Retinoid X Receptor Is a Nonsilent Major Contributor to Vitamin D Receptor-Mediated Transcriptional Activation
David J. Bettoun,
Thomas P. Burris,
Keith A. Houck,
Donald W. Buck, II,
Keith R. Stayrook,
Berket Khalifa,
Jianfen Lu,
William W. Chin and
Sunil Nagpal
Gene Regulation, Bone and Inflammation Research (D.J.B., T.P.B., D.W.B., K.R.S., B.K., J.L., W.W.C., S.N.), Eli Lilly & Company, Indianapolis, Indiana 46285; and Lead Generation Biology (K.A.H.), Sphinx, Eli Lilly & Company, Durham, North Carolina 27709
Address all correspondence and requests for reprints to: Sunil Nagpal, Gene Regulation, Bone and Inflammation Research, Eli Lilly & Company, Indianapolis, Indiana 46285. E-mail: nagpal_sunil{at}lilly.com.
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ABSTRACT
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The vitamin D receptor (VDR) belongs to the thyroid hormone/retinoid receptor subfamily of nuclear receptors and functions as a heterodimer with retinoid X receptor (RXR). The RXR-VDR heterodimer, in contrast to other members of the class II nuclear receptor subfamily, is nonpermissive where RXR does not bind its cognate ligand, and therefore its role in VDR-mediated transactivation by liganded RXR-VDR has not been fully characterized. Here, we show a unique facet of the intermolecular RXR-VDR interaction, in which RXR actively participates in vitamin D3-dependent gene transcription. Using helix 3 and helix 12 mutants of VDR and RXR, we provide functional evidence that liganded VDR allosterically modifies RXR from an apo (unliganded)- to a holo (liganded)-receptor conformation, in the absence of RXR ligand. As a result of the proposed allosteric modification of RXR by liganded VDR, the heterodimerized RXR shows the "phantom ligand effect" and thus acquires the capability to recruit coactivators steroid receptor coactivator 1, transcriptional intermediary factor 2, and amplified in breast cancer-1. Finally, using a biochemical approach with purified proteins, we show that RXR augments the 1,25-dihydroxyvitamin D3-dependent recruitment of transcriptional intermediary factor 2 in the context of RXR-VDR heterodimer. These results confirm and extend the previous observations suggesting that RXR is a significant contributor to VDR-mediated gene expression and provide a mechanism by which RXR acts as a major contributor to vitamin D3-dependent transcription.
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INTRODUCTION
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VITAMIN D RECEPTOR (VDR), a ligand-dependent transcription factor that belongs to the class II nuclear receptor subfamily, mediates the biological actions of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. The class II nuclear receptor subfamily includes VDR, retinoic acid receptors (RARs), retinoid X receptors (RXRs), thyroid hormone receptors (TRs), peroxisome proliferator-activated receptors (PPARs), liver X receptors (LXRs), and farnesoid X receptor (FXR). RXR plays a pivotal role in mediating the functions of these receptors by acting as their obligate partner. Therefore, VDR, RAR, TR, PPAR, LXR, and FXR function as heterodimers with RXR (1). Heterodimers PPAR-RXR, LXR-RXR, and FXR-RXR are permissive because both heterodimeric partners can bind their cognate ligands and induce transcription. In contrast, RXR-VDR, RXR-RAR, and RXR-TR heterodimers have been thought to be nonpermissive because they neither bind nor show activation by RXR ligands (2). Recently, ligand binding by RXR has been demonstrated in the context of RAR and TR heterodimers, thus raising the possibility of permissiveness for RXR-RAR and RXR-TR heterodimers (3, 4). In the context of the RXR-VDR heterodimer, even though RXR does not bind to its cognate ligand, the integrity of its activation function 2 (AF-2) domain is required for vitamin D3-dependent gene transcription. Dominant negative as well as AF-2 mutants of RXRs inhibited ligand-dependent transcription via VDR, thus indicating RXR to be a subordinate but essential partner in RXR-VDR-mediated gene expression (5, 6). Further, LXXLL peptides, which interacted with RXR but not VDR, inhibited 1,25-(OH)2D3-dependent transcription of osteocalcin gene, thus demonstrating that RXR may actively participate in a vitamin D3-mediated response (7). In this manuscript we confirm the previous observations that RXR is an essential transcriptional partner in vitamin D3-mediated gene expression and extend this finding by providing a molecular mechanism by which RXR acts as an active and major contributor to VDR-dependent transcription. We provide evidence for an unique aspect of ligand-mediated intermolecular RXR-VDR interaction and show by genetic (mammalian two-hybrid and three-hybrid assays) and biochemical (coactivators interaction assay) approaches that RXR actively participates in vitamin D3-dependent gene expression by directly recruiting coactivators in the absence of an RXR-specific ligand. Our results demonstrate an allosteric modification of RXR by liganded VDR, whereby RXR acquires the holoreceptor conformation in the absence of its cognate ligand (phantom ligand effect) and gains the ability to recruit coactivators. These results provide evidence for RXR as a functionally active, nonsilent partner in vitamin D3-mediated RXR-VDR-dependent gene expression.
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RESULTS AND DISCUSSION
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RXR Actively Contributes to 1,25-(OH)2D3-Dependent VDR-Mediated Transactivation
VDR belongs to the class II subfamily of the nuclear receptors and functions as a heterodimeric partner with RXR. Because several reports described conflicting results when assessing the role of RXR as part of an RXR-VDR heterodimer bound to a VDRE (8, 9), we were interested in evaluating the contribution of RXR to vitamin D3-dependent transactivation. To address whether RXR contributes to VDR-dependent transactivation, we used a transcriptionally inactive dominant-negative mutant of RXR
(dnRXR
) that lacks AF-2 but still heterodimerizes with VDR and other class II nuclear receptors (10). We examined the ability of dnRXR
to interfere with VDR-dependent activation on a VDRE. Upon 1,25-(OH)2D3 treatment, a 10-fold increase in luciferase activity was observed in HeLa cells transfected with a VDR expression vector and pVDRE3-tk-Luc. Cotransfection with pSG5-dnRXR
reduced 1,25-(OH)2D3-mediated induction to 2-fold (Fig. 1A
). These results confirm the reported observations, in which either dominant negative or AF-2 point mutants of RXRs inhibited VDR-mediated gene expression (5, 6). These data along with the reported results (5, 6) supported the idea of an active involvement of RXR in gene activation by vitamin D3. To further confirm the importance of RXR, we performed an experiment using a one-hybrid system. Transfection of HeLa cells with a Gal4-VDR-ligand-binding domain (LBD) expression plasmid and a (UAS)5-TATA-Luciferase reporter plasmid led to a 50-fold increase in luciferase activity in the presence of 1,25-(OH)2D3 (Fig. 1B
). Cotransfection with dnRXR
expression vector significantly reduced 1,25-(OH)2D3-dependent transactivation by Gal4-VDR-LBD (Fig. 1B
). These results show that RXR is required and appears to be a major contributor to 1,25-(OH)2D3-dependent transcriptional activation.

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Fig. 1. RXR Contributes to VDR-Mediated Transactivation
A, Expression of dnRXR interferes with VDR-mediated transactivation. Cells were transfected with a pVDRE3-tk-Luc, pSG5-VDR, and either dnRXR or pSG5 empty expression vector. Luciferase activity measured in the presence or absence of ligand is expressed as arbitrary light units ± SD. B, dnRXR interferes with VDR-dependent gene expression in mammalian one-hybrid system. Luciferase activity (±SD) obtained after transfection of HeLa cells with pGal4-VDR-LBD, without (black bars) or with dnRXR (gray bars), in the presence or absence of 1,25-(OH)2D3 (10-7 M). Fold activation in the presence of the ligand is represented by the numbers above the bars. C, Contribution of RXR to VDR-dependent gene expression in a mammalian two-hybrid system. Luciferase activity (±SD) of cells transfected with pGal4-RXR -LBD (bars 14, 9, and 10) or pGal4-dnRXR -LBD (bars 5, 6, 11, and 12) and pVP16-VDR-LBD (bars 38) or pSG5-VDR-LBD (bars 912) in the presence or absence of 1,25-(OH)2D3 (10-7 M) is shown. D, VDR helix 12 and helix 3 mutants do not interact with coactivators. Luciferase activity (±SD) of HeLa cells transfected with pGal4-SRC1, -TIF2, and -AIB1 along with empty pVP16 vector (white bars), pVP16-VDR-LBD (black bars), pVP16-VDR-LBDL417A (dark gray bars), or pVP16-VDR-LBDY236A (light gray bars) in the presence of 1,25-(OH)2D3 (10-7 M) is shown. Results shown are in arbitrary light units and are representative of at least four independent experiments performed in triplicate.
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To support further the idea that RXR is actively contributing to 1,25-(OH)2D3-dependent transactivation, mammalian two-hybrid experiments were performed using VP16-VDR-LBD and Gal4-RXR
-LBD or Gal4-dnRXR
-LBD. If RXR merely heterodimerizes with VDR, and does not contribute to 1,25-(OH)2D3-dependent transactivation, then the same level of activation should be observed irrespective of whether RXR
or dnRXR
is used in a two-hybrid system. On the other hand, if RXR participates in 1,25-(OH)2D3-mediated transactivation, then heterodimerization of VDR-LBD with dnRXR
should not lead to the same level of activation as that observed with VDR-LBD and RXR heterodimer. Transfection of HeLa cells with Gal4-RXR
-LBD and VP16-VDR-LBD (Fig. 1C
, bars 3 and 4) resulted in a 60-fold increase in luciferase activity in the presence of 1,25-(OH)2D3 (10-7 M). In contrast, no significant activity was observed when cells were transfected with either bait (Fig. 1C
, bars 1 and 2) or prey (Fig. 1C
, bars 7 and 8) constructs alone in the absence or presence of the VDR ligand. Consistent with an active role for RXR, a significant reduction in 1,25-(OH)2D3-dependent transactivation of reporter was observed when cells were transfected with VP16-VDR-LBD and Gal4-dnRXR
-LBD (Fig. 1C
, bars 5 and 6). Moreover, 1,25-(OH)2D3-dependent transactivation of the reporter was not completely abrogated in the context of pGal4-dnRXR
-LBD and pVP16-VDR-LBD cotransfection. This suggested that either the VDR-LBD or the VP16 moiety had a transcriptional effect. Therefore, similar two-hybrid experiments were performed using a VDR-LBD expression vector instead of the VP16-VDR-LBD. Cotransfection of HeLa cells with pGal4-RXR
-LBD and VDR-LBD (Fig. 1C
, bars 9 and 10) resulted in a 46-fold increase in luciferase activity in the presence of 1,25-(OH)2D3 (10-7 M). Interestingly, when pGal4-dnRXR
-LBD was used, a mere 4-fold induction of reporter gene activity was observed in the presence of 1,25-(OH)2D3 (Fig. 1C
, bars 11 and 12). These data also show that, in a two-hybrid system, the AF-2 of RXR and the VP16 moiety both contributed to transactivation, whereas VDR input was only marginal. Therefore it appears that, in the presence of 1,25-(OH)2D3, VDR heterodimerization affects RXR in a way that involves the AF-2 of RXR to participate in transcriptional activation. Significant loss of 1,25-(OH)2D3-induced transactivation in the absence of RXR AF-2 was also observed with full-length pVP16-VDR or pSG5-VDR (data not shown).
AF-2 and Helix 3 Mutants of VDR Disrupt Its Interaction with p160 Coactivators
To understand how VDR ligand could affect RXR, we addressed whether the effect of VDR-LBD on RXR depends on the ability of VDR to interact with coactivators. To answer this question, two point mutants of VP16-VDR-LBD and VDR-LBD, namely L417A and Y236A, were prepared. These mutants have been shown previously to prevent recruitment of coactivators without affecting the protein stability and heterodimerization with RXR (11, 12, 13). To verify that mutated VP16-VDR-LBD proteins do not recruit coactivators, mammalian two-hybrid analysis was performed using Gal4-coactivator chimeric constructs. Coexpression of Gal4-steroid receptor coactivator 1 (SRC1), Gal4-transcriptional intermediary factor 2 (TIF2), or Gal4-amplified in breast cancer-1 (AIB1) fusion protein with VP16-VDR-LBD led to a significant induction of luciferase activity in the presence of 1,25-(OH)2D3 (Fig. 1D
). As expected, no increase in reporter gene activity was observed in the absence of ligand (data not shown) or when the cells were cotransfected with either the empty vector pVP16, pVP16-VDR-LBDL417A, or pVP16-VDR-LBDY236A, confirming that these mutants do not interact with p160 coactivators (Fig. 1D
).
Vitamin D3-Dependent Recruitment of p160 Coactivators by RXR-Helix 3 Mutated VDR Heterodimer
Interestingly, although indistinguishable with respect to their lack of binding to coactivators, VDR-LBD helix 12 (L417A) and helix 3 (Y236A) mutants behaved differentially in the context of VDR-LBD/RXR
-LBD mammalian two-hybrid assays. In Fig. 2A
, cells were cotransfected with pVP16-VDR-LBD or pSG5-VDR-LBD or one of their helix 12 or helix 3 mutants along with pGal4-RXR
-LBD, whereas in Fig. 2B
, pGal4-dnRXR
-LBD was used along with VDR or one of its mutants. Transfection of cells with either the prey construct alone (Fig. 2A
, bar C) or the bait construct in the presence of the empty pVP16 or pSG5 expression vectors (Fig. 2A
, bars 1 and 5; Fig. 2B
, bars 9 and 13) did not show any significant activity in the presence of the ligand. Under conditions in which RXR/VDR heterodimerization was not affected, mutation in helix 12 (VDR-LBDL417A) failed to activate RXR in the presence of 1,25-(OH)2D3 (Fig. 2A
, compare bar 2 with bar 3 and bar 6 with bar 7). These results suggested that activation of RXR by liganded VDR-LBD requires a functional helix 12 of VDR. Interestingly, mutation in helix 3, while totally preventing coactivators recruitment to VDR (Fig. 1D
), only partially blunted RXR activation in response to the VDR ligand (Fig. 2A
, compare bar 4 with bar 2 and bar 8 with bar 6). This total or partial loss of RXR activation was also observed in the absence of VP16 (Fig. 2A
, bars 68). Upon examining in the context of dnRXR
, VDR-LBDY236A failed to activate RXR with reporter activity similar to that observed with VDR-LBDL417A. Thus, the simultaneous mutation of Y236A in VDR and AF-2 in RXR resulted in a transcriptionally inactive heterodimer (Fig. 2B
, compare bars 15 and 16 to bar 14, and bars 11 and 12 to bar 10). These results suggest that although VDR-mediated allosteric activation of RXR requires a functional VDR AF-2, coactivator recruitment to VDR is not required, because a VDR helix 3 mutant that fails to recruit coactivators can still affect RXR and cause it to activate transcription. It should be pointed out that the helix 12 of liganded VDR still likely recruits coactivators and/or participates in vitamin D receptor-interacting protein (DRIP)205-mediator interaction, and it may also function to displace a corepressor from RXR as wells as to conformationally activate it. Of note, the induction of gene expression observed when cotransfecting pGal4-RXR
-LBD and pVP16-VDR-LBDL417A is similar to that observed when cotransfecting pGal4-dnRXR
-LBD with either VP16-VDR-LBD mutants. This result demonstrates that RXR
and dnRXR
interact similarly with VDR-LBD (Fig. 2
, A and B, compare lanes 11 and 12).

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Fig. 2. Allosteric Effect of Ligand-Occupied VDR on RXR
A and B, Helix 3 mutant displays vitamin D3-dependent gene expression. Mammalian two-hybrid analysis was performed by measuring luciferase activity (±SD) in HeLa cells cotransfected with pGal4-RXR -LBD (panel A) or pGal4-dnRXR -LBD (panel B), along with pVP16 (bars 1 and 9), pVP16-VDR-LBD (bars 2 and 10), pVP16-VDR-LBDL417A (bars 3 and 11), pVP16-VDR-LBDY236A (bars 4 and 12), pSG5 (bars 5 and 13), pSG5-VDR-LBD (bars 6 and 14), pSG5-VDR-LBDL417A (bars 7 and 15), or pSG5-VDR-LBDY236A (bars 8 and 16) in the presence of 1,25-(OH)2D3 (10-7 M). Numbers shown above the bars represent fold-activation compared with vector-transfected control. As controls, activity obtained with the prey alone (panel A, bar C) or the bait along with empty pVP16 or pSG5 expression vectors (panel A, bars 1 and 5; panel B, bars 9 and 13) is also shown. Results presented in panels A and B were obtained in the same experiment. A schematic of the experiment is also shown. C, RXR AF-2 and helix 3 RXR mutants do not recruit coactivators. Luciferase activity (±SD) of HeLa cells cotransfected with pGal4-SRC1, -TIF2, or -AIB1 and pVP16 (white bars), pVP16RXR AB (black bars), pVP16-dnRXR Aß (light gray bars), or pVP16RXR K284E (dark gray bars) is shown in the presence of 9-cis-RA (10-7 M). D, VDR-dependent transcriptional is equally affected by mutations in the AF-2 or in helix 3 domains of RXR . Cells were cotransfected with pGal4-RXR -LBD, pGal4-dnRXR -LBD, or pGal4-RXR K284E along with pVP16 (white bars), pVP16-VDR-LBD (black bars), or pVP16-VDR-LBDY236A (gray bars) in the presence of 1,25-(OH)2D3 (10-7 M). Numbers on top of bars represent fold-induction over pVP16-transfected cells. Experiments were performed in triplicate; luciferase activity, expressed in arbitrary light units, is representative of at least three independent experiments.
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AF-2 and Helix 3 Mutants of RXR Do Not Support VDR-Mediated Transactivation
Deletion of the RXR helix 12 or point mutation in the AF-2 region of RXR
(RXR
L455A) has been shown to constitutively recruit corepressors (6, 14). To rule out that a decrease in transcriptional activity observed with pGal4-dnRXR
-LBD (Figs. 1C
and 2B
) was due to corepressor recruitment to the dnRXR/VDR heterodimer, we repeated the experiments shown in Fig. 2A
using a K284E RXR
(helix 3 mutant). This mutation in RXR
helix 3 prevents recruitment of p160 coactivators, DRIP205, and corepressor (15). Mammalian two-hybrid analysis involving Gal4-SRC1, -TIF2, or -AIB1 and VP16-RXR
AB or one of the RXR
mutants confirmed that both VP16-RXR
-K284E and VP16-dnRXR
were equally defective in recruiting coactivators (Fig. 2C
). When assayed with wild-type or mutated VP16-VDR-LBD constructs, pGal4-RXR
-K284E behaved essentially like pGal4-dnRXR
-LBD (Fig. 2D
). While further emphasizing that RXR is a major player in the context of VDR/RXR heterodimer, these data ruled out the possibility that the decreased transcriptional activation observed with dnRXR
results from corepressor recruitment to AF-2-deleted RXR.
VDR Allosterically Affects RXR to Recruit p160 Coactivators in a 1,25-(OH)2D3- Dependent Manner
Our observation that VDR-LBD-L417A fails to activate RXR when, under the same conditions, VDR-LBD-Y236A causes a partial activation is particularly puzzling because these two mutants are equally incapable of recruiting coactivators (Fig. 1D
). The fact that pGal4-RXR
-K284E behaved essentially like pGal4-dnRXR
-LBD suggested that the H3 and H12 VDR-LBD mutants differ in their ability to affect allosterically RXR to recruit coactivators. To test this hypothesis, we used a mammalian trihybrid approach, in which we tested the ability of VDR-LBD to recruit VP16-RXR
AB fusion protein to Gal4-SRC1, -TIF2 or, -AIB1 in the presence of 1,25-(OH)2D3 (Fig. 3B
) As a control, bait construct Gal4-AIB1 alone in the absence of the prey (VP16-RXR
AB) and VDR-LBD did not show any significant activity (Fig. 3A
, bar C). Similar results were obtained with Gal4-SRC1 and Gal4-TIF2 (data not shown). As expected, VP16-RXR
AB could not be recruited to coactivators in the absence of either 1,25-(OH)2D3 (data not shown) or in the presence of the bait construct but absence of VDR-LBD (Fig. 3A
, bars 1, 5, and 9), whereas it was efficiently recruited by Gal4-SRC1 (bar 2), -TIF2 (bar 6), and -AIB1 (bar 10) in the presence of both VDR-LBD and 1,25-(OH)2D3. When VDR-LBDL417A was used, no recruitment of VP16-RXR
AB was observed as indicated by luciferase activities similar to those obtained with control vector-transfected cells (Fig. 3A
, compare bars 2, 6, and 10 with bars 3, 7, and 11). Strikingly, VDR-LBDY236A, while also incapable of binding coactivators (Fig. 1D
), could still activate transcription of the reporter gene to nearly 50% of wild-type VDR-LBD (bars 4, 8, and 12). Taken together, these data further support the notion that ligand-bound VDR-LBD allosterically modifies RXR, thus resulting in cofactor recruitment and transactivation by RXR (Fig. 3B
). The fact that VDR-LBDY236A failed to recruit coactivators, yet still had a partial effect on Gal4-RXR
-LBD-mediated transcription, suggests that the allosteric effect on RXR is not mediated by recruitment of coactivators to VDR-LBDY236A. These data rather suggest a direct proximity effect by which liganded VDR allosterically affects RXR structure. In the context of wild-type VDR, cooperative protein-protein interactions have also been postulated between the RXR-bound and VDR-bound coactivators (16). Such postulated protein-protein interactions between the RXR-bound and VDR-bound coactivators would be lost in the context of RXR-VDR-LBDY236A heterodimer and therefore may explain why VDR helix 3 mutant showed 50% of the wild-type VDR-LBD activity instead of 7080% activity as expected from transactivation results presented in Fig. 1C
.

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Fig. 3. Allosteric Activation of RXR by Liganded VDR, But Not by TR, Causes Recruitment of Coactivators to RXR in the Presence of VDR Ligand
A, Trihybrid analysis of RXR recruitment to coactivators in the presence of the VDR ligand. Cells were transfected with pGal4-SRC1, -TIF2, or -AIB1 along with pVP16-RXR AB and pSG5 (bars 1, 5, and 9), pSG5-VDR-LBD (bars 2, 6, and 10), pSG5-VDR-LBDL417A (bars 3, 7, and 11) or pSG5-VDR-LBDY236A (bars 4, 8, and 12). After 1,25(OH)2D3 treatment (10-7 M), luciferase activity (± SD) was measured and shown here as light units. Bar C represents control, where cells were transfected with only bait Gal4-AIB1 in the presence of the ligand. B, Schematic representation of panel A data. Allosterically modified VP16-RXR AB is recruited to the coactivator (top panel). Mutation in VDR helix 12 prevents allosteric effect and also coactivator recruitment (middle panel). VDR mutated in its helix 3 mutation can still cause RXR to contact coactivators (bottom panel). C and D, RXR also contributes to TRß-dependent gene transcription. HeLa cells were cotransfected with pGal4-RXR -LBD or one of its mutants and pVP16-TRß (panel C) or pGal4-RXR -LBD or one of its mutant constructs and pVP16-TRß-LBD (panel D) in the presence of various concentrations of T3. Results are expressed in luciferase activity (±SD) in arbitrary light units and are representative of two separate experiments performed in triplicate.
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TR Also Shows an Allosteric Effect on RXR
We next examined whether the recruitment of coactivators to RXR in the absence of its cognate ligand is unique to VDR or whether it also happens in the context of RXR-TR heterodimer. We therefore assayed TR for any loss of transcriptional activation in the context of a mammalian two-hybrid assay with Gal4-dnRXR
-LBD or Gal4-RXR
-LBDK284E vs. Gal4-RXR
-LBD. T3 induced the expression of the reporter in a dose-dependent manner, when the cells were cotransfected with both Gal4-RXR
-LBD and pVP16-TRß (Fig. 3C
) or pVP16-TRß-LBD (Fig. 3D
). Cotransfection of the cells with Gal4-dnRXR
-LBD or Gal4-RXR
-LBDK284E, along with the pVP16-TRß-LBD (Fig. 3C
) or pVP16-TRß (Fig. 3D
) expression vector, did not show any induction of the reporter in the presence of various concentrations of T3. These observations suggest that TR also requires RXR for gene expression and may allosterically activate RXR
.
Biochemical Evidence for the Involvement of RXR in 1,25-(OH)2D3-Dependent Coactivator Recruitment
Finally, The genetic evidence for the active involvement of RXR in vitamin D3-mediated coactivator recruitment by RXR-VDR heterodimer was further strengthened by a biochemical coactivator recruitment (Alphascreen) assay, which utilizes a nickel-coated donor bead containing immobilized his-tagged VDR-LBD and an anti-glutathione-S-transferase (GST) acceptor bead coated with GST-TIF2-purified proteins. In this assay, 1,25-(OH)2D3 brings the donor and acceptor pair of protein-coated beads in proximity by ligand-mediated biomolecular interaction between VDR and TIF2 (Fig. 4A
). Alphascreen signal in counts per second indicates the recruitment of TIF2 by VDR in the absence or presence of RXR
protein. As a control, RXR
alone in the absence of VDR-LBD failed to recruit TIF2 in a 1,25-(OH)2D3-dependent manner, while TIF2 was recruited by VDR-LBD in the presence of its cognate ligand (Fig. 4B
). Addition of RXR
to the assay mixture resulted in increased 1,25-(OH)2D3- dependent recruitment of TIF2 to RXR-VDR heterodimer, thus confirming the allosteric activation and cofactor recruitment by apo-RXR by a unique intermolecular signaling in response to ligand occupancy of VDR (Fig. 4B
). Similarly, RXR enhanced the binding of transcription factor IIB and nuclear coactivator-62 to VDR, presumably by forming ternary complexes (16, 17). The effect of RXR on TIF2 recruitment was modest (2-fold), whereas RXR accounted for 7080% of vitamin D3-mediated transactivation (Fig. 1C
). This discrepancy could be explained from the absence of cooperative interactions between VDR-occupied and RXR-occupied coactivators (16).

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Fig. 4. RXR Augments 1,25-(OH)2D3-Dependent Recruitment of TIF2 to VDR
A, Schematic representation of Alphascreen coactivator recruitment assay. In this assay, a donor and an acceptor pair of beads are brought into proximity by interaction between VDR-LBD and TIF2. Excitation with a laser beam (680 nm) induces the formation of a singlet oxygen resulting in activation of fluorophores in the acceptor bead and emission of light at 520620 nm. B, In vitro coactivator recruitment by apo-RXR in response to ligand-occupied VDR. An Alphascreen cofactor interaction assay was performed using polystyrene beads coated with purified VDR-LBD and GST-TIF2 proteins in the presence of absence of purified RXR protein. Dose-response curves involving 12 different concentrations of 1,25-(OH)2D3 are shown for the recruitment of TIF2 by VDR (red) or RXR-VDR (blue). As a control, RXR alone did not recruit TIF2 (black diamond) in the presence of 1,25-(OH)2D3 (400 nM). Each data point was assayed in quadruplicate, and the results shown are representative of at least three separate experiments.
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CONCLUSIONS
Permissive heterodimers, PPAR-RXR, LXR-RXR, and FXR-RXR, can simultaneously respond to cognate ligands of both receptors. In the case of PPAR
-RXR
, permissiveness is associated with the discrete recruitment of DRIP205 by PPAR
, and p160 coactivators by RXR (18). In contrast, RXR-VDR is a nonpermissive heterodimer and therefore does not support activation by RXR ligand. For this reason, RXR is referred to as a silent partner of the RXR-VDR heterodimer. Our results demonstrating the interference of Gal4-VDR-LBD transcriptional activation by dnRXR
(Fig. 1B
) along with the previously reported studies (5, 6, 7) show that RXR actively participates in vitamin D3-induced transcriptional activation. Two-hybrid experiments showed that RXR accounted for as much as 7080% of vitamin D3-dependent gene expression, because dnRXR
-VDR-driven reporter activity was only 2030% of that observed with RXR
-VDR (Fig. 1
, AC). In contrast, others have shown that PPAR
-RXR
heterodimer responsiveness to PPAR
ligand was not affected by the use of AF-2 mutants of RXR (18, 19).
The classical model of vitamin D3 action states that the interaction of 1,25-(OH)2D3 with VDR conformationally restricts RXR so that it no longer interacts with its specific ligand, 9-cis-retinoic acid (20). We extend this classical model of vitamin D3 interaction and provide a unique mode of intermolecular RXR-VDR interaction, wherein 1,25-(OH)2D3-bound VDR allosterically modifies RXR into an active conformation that is receptive to interaction with coactivators. The phantom ligand effect on RXR in the context of 1,25-(OH)2D3-occupied heterodimer is clearly demonstrated by using VP16-VDR-LBDY236A (helix 3 mutant) and Gal4RXR
or Gal4-RXR
K284E (helix 3 mutant) in a mammalian two-hybrid assay (Fig. 2D
). This 1,25-(OH)2D3-mediated phantom ligand effect was further confirmed in a mammalian three-hybrid system, in which a VDR-LBDY236A-VP16-RXR
heterodimer could recruit coactivators, as well as using a biochemical approach (Figs. 3A
and 4B
). Our results also support the postulation of Jurutka et al. (21), which states that ligand-occupied VDR functions as an allosteric regulator of RXR and transforms RXR into an active conformation that is receptive for coactivator binding even in the absence of 9-cis-retinoic acid, an RXR ligand (21). Similar allosteric control of heterodimeric partner activity by a synthetic RXR ligand in the context of RXR-RAR heterodimer has been described as a phantom ligand effect (22). In summary, we describe a unique and unexpected facet of intermolecular cross-talk between VDR and RXR and demonstrate that RXR actively participates in RXR-VDR-mediated gene transcription by directly recruiting coactivators in response to 1,25-(OH)2D3.
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MATERIALS AND METHODS
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Cell Culture and Transfection
HeLa cells, maintained in DMEM supplemented with 10% fetal bovine serum, were plated at 5000 cells per well in a 96-well plate. The next day, cells were transfected using 0.5 µl of Fugene (Roche Diagnostics, Indianapolis, IN), 100 ng of luciferase reporter vector pFR-Luc (Stratagene, La Jolla, CA), and 10 ng of receptor or cofactor expression vector per well. Total DNA amount was kept constant by adding empty vector when needed. Cells were treated with the ligand 24 h after transfection, and luciferase activity was quantitated the next day using Steady-Glo luciferase detection reagent (Promega Corp., Madison, WI).
Vector Construction
pVDRE3-tk-Luc was constructed by inserting a BamHI- and XhoI-flanked oligonucleotide containing three copies of the osteopontin VDRE upstream of the herpes simplex virus-thymidine kinase-luciferase cassette of the pT109luc plasmid (23, 24). Gal4-DNA-binding domain chimera of SRC1, TIF2, or AIB1 have been reported (25). Construction of pGal4-RXR
-LBD, pGal4-dnRXR
-LBD, pSG5-dnRXR
, and pVP16-RXR
AB has been described (10). Vectors pGal4-VDR-LBD, pVP16-VDR-LBD, and pSG5-VDR-LBD were constructed by inserting a PCR-generated fragment of VDR (89427) into the pM vector (CLONTECH, Palo Alto, CA) in frame with the Gal4-DNA-binding domain, in the pVP16-vector in frame with the VP16 activation domain (CLONTECH), or into the pSG5 expression vector (Stratagene). pVP16-TR-LBD or pVP16-RAR-LBD were generated by inserting PCR-generated fragments corresponding to the same region as VDR-LBD into pVP16-VDR-LBD, and RXR mutants were generated by site-directed mutagenesis of the parent vectors using QuikChange XL mutagenesis kit (Stratagene).
Coactivator Interaction Assay
Interaction between the VDR/RXR-VDR heterodimer and the coactivator TIF2 was assayed using Alphascreen (amplified luminescent proximity homogenous assay) technology (Packard BioScience, Meriden, CT). The assay was performed in white, low-volume, 384-well plates utilizing a final volume of 15 µl containing final concentrations of 20 nM of His-tagged bacterially expressed VDR-LBD with or without baculovirus-expressed, full-length RXR
protein (5 nM), 5 nM of GST-TIF2 protein that contained the entire nuclear receptor-interacting domain of TIF2 (594766 amino acids of mouse TIF2) fused to GST and 10 µg/ml of both nickel chelate donor beads and anti-GST acceptor beads. The assay buffer contained 25 mM HEPES (pH 7.0), 100 mM NaCl, 0.1% BSA, and 2 mM dithiothreitol. Drugs were added to the assay plate at 5x concentrations in 5% dimethylsulfoxide resulting in the final concentrations of compounds as indicated in the figure legends and a final assay concentration of 1% dimethylsulfoxide. Drugs (3 µl of 5x) were initially added to the assay plate followed by 12 µl of a master mix containing the protein and assay beads. Assay plates were covered with a clear seal and incubated in the dark for 2 h. Plates were read (1 sec/well) in a Packard BioScience Fusion microplate analyzer using the manufacturers detection protocol.
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ACKNOWLEDGMENTS
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We thank Professor Shigeaki Kato for critical comments on the manuscript and for the gifts of Gal4-coactivator constructs.
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FOOTNOTES
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Abbreviations: AF-2, Activation function 2; AIB1, amplified in breast cancer-1; dnRXR
, dominant-negative mutant of RXR
; DRIP, vitamin D receptor-interacting protein; FXR, farnesoid X receptor; GST, glutathione-S-transferase; LBD, ligand-binding domain; LXR, liver X receptor; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; PPAR, peroxisome proliferator activated receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; SRC1, steroid receptor coactivator 1; TIF2, transcriptional intermediary factor 2; TR, thyroid hormone receptor; VDR, vitamin D receptor; VDRE, vitamin D response element.
Received for publication April 21, 2003.
Accepted for publication July 24, 2003.
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