept the inducibility by the natural hormone on the same high level (6.2-fold, lane 17), but reduced the induction by Gemini to 5.1-fold (lane 18). The combined overexpression of both deletion mutants (VDR{Delta}H12 and RXR{Delta}H12) kept the basal activity low (36% of reference, lane 19), the induction by 1{alpha},25(OH)2D3 blunted (1.1-fold, lane 20), and attenuated the stimulation by Gemini (1.9-fold, lane 21). The overexpression of NCoR reduced the basal activity even more (20% of reference, lane 22), kept the natural hormone silent (1.1-fold induction, lane 23), and again reversed Gemini from a (weak) agonist to an inverse agonist (1.6-fold reduction, lane 24). Taken together, at standard conditions Gemini was at least as potent a VDR agonist as 1{alpha},25(OH)2D3, but in excess of CoR it has a clearly reduced agonistic potential. Confirming previous reports (30, 31), helix 12 of VDR proved to be essential for the agonistic action of the natural hormone. For the agonism of Gemini, helix 12 is also important but not as critical as for that of 1{alpha},25(OH)2D3. Remarkably, the lack of VDR’s helix 12 combined with a CoR excess allows one to detect the shift of Gemini from agonism to an inverse agonism. In contrast, helix 12 of RXR appears to inhibit the repression potential of VDR and explains why the overexpression of RXRwt increased the basal activity of the model VDRE (see Fig. 1AGo). Similar observations were already reported for other NRs, such as the retinoid acid receptor (32) and the thyroid hormone receptor (33). As compared with these reports it can be assumed that helix 12 of RXR reduces the interaction potential of VDR-RXR heterodimers with NCoR.

Earlier reports on VDR (30, 31) and comparisons with other members of the NR superfamily, such as CAR (17), have indicated that two amino acid of helix 12 are most critical for the agonist action on the NR. The first is the highly conserved glutamate residue (E420 in VDR) that is part of the charge clamp for CoA interaction. The second is a hydrophobic amino acid, which is found in most NRs as the second carboxy-terminal amino acid to the glutamate (F422 in VDR). This hydrophobic amino acid serves as a ligand-triggered lock of helix 12 and stabilizes the helix in a position that is optimal for the charge clamp-controlled CoA interaction (see also Fig. 4AGo). The reporter gene assays in Fig. 1CGo assessed the contribution of these two amino acids to the different functional profiles of 1{alpha},25(OH)2D3 and Gemini. The basal activities mediated by VDRwt-RXRwt and VDR{Delta}H12-RXRwt heterodimers (lanes 1 and 3) were found to be nearly identical and approximately 2-fold repressed in reference to the derepressed state of the VDRE (lanes 2 and 4). The functional profile of VDR{Delta}H12-RXRwt heterodimers was found to be identical to that described in the previous experimental series (compare lanes 3 and 5–9 in Fig. 1CGo with lanes 13–18 in Fig. 1BGo). The shift of Gemini from agonist to inverse agonist could not be observed with VDRE420A-RXRwt heterodimers (compare lanes 12 and 15). Moreover, the overexpression of NCoR had no significant effect (compare lanes 10–12 with lanes 13–15). In contrast, the functional profile of VDRF422A-RXRwt heterodimers (lanes 16–21) was identical to that of VDR{Delta}H12-RXRwt heterodimers (lanes 3 and 5–9). This indicates that the amino acid F422 is critical for the helix 12-dependent profile of Gemini, i.e. its ability to shift from agonist to inverse agonist.



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  		Fig. 4. MD Simulations of Gemini-VDR-LBD Complexes

A, View of the whole VDR-LBD complexed with Gemini in two alternative positions after MD simulations (25 ). The {alpha}-helices are represented by ribbons of C{alpha}-atoms, and the side chains of only the four most critical amino acids, K246, H397, E420, and F422, are shown. In both positions the location of one side chain of Gemini is identical to that of the single side chain of 1{alpha},25(OH)2D3 (7 ), whereas the location of the extra side chain in position 1 is shown in blue and of that in position 2 in green. The dihedral angles of C16-C17-C20-C21 of Gemini in position 1 are -29.5° for the first side chain and 102.7° for the extra side chain and in position 2 -69.8° and 155.6°, respectively (for 1{alpha},25(OH)2D3 it is -32.6°). B, Distance between the C{alpha}-atoms of K246 and E420 in the complexes between the LBD and 1{alpha},25(OH)2D3 or Gemini in VDRwt during 2-nsec MD simulations at 340 K. C, The same distance in VDRF422A during 5-nsec MD simulations at 340 K.

 
NCoR is known to inhibit in combination with HDAC3 the Jun kinase pathway (34), so that the AP-1 site overlaying the osteocalcin VDRE (Fig. 1AGo) may contribute to the effects observed in Fig. 1Go, B and C. We therefore performed reporter gene assays on four copies of the DR3-type VDRE of the rat atrial natriuretic factor (ANF) gene, which does not contain an overlaid AP-1 site (Fig. 1DGo). When using VDRwt, TSA activated this VDRE multimer by a factor of 4.3 (lane 2), which is rather low compared with approximately 40-fold stimulation by 1{alpha},25(OH)2D3 and Gemini (lanes 3 and 5). TSA and the two VDR ligands showed no synergistic or additive effects (lanes 4 and 6). The overexpression of NCoR caused a slight reduction of basal activity (lane 7) and of inducibility with 1{alpha},25(OH)2D3 (32.1-fold, lane 9). Comparable with the observation of the osteocalcin VDRE (Fig. 1BGo, lane 6), stimulation with Gemini was significantly reduced in the presence of NCoR (lane 11). Interestingly, this reduction was reversed by a costimulation with TSA (lane 12). This could be explained by the assumption that some VDR molecules are stabilized by Gemini in a conformation that recruits NCoR and mediates repression via HDACs, whereas the remaining VDR subpopulation is responsible for the reduced activation. TSA inhibits the Gemini-induced repression and full activity is restored. When using VDRF422A, the stimulation by 1{alpha},25(OH)2D3 is drastically reduced (2.3-fold, lane 15) and that of Gemini is moderately reduced (8.1-fold, lane 17). Compared with VDRwt (lane 2), the stimulation by TSA alone (lane 14) is not significantly affected, but again in combination with the two VDR ligands no synergistic or additional effect was observed (lanes 16 and 18). In confirmation of the effects that were observed on the osteocalcin VDRE (Fig. 1CGo, lane 21), the overexpression of NCoR turned Gemini from a weak agonist (lane 17) to an inverse agonist (lane 23). This inverse agonistic effect was partially reversed by cotreatment with TSA (lane 24). In conclusion, on both VDRE types the same effects were observed. Therefore, the possibility of a contribution of AP-1 to the inverse agonistic action of Gemini is unlikely. TSA is able to reverse the conditional inverse agonism of Gemini, which suggests that HDACs are involved in this phenomenon.

Next we used glutathione S-transferase (GST)-pull-down assays to compare the ligand-triggered interaction of NCoR with VDRwt and its mutants in solution (Fig. 2AGo). GST-NCoR1649-2453 precipitated similar amounts of VDRwt, VDR{Delta}H12, VDRE420A, and VDRF422A input (~10%, compare lanes 1, 4, 7, and 10). VDRwt and VDRE420A showed a nearly identical profile, which is characterized by an equal reduction of NCoR-VDR interaction after the addition of 1{alpha},25(OH)2D3 or Gemini (compare lanes 2, 3, 8, and 9). This supports earlier reports on 1{alpha},25(OH)2D3-triggered dissociation of NCoR-VDR complexes (13, 14). Interestingly, VDR{Delta}H12 and VDRF422A formed another pair of identical NCoR interaction profiles (compare lanes 4–6 with lanes 10–12). However, in this case Gemini significantly increased the VDR-NCoR complex formation (lanes 6 and 12) but 1{alpha},25(OH)2D3 showed no effect (lanes 5 and 11). To support the results from this in vitro assay a similarly designed mammalian two-hybrid assay was performed in MCF-7 cells (Fig. 2BGo). In this assay the effect of expressing a VP16-NCoR1552-2453 fusion protein on the solvent, 1{alpha},25(OH)2D3- and Gemini-triggered activity of wild-type and mutant versions of GAL4DBD-VDRLBD was assessed. This cell-based assay provided essentially the same results as the GST-pull-down assay. VDRwt and VDRE420A form a pair with a similar profile, in which in the absence of ligand a protein-protein interaction between the VDR-LBD and NCoR is observed (lanes 4 and 10) that is abolished after ligand treatment (lanes 5, 6, 11, and 12). VDR{Delta}H12 and VDRF422A also showed a similar profile, in which Gemini (lanes 9 and 12), but not 1{alpha},25(OH)2D3 (lanes 8 and 11), significantly increased the interaction between VDR and NCoR. Taken together, both types of protein-protein interaction assays support the result of the functional assays (Fig. 1CGo) indicating that F422, but not E420, is the critical amino acid for a Gemini-triggered interaction between VDR and NCoR.



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  		Fig. 2. Ligand-Triggered VDR-NCoR Interaction

A, GST-pull down assays were performed with bacterially expressed GST-NCoR1649-2453 and full-length in vitro translated, 35S-labeled wild-type or mutant VDR in the presence of DMSO (as solvent control), 1{alpha},25(OH)2D3, or Gemini (each 1 µM). After precipitation and washing the samples were electrophoresed through 10% SDS-polyacrylamide gels, and the percentage of precipitated VDR proteins with respect to input was quantified using a Fuji FLA3000 reader. Representative gels are shown. B, Mammalian two-hybrid assays were performed in MCF-7 cells that were transiently transfected with a reporter gene construct driven by three copies of the GAL4 binding site and the expression vector for GAL4DBD (as a control) or GAL4DBDVDRLBD fusion protein (wild type or mutant) in the presence or absence of the expression vector for VP16-NCoR1552-2453. The cells were treated for 16 h with ethanol (0.1%), 1{alpha},25(OH)2D3, or Gemini (each 100 nM). The stimulation of luciferase activities due to the overexpression of VP16-NCoR1552-2453 was calculated. Columns represent the mean of at least three experiments, and the tips of the bars indicate the SD values. Statistical analysis was performed by two-tailed, paired Student’s t test, and P values were calculated in reference to NCoR interaction in the presence of solvent (*, P < 0.05; **, P < 0.01).

 
Limited protease digestion allowed us to monitor the functional conformations of NRs (6). VDR agonists are known to stabilize the VDR in the active conformation c1 (30), in which the receptor is able to interact with CoA proteins. The VDR LBD was crystallized in this conformation (7), which is characterized by helix 12 in the lid-closed position as schematically depicted in Fig. 3Go. In contrast, in the silent conformation c3 (24) helix 12 is in the open-lid position. Most VDR agonists stabilize the latter conformation only to a minor extent. The antagonistic conformation c2 can be observed only with the few presently known VDR antagonists (35) but neither with 1{alpha},25(OH)2D3 nor Gemini. To compare 1{alpha},25(OH)2D3 and Gemini with respect to their potential to stabilize the active and the silent conformation in VDRwt, VDR{Delta}H12, VDRE420A, and VDRF422A, limited protease digestion assays were performed with the receptor alone. VDR-RXR heterodimers bound to DNA and VDR-RXR heterodimers on DNA in the presence of NCoR (Fig. 3Go). Under the experimental conditions of these assays the solvent dimethylsulfoxide (DMSO) stabilized only 5% or less (lanes 2, 5, 8, 12, 15, 18, 22, 25, 28, 32, 35, and 38) of receptor input (lanes 1, 11, 21, and 31) in approximately equal proportions of c1 and c3. Consistent with results of the functional and interaction assays (Figs. 1CGo and 2Go), VDRwt and VDRE420A displayed a similar profile. This is characterized by preferential stabilization of the LBD in c1, when the natural ligand was applied (lanes 3, 6, 9, 23, 26, and 29), and in c3, when Gemini was used (lanes 4, 10, 24, and 30). In the presence of RXR and DNA, but in the absence of NCoR, Gemini stabilized the receptor to approximately equal amounts in c1 and c3 (lanes 7 and 27). With VDR in solution, the profiles of VDR{Delta}H12 and VDRF422A resembled each other, i.e. both 1{alpha},25(OH)2D3 and Gemini stabilized the LBD in c3 (lanes 13, 14, 33, and 34). A similar profile was observed with VDRF422A in the presence of RXR and DNA (lanes 36 and 37), whereas under the same conditions with VDR{Delta}H12 relatively low amounts of the LBD were stabilized to a slightly higher extent in c1 than in c3 (lanes 16 and 17). However, the most prominent difference between VDR{Delta}H12 and VDRF422A was found in the presence of NCoR. With VDR{Delta}H12, again only relatively low amounts of the LBD were stabilized to approximately equal proportions in c1 and c3 (lanes 19 and 20), whereas with VDRF422A 1{alpha},25(OH)2D3 stabilized significant amounts of the receptor both in c1 and c3 (lane 39) and Gemini preferentially in c3 (lane 40). In summary, in solution, Gemini stabilizes the silent open-lid VDR conformation c3, whereas the natural agonist stabilizes the active closed-lid conformation c1. The comparison of the conformation profiles confirms that, in solution, F422 is a more critical amino acid of helix 12 than E420. The presence of RXR and DNA turns Gemini from an inverse agonist to an agonist, which can be specifically blocked by mutating F422. Interestingly, in the presence of NCoR, Gemini shifts back to an inverse agonist, which explains the observations of the functional assays (Fig. 1CGo). Moreover, the limited protease digestion assay appears to be more sensitive than the reporter gene assay (Fig. 1Go) and the GST-pull down assay (Fig. 2Go), because it monitors the conversion of Gemini from agonist to inverse agonist and vice versa already with VDRwt and not only with VDRF422A.



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  		Fig. 3. Stabilization of VDR Conformations by 1{alpha},25(OH)2D3 and Gemini

Limited protease digestion assays were performed by preincubating in vitro translated 35S-labeled wild-type or mutant VDR with DMSO (as solvent control) and saturating concentrations (1 µM) of 1{alpha},25(OH)2D3 or Gemini. The assays were performed with either VDR alone or in combination with unlabeled, in vitro translated RXR and unlabeled VDRE or in combination with RXR, VDRE, and bacterially expressed GST-NCoR1679-2453. After digestion with trypsin, the ligand-stabilized VDR conformations c1 (active) and c3 (silent) were electrophoresed through 15% SDS-polyacrylamide gels. Representative experiments are shown. VDR conformations that are represented by c1 and c3 are schematically depicted.

 
As a preparation for MD simulations, Gemini was docked into the crystal structure of the VDR-LBD (7), which has been completed by molecular modeling with the amino acid residues 118, 119, 375–377, and 424–427. The conformational analysis of Gemini showed that the first side chain keeps the same position as the single side chain of 1{alpha},25(OH)2D3 and that there are two approximately equal positions of the extra side chain of Gemini (25) (see the whole VDR-LBD view in Fig. 4AGo). In position 1 the extra side chain of Gemini (blue in Fig. 4AGo) points in the same direction as the methyl-group (C21) at C20 of the 1{alpha},25(OH)2D3-VDR-LBD crystal structure (7), whereas in position 2 (green) the second side chain is rotated by 120° in relation to its location in position 1. Ligand-triggered conformational changes of the VDR-LBD, as observed by limited protease digestion (Fig. 3Go), can be reflected on a structural level by a change of the distance of critical amino acids, such as the charge clamp residues K246 and E420 (Fig. 4AGo). MD simulations of VDRwt complexed with either the natural hormone or Gemini were performed for 2 nsec, and the distance between the C{alpha}-atoms of K246 and E420 was measured every 10 psec (Fig. 4BGo). Within this relatively long simulation period no significant deviation from the optimal distance of 19 Å could be observed, i.e. with both ligands helix 12 stayed in its agonistic position. This is in contrast to the results of the in vitro conformational analysis by limited protease digestion (Fig. 3Go) and suggests that because crystal data of apo-VDR or VDR-CoR complexes are lacking, the potential of MD simulations with VDRwt is restricted. It is likely that very long MD simulations of tens of nanoseconds are required to observe the changes between different VDRwt conformations. To avoid these restrictions, MD simulations were repeated with VDRF422A and both ligands and monitored over the rather long period of 5 nsec (Fig. 4CGo). Interestingly, with Gemini the distance between K246 and E420 was found to be decreased to an average of 17.5 Å, whereas it stayed with 1{alpha},25(OH)2D3 constant at 19 Å. This demonstrates that Gemini, but not the natural hormone, can push helix 12 into a position that prohibits interaction with CoA proteins and may favor complex formation with CoRs. When the extra side chain of Gemini is located in position 1 (blue in Fig. 4AGo), it is in direct contact with H397 and L309. The side chain pushes H397 to partially fill the empty space of the missing aromatic group of the F422A mutant. At the same time, the C-terminal portion of helix 12 is rotated counterclockwise, resulting in a movement of 3 Å for the C{alpha} atom of A422 and a movement of 2 Å for the C{alpha} atom of L421 as compared with their positions in the x-ray structure (7), when helix 12 is in the agonistic position. However, in the absence of CoR protein the tension through Gemini’s second side appears not to be strong enough to break the interaction between H397 and F422 in VDRwt. Therefore, in MD simulations, the effect can be observed only when the H397-F422 interaction is neutralized by a mutation of F422.


   Conclusion
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 Conclusion
MATERIALS AND METHODS
 REFERENCES
 
This study demonstrates that endocrine members of the NR superfamily, such as the VDR, can be inversely antagonized by synthetic ligands, such as Gemini. Due to its second side chain, Gemini seems to be the most flexible known VDR ligand. At endogenous CoA/CoR ratios, Gemini acts as an agonist and is at least as potent as the natural hormone. However, at CoR excess Gemini is clearly less active than 1{alpha},25(OH)2D3. Under the latter conditions, a subpopulation of the VDR pool may still be stabilized by Gemini in the active conformation c1, binds CoA proteins, and is responsible for the residual agonistic activity. The remaining VDR molecules also bind Gemini, but in the silent conformation c3, and do not contribute to gene activation. In contrast, as an inverse agonist, Gemini actively recruits CoR proteins to the VDR and induces superrepression. The CoR-driven shift of Gemini from agonist to inverse agonist is most pronounced when helix 12 is deleted or F422 is mutated. This suggests that the position of helix 12, which under control of its lock F422, plays an important role in determining whether Gemini acts as an agonist or an inverse agonist. On one hand, the second side chain of Gemini provides extra stability to the receptor-ligand complex, but on the other it also induces some tension to the receptor structure. Due to this yin-yang nature, Gemini is much more sensitive to changes of the CoA and CoR expression level than other VDR ligands. Although not yet studied in detail, the CoA/CoR ratio is assumed to be an important cell-specific characteristics and can be specifically sensed by Gemini.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 Conclusion
 MATERIALS AND METHODS
REFERENCES
 
Compounds
Gemini is a 21-(3-methyl-3-hydroxy-butyl) derivative of 1{alpha},25(OH)2D3, which carries two identical side chains at carbon 20 (22, 23). Both VDR ligands were a gift of LEO Pharmaceuticals (Ballerup, Denmark). They were dissolved in 2-propanol; further dilutions were made either in DMSO (for in vitro experiments) or in ethanol (for cell culture experiments). The HDAC inhibitor TSA was purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA) and dissolved in ethanol.

DNA Constructs
The full-length cDNAs for human VDR (27) and human RXR{alpha} (36) were subcloned into the simian virus 40 promoter-driven pSG5 expression vector (Stratagene, La Jolla, CA). These constructs are also suitable for T7 RNA polymerase-driven in vitro transcription/translation of the respective cDNAs. The DNA-binding domain of the yeast transcription factor GAL4, spanning amino acids 1–147, was fused with the cDNA of the human VDR LBD, spanning amino acids 109–427 (19). All point mutants were generated using the QuikChange point mutagenesis kit (Stratagene) and confirmed by sequencing. The helix 12 deletion mutants of VDR and RXR (VDR{Delta}H12 and RXR{Delta}H12) were generated by introducing a stop codon at positions 413 and 444, respectively. The full-length cDNA of mouse NCoR (37) was subcloned into the cytomegalovirus promoter-driven CMX expression vector pCMX. The luciferase reporter gene was driven by one copy of the complex VDRE from the human osteocalcin gene promoter (27), four copies of the rat ANF gene promoter (38), or three copies of a GAL4 binding site fused to the tk promoter. A cDNA fragment of mouse NCoR (spanning amino acids 1679–2453) was subcloned into the GST fusion vector pGEX (Amersham-Pharmacia, Uppsala, Sweden) and a slightly larger cDNA fragment (spanning amino acids 1552–2453) was fused with the activation domain of VP16 (39).

Transfection and Luciferase Reporter Gene Assay
MCF-7 human breast cancer cells were seeded into six-well plates (105 cells/ml) and grown overnight in phenol red-free DMEM supplemented with 5% charcoal-treated fetal bovine serum. For regular reporter gene assays, liposomes were formed by incubating each 1 µg of osteocalcin or ANF VDRE-driven reporter plasmid, pSG5-based receptor expression vectors for VDR and RXR, and pCMX-based expression vector for NCoR (as indicated) with 10 µg N-[1-(2, 3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (Roth, Karlsruhe, Germany) for 15 min at room temperature in a total volume of 100 µl. After dilution with 900 µl phenol red-free DMEM, the liposomes were added to the cells. Phenol red-free DMEM supplemented with 15% charcoal-treated fetal bovine serum (500 µl) was added 4 h after transfection. At this time, VDR ligands or TSA were also added. For mammalian two-hybrid assays the same cells and the same transfection and stimulation procedure were applied, but different reporter and expression plasmids were used. These were a luciferase reporter gene plasmid driven by three copies of a GAL4 binding site and expression vectors for wild-type and mutant GAL4-VDR and VP16-NCoR fusion proteins. For both types of assays the cells were lysed 16 h after onset of stimulation using the reporter gene lysis buffer (Roche Diagnostics, Mannheim, Germany) and the constant-light signal luciferase reporter gene assay was performed as recommended by the supplier (Canberra-Packard, Groningen, The Netherlands). The luciferase activities were normalized with respect to protein concentration and relative activities were calculated.

In Vitro Protein Translation and Bacterial Fusion Protein Overexpression
In vitro translated VDR and RXR proteins were generated by transcribing their respective cDNA expression vector with T7 RNA polymerase and translating these RNAs in vitro using rabbit reticulocyte lysate as recommended by the supplier (Promega Corp., Madison, WI). 35S-labeled VDR was generated by translation in the presence of [35S]methionine. Bacterial overexpression of GST-NCoR1679-2453 was facilitated in the Escherichia coli BL21(DE3)pLysS strain (Stratagene) by induction with isopropyl-ß-D-thio-galactopyranoside (0.25 mM) for 5 h at 25 C.

GST Pull-Down Assay
The GST pull-down assay was performed with 40 µl of a 50% Sepharose bead slurry of GST-NCoR1679-2453 (preblocked with 1 µg/µl BSA) and 20 ng of in vitro translated, 35S-labeled VDR (wild type or mutant) in the presence of indicated ligands. VDR proteins were incubated in immunoprecipitation buffer [20 mM HEPES (pH 7.9), 200 mM KCl, 1 mM EDTA, 4 mM MgCl2, 1 mM dithiothretiol, 0.1% Nonidet P-40, and 10% glycerol] for 20 min at 30 C. In vitro translated proteins that were not bound to GST-fusion proteins were washed away with immunoprecipitation buffer. GST-fusion protein-bound 35S-labeled VDR proteins were resolved by electrophoresis through 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and quantified on a Fuji FLA3000 reader (Tokyo, Japan) using Image Gauge software (Fuji).

Limited Protease Digestion Assay
In vitro translated, 35S-labeled VDR protein (2 µl), nonlabeled RXR (2 µl), nonlabeled NCoR protein (1 µl), or respective amounts of nonprogrammed reticulocyte lysate and 1 ng of unlabeled VDRE (as indicated) were incubated with ligand for 15 min at room temperature in 20 µl binding buffer [10 mM HEPES (pH 7.9), 1 mM dithiothretiol, 0.2 µg/µl poly(dI-C), and 5% glycerol]. The buffer was adjusted to 150 mM of monovalent cations by addition of KCl. Trypsin (Promega Corp., final concentration, 8.3 ng/µl) was then added, and the mixtures were further incubated for 15 min at room temperature. The digestion reactions were stopped by adding 25 µl protein gel loading buffer [0.25 M Tris (pH 6.8), 20% glycerol, 5% mercaptoethanol, 2% SDS, 0.025% bromophenol blue]. The samples were denatured at 85 C for 3 min and electrophoresed through 15% SDS-polyacrylamide gels. The gels were dried, exposed to a Fuji MP2040S imager screen, and monitored on a Fuji FLA3000 reader.

MD Simulations
The initial coordinates for the MD simulations were obtained from the x-ray crystal structure of the 1{alpha},25(OH)2D3-VDR-LBD complex (Brookhaven Protein Data Bank code 1DB1) determined to 1.8 Å resolution (7). The missing amino acid residues of the x-ray structure (residues 118, 119, 375–377, and 424–427) were built using the Quanta98 molecular modeling package (Molecular Simulations, Inc., San Diego, CA). The four residues missing from the C terminus (424–427) were built in an {alpha}-helical conformation ({phi} = -57°, {psi} = -47°). The F422A mutant was generated from the wild-type VDR by placing the Cß of alanine to the position it has in phenylalanine. Crystallographic water molecules were included in simulation systems neutralized by placing four Na+ ions in the positions of largest electrostatic potential as determined by the program CION of the AMBER6.0 simulation package (University of California, San Francisco, CA). Gemini was docked to the ligand-binding pocket of the LBD using the locally enhanced sampling method, which is a mean-field technique providing ability to focus on the interesting part of the system (40). In practice, multiple copies are employed on the part of the system where conformational sampling may be critical, and the rest of the system is treated as a single structure. For the locally enhanced sampling calculations, the protein-ligand complexes were solvated by adding a sphere of TIP3P water molecules with a 25 Å radius from the mass center of the ligand. This resulted in 764 water molecules for the complexes. Only residues having atoms within 12 Å from the atoms of the ligand were allowed to move. The water molecules of the complex were first energy minimized for 2000 steps, heated to 300 K in 5 psec, and equilibrated by 30 psec at a constant temperature of 300 K. After that, the moving part of the protein-ligand complex was minimized for 2000 steps, the temperature of the system was increased to 300 K in 5 psec and equilibrated for 75 psec using a time step of 2 fsec. The system was then heated to 400 K in 5 psec and equilibrated for 40 psec using a time step of 1 fsec and applying harmonic positional restraint of 2.0 kcal/mol. After that, the positional restraints were removed, velocities of the atoms were assigned to correspond to 340 K, and the system was cooled from 340 to 0 K in 160 psec.

For periodic box simulations, the LBD-ligand complexes were solvated by 10,778 TIP3P water molecules in a periodic box of 69x61x87 Å. The minimization and equilibration of water molecules were done as in the water cap simulations. Then the whole system was again energy minimized for 2000 steps, heated to 300 K in 5 psec and equilibrated by 300 psec at 300 K and pressure of 101.325 Pa. After that, 2- and 5-nsec production simulations were started. Bonds involving hydrogen atoms were constrained to their equilibrium lengths using the SHAKE algorithm. The time step of 1.5 fsec was used in periodic box simulations. The electrostatics was treated using the particle-mesh Ewald method, and continuum model correction was applied for energy and pressure. The center of mass velocity was removed each 75 psec. All the simulations were done using the AMBER6 program and the force field described by Cornell et al. (41).


   ACKNOWLEDGMENTS
 
We thank Drs. M. Calverley and L. Binderup for providing VDR ligands and Dr. T.W. Dunlop for critical reading of the manuscript.


   FOOTNOTES
 
This work was supported by grants from The Academy of Finland (Grant 50319 to C.C. and Grant 74097 to M.P.).

Abbreviations: ANF, Atrial natriuretic factor; CAR, constitutive androstane receptor; CoA, coactivator; CoR, corepressor; DMSO, dimethylsulfoxide; GST, glutathione S-transferase; HDAC, histone deacetylase; LBD, ligand-binding domain; MD, molecular dynamics; NCoR, nuclear receptor corepressor; NR, nuclear receptor; 1{alpha},25(OH)2D3, 1{alpha},25-dihydroxyvitamin D3; RXR, retinoid X receptor; RXRwt, wild-type RXR; SDS, sodium dodecyl sulfate; TSA, trichostatin A; VDR, 1{alpha},25(OH)2D3 receptor; VDRwt, wild-type VDR; VDRE, 1{alpha},25(OH)2D3 response element.

Received for publication March 4, 2003. Accepted for publication June 26, 2003.


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NURSA Molecule Pages Link:

Nuclear Receptors:   VDR
Coregulators:   NCOR
Ligands:   Calcitriol



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