Retinoid X Receptor Dominates the Nuclear Import and Export of the Unliganded Vitamin D Receptor

Kirsten Prüfer and Julia Barsony

Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Dr. Julia Barsony, LCBB, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 8 Center Drive, Room 422, Bethesda, Maryland 20892. E-mail: jul{at}helix.nih.gov.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Liganded and unliganded vitamin D receptors (VDRs) carry out distinct functions; both types of functions require heterodimerization with retinoid X receptors (RXRs). Our recent studies with fluorescent protein chimeras of VDR and RXR, termed GFP-VDR, YFP-RXR, and RXR-BFP, indicated that RXR regulates VDR functions in part by regulating subcellular localization. Here we explored the mechanisms of this regulation. Photobleaching experiments demonstrated that YFP-RXR and both unliganded and liganded GFP-VDR shuttle constantly between nucleus and cytoplasm. To characterize RXR import, we identified a nuclear localization sequence (NLS) in the DNA-binding domain. Mutations in this NLS caused predominant cytoplasmic localization of nlsYFP-RXR and prevented transcriptional activity. The nlsRXR-BFP retained unliganded GFP-VDR in the cytoplasm and reduced baseline transcriptional activity. After calcitriol exposure, however, both GFP-VDR and nlsRXR-BFP entered the nucleus. We characterized receptor export rates and mechanisms using permeabilization experiments. Mutations in the calreticulin binding region slowed both GFP-VDR and YFP-RXR export. Coexpression of RXR-BFP slowed the export of unliganded GFP-VDR, whereas calcitriol treatment tripled the rate of GFP-VDR export. Treatment with leptomycin B, an inhibitor of CRM-1 receptor-mediated export, inhibited export of unliganded GFP-VDR but did not influence export of liganded GFP-VDR or YFP-RXR. Leptomycin B added before calcitriol similarly decreased hormone-induced luciferase activity but was ineffective when added subsequent to calcitriol. These results indicate that the unliganded and liganded VDR interact differently with the import and export receptors and with RXR. Most likely, the regulation of VDR nuclear import by RXR is essential for ligand-independent functions.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
VITAMIN D RECEPTORS (VDRs) carry out hormone-dependent and independent functions. Calcitriol-dependent functions of VDRs regulate calcium homeostasis, immune functions, endocrine functions, vitamin D metabolism, and cellular differentiation. It is well known that dimerization with retinoid X receptors (RXRs) plays an important role in calcitriol-dependent functions of VDR (1, 2). Calcitriol promotes the binding of VDR/RXR heterodimers to target DNA sequences and promotes the interaction of VDR/RXR heterodimers with coactivators, corepressors, and with proteins of the transcription machinery (3, 4). Consequently, defects in either VDR or RXR functions can result in rickets, a disease also caused by vitamin D deficiency.

Ligand-independent effects of VDR on promoters of target genes can be measured in reporter assays as baseline activity. The functional significance of these reporter activities, however, remains to be defined. The most convincing reporter assay showing ligand-independent activation was published by Tolon et al. (5); they demonstrated that without calcitriol VDR/RXR heterodimers activate a reporter driven by the prolactin promoter, and this activation required the presence of Ets-1 protein. More importantly, the ligand-independent function of VDR to promote the hair cycle became evident recently. Defects in this function causes alopecia in patients with hereditary VDR defects and in mice harboring a targeted deletion of the VDR gene (6, 7). Neither vitamin D deficiency (8) nor targeted deletion of the 1{alpha}-hydroxylase gene in mice causes alopecia (9). However, targeted deletion of the RXR{alpha} gene in the skin did result in alopecia (10). These findings indicate that dimerization with RXR is likely to be involved in ligand-independent functions, as well.

RXRs have many VDR-independent functions, as they form heterodimers with a variety of nuclear receptors. Dimerization interfaces of RXR were identified in the DNA-binding domain (DBD) and in the identity box, a 40-amino acid subregion within the ligand-binding domain (11, 12). RXR partners include thyroid hormone receptors (TRs), retinoic acid receptors (RARs), peroxisome proliferator-activated receptor, several constitutive active orphan nuclear receptors (e.g. nuclear growth factor I-B), oxysterol receptors, and constitutive androstane receptors. RXRs also form homodimers to mediate the effects of 9-cis-retinoic acid (9-cRA). Depending on these protein-protein interactions, RXR-containing complexes have distinct ligand-dependent and constitutive functions.

In recent years, mechanisms have been described that regulate transcriptional activities of nuclear proteins through regulation of subcellular localization. Well studied examples of transcription factors that move from the cytoplasm into the nucleus under the control of cell surface receptors include tubby proteins, nuclear factor of activated T cells, nuclear factor-{kappa}B, SMADs, serum response element binding protein, and signal transducers and activators of transcription (reviewed recently in Ref. 13). Regulation of nuclear localization through protein interactions has been shown for the glucocorticoid receptors (GRs) (14, 15), mineralocorticoid receptors (16), MST1 (17), MIZ-1 transcription factor (18), RXR (19), TR (20), histone deacetylases (21), androgen receptors (22), and constitutive androstane receptors (23). We have shown previously that heterodimerization with RXR promotes VDR steady-state nuclear accumulation (24).

Many nuclear proteins shuttle between the cytoplasm and the nucleus; hence their localization is controlled by their interactions with the nuclear import and export machinery. Nuclear import of several transcription factors is mediated by importin {alpha} through binding of their nuclear localization sequences (NLS). Other transcription factors, such as the cAMP response element binding protein and activator protein 1, bind directly to importin ß through their NLS (25). Deletions or mutations within these regions prevent nuclear entry of these proteins. Heterokaryon experiments have shown that GRs (26), progesterone receptors (27), TRs (20, 28), and estrogen receptors (ERs) (29) shuttle across the nuclear membrane within 1–8 h, which implies that they are not only imported into the nucleus but also are exported from the nucleus. Export from the nucleus is often mediated by nuclear export receptors that bind to specific export signal sequences (NES). Several export receptors have been identified. The most common type is CRM-1 export receptor, which binds to leucine-rich NES, and this binding is inhibited by leptomycin B (LMB). Nuclear receptors such as GR, progesterone receptor, androgen receptor, and TR do not contain leucine-rich NES, and their export is not inhibited by LMB. Instead, calreticulin serves as an export adapter through binding to their DBD. Calreticulin also supports the export of DBD isolated from ER, RAR, RXR, or VDR (30). Another type of NES, KNS and HNS sequences, require serine and acidic residues for export activity (31), and this export is also not inhibited by LMB. Export of nuclear receptors can occur through multiple alternative pathways, as indicated for RAR export (30). To gain insight into the mechanisms of VDR and RXR transport across the nuclear pore, both nuclear import and export processes require exploration.

We have previously shown that RXR promotes nuclear accumulation of the unliganded VDR. An NLS within the DBD of VDR has been identified (32), and we have shown that green fluorescent protein (GFP)-VDR harboring this NLS mutation (nlsGFP-VDR) is predominantly cytoplasmic. Coexpression of RXR, however, promoted a nuclear accumulation of nlsGFP-VDR and restored VDR-dependent transcriptional activity (17). Although the interaction of RXR with the nuclear import machinery has not been explored, a plausible explanation for RXR effect on nlsGFP-VDR import is a piggyback mechanism, similar to the mechanism demonstrated for the homodimerizing PR (33). An alternate possibility, which is more likely when RXR is not exported from the nucleus, is that RXR retains the VDR in the nucleus by inhibiting VDR nuclear export or by promoting nuclear retention.

Here we analyzed the complex mechanisms of VDR and RXR nuclear import and export to gain insight into the regulation of VDR localization by RXR and the impact of this regulation on transcriptional activities of VDR. Our studies show that RXRs influence constitutive VDR effects by regulating VDR subcellular localization and clarify differences between nuclear import and export mechanisms for the liganded and the unliganded VDR.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RXR and VDR Shuttle between Nucleus and Cytoplasm
There are at least two possible explanations for the steady-state nuclear localization of RXR and liganded VDR. One possibility is that receptors translocate into the nucleus immediately after synthesis and remain there until they are degraded. Alternatively, the same localization could be generated when the receptors shuttle between cytoplasm and nucleus, but the residence time in the nucleus is longer than the residence time in the cytoplasm. To distinguish between these two possibilities, we conducted fluorescence recovery after photobleaching (FRAP) experiments in transiently transfected COS-7 cells.

For these FRAP experiments, fluorescing binucleated cells were selected (Fig. 1AGo, a and d), and within one of the nuclei a 4 x 4 pixel area was exposed to an intense laser illumination for 1 sec/pixel duration. This photoexposure resulted in a complete loss of fluorescence within the exposed nucleus (Fig. 1AGo, b and e). This loss of fluorescence indicates the rapid movement of VDR and RXR within the nucleus of living cells. In contrast, photobleaching in fixed cells caused loss of fluorescence only in the small bleached area and did not result in any loss of fluorescence in other parts of the nucleus (not shown). Detailed analysis of this intranuclear receptor movement is the subject of our ongoing studies, but in this paper we focus on exploring receptor movement across the nuclear membrane.



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Figure 1. Experiments with Fluorescence Recovery after Photobleaching Demonstrate that Both YFP-RXR and GFP-VDR Shuttle between the Nucleus and the Cytoplasm

A, RXR and VDR exchange rapidly between the two nuclei. A representative image of a binucleated COS-7 cell expressing YFP-RXR is shown in the upper panels. A 2-µm2 spot within one of the nuclei (arrow) was exposed to a focused laser beam at full power (a), which caused irreversible photobleaching (darkening) of all the receptors within this nucleus (b). An image after complete recovery (30 min) is shown in panel c. Fluorescence recovery (brightening) within this exposed nucleus indicates import from other cell compartments. The darkening of the other nucleus indicates that the bright receptors from the unbleached nucleus move into the exposed nucleus. Because the exposed and the unexposed nuclei were not connected, exchange of fluorescing and nonfluorescing YFP-RXR molecules occurs by passage through the cytoplasm. The fluorescence intensities of nearby cells remained unaffected during the photoexposure and the recovery periods. Similar experiments for the unliganded GFP-VDR are shown in the lower panels. A laser beam was focused onto the desired small area within one of the two nuclei (d), and photoexposure resulted in a complete photobleaching (darkening) of all the receptors within this nucleus (e). An image after complete recovery (30 min) is shown in panel f. Bar, 10 µm. B, Graphs show signal intensity changes within cell compartments from representative FRAP experiments. Mean brightness values over areas within the exposed (solid black line) and unexposed nuclei (solid gray line), and the cytoplasm (dashed line) were determined and plotted against time. Representative FRAP curves show that the recovery half-time for the YFP-RXR is about 20 min (a), for the unliganded GFP-VDR is about 20 min (b), and for the liganded GFP-VDR is about 5 min (c).

 
From yellow fluorescent protein (YFP)-RXR-expressing cells, serial images taken during the recovery phase showed that YFP-RXR fluorescence increases in the exposed nucleus and decreases concomitantly in the unbleached nucleus of the same cell (pictures from a typical experiment are shown on Fig. 1AGo, a–c). The rates of change in fluorescence intensities in the bleached and the unbleached nuclei and in the cytoplasm were plotted, thereby providing for determination of recovery half-time. Ten experiments showed that half of YFP-RXR exchanged place between the two nuclei within 20 to 30 min (a typical recovery curve is shown on Fig. 1BGo, a). This exchange of fluorescing and nonfluorescing YFP-RXR between the bleached and the unbleached nuclei requires the export of fluorescing and nonfluorescing receptors from each nucleus into the cytoplasm and their import into both nuclei.

We performed several control measurements to assure that the results accurately reflect receptor shuttling. Images taken immediately after photoexposure showed that fluorescence in the unbleached nucleus within the same cell, and in the neighboring cells, remains intact during photobleaching. To exclude the contribution of newly synthesized receptors, experiments were carried out after cycloheximide pretreatment. These experiments showed a similar YFP-RXR recovery half-time (20–30 min) (not shown). Control recordings of recovery in cells with a single nucleus after photobleaching showed a minimal, 1–5% recovery after 30 min from newly synthesized or cytoplasmic receptor import (not shown). In the binucleated cells, we determined the separation of the nuclei before each experiment by differential interference contrast imaging. The lack of connection was further supported by the loss of fluorescence exclusively in the bleached nucleus. Our preliminary experiments showed that in some cells, where the two nuclei were not completely separated, both nuclei became dark during photobleaching. These experiments demonstrated that FRAP experiments reliably detect receptor shuttling between the cytoplasm and the nucleus.

We conducted similar FRAP experiments on GFP-VDR expressing binucleated COS-7 cells. Consistent with our earlier findings, unliganded GFP-VDR distributed evenly between the cytoplasm and the nucleus (Fig. 1AGo, d) (34). The brief photoexposure caused a complete loss of GFP-VDR fluorescence within the exposed nucleus, without affecting the fluorescence of the other nucleus within the same cell (Fig. 1AGo, e). During the recovery phase, the brightness of the exposed nucleus increased, and the brightness of the unbleached nucleus decreased until their fluorescence intensities equalized (Fig. 1AGo, f). The brightness of the cytoplasm remained the same. In six experiments recovery half-times were between 15 and 30 min (Fig. 1BGo, b.), a finding consistent with active transport. In contrast, when FRAP was done in cells expressing GFP, which is known to diffuse freely across the nuclear membrane, half-time of recovery was about 30 sec (not shown).

Further FRAP experiments demonstrated that the liganded VDRs also shuttle between the cytoplasm and the nucleus. After calcitriol (10 nM) addition for 30 min, GFP-VDR was predominantly nuclear. FRAP experiments showed that the liganded GFP-VDR also redistributes rapidly between the two nuclei. The recovery half-time in eight experiments was between 5 and 15 min (a typical recovery curve is shown on Fig. 1BGo, c). This recovery time is faster than the recovery time for the unliganded GFP-VDR (P < 0.05). These experiments demonstrated that both unliganded and liganded GFP-VDR shuttle between the cytoplasm and nucleus and that calcitriol accelerates shuttling speed of VDR.

GFP-VDR and YFP-RXR recovery happened only at physiological temperatures. When FRAP experiments were performed at room temperature in COS-7 cells expressing either YFP-RXR or GFP-VDR, only 5–10% of fluorescence was recovered in the bleached nucleus within 30 min after bleaching. This temperature sensitivity is also consistent with active transport of these receptors.

The realization that both RXR and VDR are in constant movement across the nuclear membrane with rates consistent with receptor-mediated import and export led us to investigate these mechanisms and to study the influence of dimerization on import and export.

RXR Import Is Mediated by Import Receptors
To study the import process of RXRs, we generated point mutations within basic amino acid-rich sequences. The search for candidate NLS sequences in the DBD of the RXR revealed a putative NLS between amino acids 160 and 165 (Fig. 2AGo). We mutated two lysines and two arginines in this sequence and expressed the mutant nlsYFP-RXR in COS-7 and CV-1 cells. Whereas steady-state localization of the wild-type YFP-RXR was predominantly nuclear (Fig. 2BGo, a), localization of nlsYFP-RXR was predominantly cytoplasmic (Fig. 2BGo, b) in both cell lines. Similar mutation in the RXR-blue fluorescent protein (BFP) sequence also caused a nuclear import defect (Fig. 3AGo, e). Addition of 100 nM 9-cRA did not change cytoplasmic localization of nlsYFP-RXR or nlsRXR-BFP (not shown).



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Figure 2. Mutation of the NLS in the YFP-RXR (nlsYFP-RXR) Results in a Predominantly Cytoplasmic Localization

A, Schematic representation of the RXR{alpha} DNA binding domain. Amino acids 160, 161, 164, and 165, representing the NLS, are shaded. The respective sequences of other type II nuclear receptors are aligned and compared in the lower portion of the figure, and amino acids representing putative NLS are shaded. B, COS-7 cells were transiently transfected with YFP-RXR (a) and nlsYFP-RXR (b). The wild-type YFP-RXR is predominantly nuclear (a), whereas the nlsYFP-RXR is predominantly cytoplasmic (b). Bar, 10 µm.

 


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Figure 3. Dominant Negative Effects of nlsRXR-BFP on the Nuclear Import and Transcriptional Activity of GFP-VDR

A, COS-7 cells express GFP-VDR alone (a and b) or coexpress GFP-VDR and nlsRXR-BFP (c–f). GFP-VDR (c) is retained in the cytoplasm in the presence of nlsRXR-BFP (e). Addition of 10 nM calcitriol for 30 min causes a complete translocation of GFP-VDR into the nucleus (b). Calcitriol causes a similar translocation of GFP-VDR (d) in the presence of nlsRXR-BFP, and a partial translocation of nlsRXR-BFP (f) in the same cell. Bar, 10 µm. B, Coexpression of RXR-BFP increases and coexpression of nlsRXR-BFP decreases both basal transcriptional activity and calcitriol-induced activity of GFP-VDR. COS-7 cells were transiently transfected with receptor expression plasmids and a 24-OH/Luc reporter as described. Normalized luciferase activities from vehicle-treated cells are shown as open bars, and from calcitriol-treated (10 nM) cells are shown as shaded bars. Luminescence data were normalized with ß-galactosidase activities, and data are expressed as mean ± 1 SD.

 
Transcriptional activities of YFP-RXR and nlsYFP-RXR were evaluated using a DR-1 luciferase reporter (DR-1/Luc) assay. These assays demonstrated that in YFP-RXR-expressing cells treatment with 100 nM 9-cRA for 24 h induces a 37-fold increase in DR-1 reporter activity. In contrast, in nlsYFP-RXR-expressing cells transcriptional activities were undetectable both in the absence and presence of 9-cRA. These data define a NLS in the DBD of RXRs and establish that RXR transcriptional activities are dependent on the integrity of this NLS.

RXR Regulates Ligand-Independent Nuclear Import of VDR
With the nlsRXR-BFP, we had a convenient tool to explore the effect of RXR on the ligand-independent and ligand-dependent nuclear import of VDR. Previously, we found that coexpression of RXR-BFP increases nuclear accumulation of an NLS mutant GFP-VDR (17). Here, we evaluated the effect of nlsRXR-BFP expression on GFP-VDR steady-state distribution in COS-7 cells. As shown in Fig. 3AGo, GFP-VDR (panel c) is more cytoplasmic in cells coexpressing nlsRXR-BFP (panel e) than in cells expressing only GFP-VDR (panel a). Addition of 10 nM calcitriol, however, caused complete translocation of GFP-VDR (panels b and d) regardless of nlsRXR-BFP expression. Calcitriol-induced translocation of GFP-VDR caused a partial translocation of nlsRXR-BFP (panel f). These observations demonstrate the effect of RXR to increase the ligand-independent nuclear import of VDR and the effect of liganded VDR to promote nuclear import of RXR.

Transactivation assays further supported the significance of RXR and VDR interactions during nuclear import. Transcriptional activities of GFP-VDR were evaluated using a 24-hydroxylase luciferase reporter (24OH/Luc). GFP-VDR dimerize with endogenous RXR in COS-7 cells (35) to activate this DR3 reporter. Coexpression of RXR-BFP increased both basal (Fig. 3BGo, open bars) and calcitriol-induced transcriptional activities of GFP-VDR (Fig. 3BGo, shaded bars). Basal activities were 41% higher and ligand-induced transcriptional activities were 49% higher in these cells than in cells expressing GFP-VDR only (Fig. 3BGo). Coexpression of nlsRXR-BFP decreased both basal and calcitriol-induced transcriptional activities of GFP-VDR. Basal activities were 20% lower (P = 0.3), and ligand-induced transcriptional activities were 40% lower (P < 0.01) in these cells than in cells expressing GFP-VDR alone (Fig. 3BGo, shaded bars). These experiments demonstrated that the localization of RXR regulates both nuclear import and basal transcriptional activities of unliganded VDR. The dominant negative effect of nlsRXR-BFP on the calcitriol-induced reporter activity is similar to the dominant negative effect of a DBD deletion mutant RXR on RAR-induced transcriptional activity (36). A DNA binding phenotype of this nlsRXR-BFP is also expected based on studies on conserved amino acids in VDR (32).

VDR and RXR Are Exported from the Nucleus
Our FRAP experiments indicated that VDR and RXR are exported from the nucleus. We explored this further using digitonin permeabilization experiments on cell lines that stably express either GFP-VDR (GL48 cells) or YFP-RXR (CYR cells). Plasma membranes of GL48 and CYR cells were selectively permeabilized with digitonin in transport buffer (TB) for 10 min on ice, and cells were than incubated in TB for 1 h at 37 C. Control cells were kept in TB without digitonin. After incubation, cells were fixed, and images were taken for brightness measurements. Microscopy showed that GFP-VDR fluorescence was predominantly nuclear in TB-treated fixed cells, in contrast to that in living cells. Similar fixation artifacts have been reported for VDR and other nuclear receptors (37). One hour after digitonin permeabilization of untreated GL48 cells, residual fluorescence was 50 ± 19% of control (Fig. 4Go, a and b). In another experiment, GL48 cells were treated with 10 nM calcitriol for 1 h before digitonin permeabilization. Under these conditions, 1 h after digitonin permeabilization the residual brightness values were only 17 ± 18% of control (Fig. 4Go, c and d) (P < 0.001). These data demonstrate that half of unliganded GFP-VDR is exported from the nucleus within 1 h and that this export is accelerated by calcitriol.



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Figure 4. VDR and RXR Are Exported from the Nucleus

Nuclear export of GFP-VDR was measured in GL48 and export of YFP-RXR was measured in CYR cells by fluorescence microscopy. GL48 cells were treated with vehicle (a and b) or with 10 nM calcitriol (c and d) for 1 h. CYR cells were treated with vehicle (e and f) or with 100 nM 9-cRA (g and h) for 1 h. Then, both GL48 and CYR cells were incubated with either TB (left panels) or permeabilized with TB containing 50 µg/ml digitonin (right panels) and then kept in TB for 1 h. After fixation and mounting, confocal laser scanning microscopy using the same brightness and contrast parameters visualized residual fluorescence in samples. Bar, 10 µm.

 
Similar experiments were performed to explore YFP-RXR export. In CYR cells 1 h after digitonin permeabilization, residual brightness values were 40 ± 16% of the control (Fig. 4Go, e and f). In another experiment, CYR cells were treated with 100 nM 9-cRA for 1 h before digitonin permeabilization. Under these conditions, residual brightness values of digitonin-treated cells were 68 ± 13% of control (Fig. 4Go, g and h); this retention was higher than the retention without hormone treatment (P < 0.001). These experiments demonstrated that the unliganded RXR is exported from the nucleus and the ligand slows this export.

To evaluate the relative contribution of nuclear docking/retention to the residual nuclear fluorescence 1 h after digitonin permeabilization, in parallel experiments we permeabilized the nuclear membrane of GL48 and CYR cells with 0.5% Triton X-100 in TB. Microscopy confirmed that the nuclear membrane stayed intact after simultaneous incubation with fluorescent wheat germ agglutinin and TB for 1 h following digitonin permeabilization (not shown). In contrast, the nuclear membrane was completely lost after Triton X-100 permeabilization (not shown). Then we compared residual nuclear GFP-VDR and YFP-RXR fluorescence after 2 h incubation with TB in Triton-permeabilized cells (nuclear matrix bound) with residual nuclear fluorescence in digitonin-permeabilized cells (bound plus soluble). In these experiments, only 2.3 ± 0.1% of GFP-VDR fluorescence was retained in the nucleus after Triton permeabilization, whereas 13 ± 0.6% of GFP-VDR was retained in the nucleus after digitonin permeabilization. For YFP-RXR, 3.8 ± 0.1% of the fluorescence was retained in the nucleus after Triton permeabilization, whereas 23 ± 1% of YFP-RXR was retained in the nucleus after digitonin permeabilization. These data demonstrate that the contribution of nuclear retention to the residual nuclear fluorescence in digitonin permeabilization experiments is minimal compared with the contribution of the receptor export kinetics.

We then evaluated the need for export proteins for the loss of receptor fluorescence from the nucleus after digitonin permeabilization. Without the addition of reticulocyte lysate to the buffer, GFP-VDR and YFP-RXR fluorescence did not decrease during incubation after digitonin permeabilization (data not shown). These experiments showed that both RXR and VDR export require proteins of the export machinery and argue against export by diffusion.

Unliganded VDR Is Exported by the CRM-1 Export Receptor
Next, we used LMB treatment to test whether the export of VDR is mediated by the CRM-1 export receptor. Because unliganded GFP-VDR normally distributes evenly between cytoplasm and nucleus, sensitivity to LMB is easily detectable by monitoring steady-state distribution. We treated GL48 cells and transiently transfected CV-1 and COS-7 cells with 2 nM LMB for 1 h and found that after this treatment GFP-VDR was predominantly nuclear (Fig. 5AGo).



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Figure 5. Export of Unliganded VDR Is LMB Sensitive

A, GFP-VDR distributes evenly between the cytoplasm and the nucleus in GL48 cells (control). Treatment with 2 nM LMB for 1 h causes accumulation of GFP-VDR in the nucleus. Bar, 10 µm. B, GL48 cells were incubated with vehicle (a and b) or with 10 nM calcitriol (c and d) for 1 h. LMB sensitivity of YFP-RXR export in CYR cells was examined without hormone pretreatment (e and f). Then, both GL48 and CYR cells were incubated with 2 nM LMB for 1 h. Subsequently, cells were incubated in either TB (left panels) or permeabilized with TB containing 50 µg/ml digitonin (right panels) and then kept in TB for 1 h. After fixation and mounting, confocal laser scanning microscopy using the same brightness and contrast parameters visualized residual fluorescence in samples. Bar, 10 µm.

 
We then explored the effect of LMB on VDR export using digitonin permeabilization experiments. GL48 cells were treated with 2 nM LMB for 1 h and then incubated in TB with or without digitonin as described above. Residual brightness values of LMB-treated permeabilized cells were 82 ± 18% of control (Fig. 5BGo, a and b). This represents a near-complete inhibition of export, as the residual brightness values were not significantly lower than values in control, nonpermeabilized cells (P = 0.1). In the same experiment, residual brightness values of vehicle-treated permeabilized cells were 50 ± 19% of controls (P < 0.05). These data indicate that export rate of the unliganded VDR is slower by 30% in LMB-treated cells than in cells treated with vehicle.

This ability of LMB to slow GFP-VDR export was also apparent in FRAP experiments. Recovery of GFP-VDR fluorescence was monitored after photobleaching as described above. From five cells treated with 2 nM LMB and five cells treated with vehicle for 1 h, we detected only 45% of the recovery in LMB-treated cells of that detected in vehicle-treated cells 30 min after photobleaching.

We then used mutational analysis to identify putative CRM-1 binding sites in the VDR. Mutations of the leucine-rich sequences did not result in a predominantly nuclear steady-state distribution of GFP-VDR, which would signify defective nuclear export. Another indicator of perturbed CRM-1 binding is a loss of LMB sensitivity due to mutations of leucine-rich sequences. We found that point mutations at leucines 320, 323, and 325 (ex5GFP-VDR) resulted in a loss of LMB sensitivity. Other leucine-to-alanine mutations did not affect LMB sensitivity. One interpretation of these results is that the NES of the VDR is located between amino acids 320 and 325. The existence of multiple export pathways for the VDR could explain why the loss of LMB sensitivity did not block VDR export to the extent that would be apparent by the altered steady-state distribution of the ex5GFP-VDR. Alternatively, mutations of these leucines could change the conformation of VDR and thus influence protein-protein interactions, ultimately leading to the loss of direct or indirect CRM-1 binding to VDR.

Unlike the export of the unliganded VDR, the export of the liganded VDR was not sensitive to LMB. Digitonin permeabilization experiments showed that the export from the nuclei of GL48 cells after treatment with 10 nM calcitriol for 1 h was the same irrespective of the addition of 2 nM LMB or vehicle for the second hour. After calcitriol and LMB, residual fluorescence over the nuclei of permeabilized cells was 11 ± 15% of control (Fig. 5BGo, c and d), whereas after calcitriol alone residual fluorescence over the nuclei of permeabilized cells was 17 ± 18% of control. These data show that the export rates of liganded VDR were insensitive to LMB. Similar results were obtained in FRAP experiments. COS-7 cells expressing GFP-VDR were pretreated with 10 nM calcitriol for 1 h, after which 2 nM LMB or vehicle was added to calcitriol for another hour. FRAP experiments on five binucleated cells showed that recovery half-times over the bleached nuclei were between 5 and 15 min, indistinguishable from recovery half-times over bleached nuclei of cells treated with vehicle (not shown). These data indicate that the export of the liganded VDR is CRM-1 independent.

We tested LMB sensitivity of RXR export in a similar manner. Treatment of CYR cells with 2 nM LMB or vehicle for 1 h was followed by incubation with TB and/or digitonin to assess loss of nuclear fluorescence due to export. Residual brightness values of LMB-treated permeabilized cells were 50 ± 13% of LMB-treated controls (Fig. 5BGo, e and f). In the same experiment, residual brightness values of vehicle-treated CYR cells 1 h after permeabilization were 40 ± 16% of control. This export rate was not statistically different from the export rate of LMB-treated CYR cells (P = 0.08). Furthermore, the difference between the export rates of LMB-treated and vehicle-treated cells was not statistically significant in FRAP experiments. In COS-7 cells expressing YFP-RXR, the recovery half-times for five LMB-treated cells were between 20 and 30 min and for three vehicle-treated cells were between 25 and 30 min. These experiments indicate that nuclear export of RXR is mediated by CRM-1-independent mechanisms.

Calreticulin Binding Is Important for VDR and RXR Export
The role of calreticulin binding in the export of nuclear receptors has been shown recently (14). To confirm this finding for the export of intact receptors, we generated the same FF-to-AA mutations in the calreticulin-binding site of the GFP-VDR (calGFP-VDR), which was critical for the export of the DBD of the VDR in a GFP reporter (30). We expressed this calGFP-VDR in COS-7 cells and found that this mutant is predominantly nuclear at steady state. Digitonin permeabilization experiments showed that export of calGFP-VDR was significantly inhibited (residual fluorescence 90 ± 9% of control) compared with export of the wild-type GFP-VDR (residual fluorescence 48 ± 9% of control) (P < 0.01). Moreover, treatment with LMB did not increase calGFP-VDR nuclear retention; residual fluorescence was 81 ± 7% of control in LMB-treated and 90 ± 1.4% of control in untreated cells. In the same experiment, treatment with LMB increased nuclear retention of the wild-type GFP-VDR; residual fluorescence was 67 ± 9% of control in LMB-treated and 48 ± 1.1% of control in untreated cells (P < 0.05). Export of liganded calGFP-VDR was accelerated by calcitriol treatment, but to a lesser degree than the wild-type GFP-VDR; residual calGFP-VDR fluorescence was 72 ± 7% of control after calcitriol. These data confirm the role of calreticulin in VDR export.

Experiments with the DBD of RXR indicated that calreticulin also plays a role in RXR export. Thus, we mutated the calreticulin-binding site in YFP-RXR (calYFP-RXR) as described by Black et al. (30). Digitonin permeabilization experiments in transfected COS-7 cells showed that the export of calYFP-RXR is inhibited; residual calYFP-RXR fluorescence was 78 ± 8% of control, and residual wild-type YFP-RXR fluorescence was 68 ± 8% of control (P < 0.001). These combined results confirmed that calreticulin plays a role in the export of intact VDR and RXR.

Export of Unliganded VDR Is Necessary for Full Transcriptional Activity
Our ability to inhibit export of the unliganded VDR by LMB treatment allowed us to investigate the impact of export on transcriptional activities. If nuclear access were a limiting step for transcriptional activity, we would expect an increase in baseline and possibly in calcitriol-induced transcriptional activity upon LMB treatment. We tested this hypothesis by measuring the ability of the endogenous VDR to induce the stably expressed 24OH/Luc activities in ROSA I cells after LMB treatment. Treatment with LMB increased baseline transcriptional activities by 1.5-fold. We compared calcitriol-induced transcriptional activities of VDR in the absence of LMB with calcitriol-induced activities after pretreatment with 2 nM LMB for 3 h, calcitriol-induced activities in the presence of LMB, and calcitriol-induced activities in the presence of LMB that was added 3 h after the calcitriol. As shown in Fig. 6Go, pretreatment with LMB decreased calcitriol-induced transcriptional activity of VDR from a 29-fold induction to a 13-fold induction; differences in luciferase activities after calcitriol were statistically significant (P < 0.001). Simultaneous treatment with LMB reduced induction to 15-fold; differences in activities were also significant (P < 0.005). LMB treatment 3 h after calcitriol did not significantly reduce the induction (P = 0.08). These results show that inhibition of VDR export results in inhibition of calcitriol-induced reporter activity. The decrease of LMB sensitivity after calcitriol treatment is consistent with the diminished LMB sensitivity of VDR export after calcitriol.



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Figure 6. LMB Treatment Increases Baseline Transcriptional Activities and Decreases Calcitriol-Induced Activities when Added Before or Simultaneously to Calcitriol

ROSA I cells, which stably express a 24-hydroxylase-luciferase reporter, were treated with vehicle (open bars) or 10 nM calcitriol (shaded bars). Vehicle (control) or LMB (2 nM) was added 3 h before calcitriol treatment (LMB-3 h), LMB was added simultaneously with calcitriol (LMB), and LMB was added 3 h after calcitriol treatment (LMB+3 h). Luminescence values are expressed as mean ± 1 SD.

 
Nuclear Export of GFP-VDR Is Inhibited by RXR-BFP
The different export rates for RXR and VDR in the digitonin permeabilization experiments and the difference in LMB sensitivity between RXR and VDR prompted the question as to whether export of GFP-VDR is influenced by the coexpression of RXR-BFP. To address this question, we transiently transfected COS-7 cells with plasmids encoding GFP-VDR and RXR-BFP and compared the loss of nuclear fluorescence within 1 h after incubation with TB and/or digitonin. For control, export within 1 h was evaluated in cells transfected with GFP-VDR and hdRXR-BFP. Figure 7Go shows GFP-VDR fluorescence (green channel) and RXR-BFP fluorescence (blue channel) from the same fields of representative images from TB-treated control and digitonin-permeabilized cells. The loss of GFP-VDR brightness over the nuclei of digitonin-treated cells was 38 ± 13% in cells coexpressing RXR-BFP (Fig. 7AGo). This export was significantly less than the GFP-VDR export in cells coexpressing hdRXR-BFP (63 ± 12%; P < 0.005) (Fig. 7 BGo). Comparable results were obtained with FRAP experiments. In six experiments on COS-7 cells cotransfected with GFP-VDR and the heterodimerization mutant hdRXR-BFP, the recovery half-times were within 10–30 min. Recovery in 14 experiments on COS-7 cells cotransfected with GFP-VDR and RXR-BFP was slower. Recovery 30 min after the photobleaching was 15% of the recovery in cells coexpressing GFP-VDR and hdRXR-BFP. These findings show that both export and shuttling of VDR are slowed down by RXR.



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Figure 7. RXR-BFP Inhibits Export of GFP-VDR

A, COS-7 cells were cotransfected with GFP-VDR and RXR-BFP and 24 h after transfection incubated with either TB (left panels) or permeabilized with TB containing 50 µg/ml digitonin (right panels) and then kept in TB for 1 h. After fixation and mounting, confocal laser scanning microscopy visualized residual GFP-VDR fluorescence (upper panels) and residual RXR-BFP fluorescence (lower panels) in the same field. Brightness and contrast parameters were kept constant. B, Control experiments were carried out using COS-7 cells cotransfected with GFP-VDR (upper panels) and hdRXR-BFP (lower panels). Bar, 25 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We demonstrated that both VDR and RXR constantly shuttle between nucleus and cytoplasm using photobleaching and permeabilization experiments. Detailed analysis of nuclear import and export revealed that RXR slows down both import and export of the unliganded VDR. We identified the NLS of the RXR and used NLS mutants of RXR to clarify that VDR/RXR dimers are formed in the cytoplasm and translocate together to the nucleus upon calcitriol binding. Further analysis showed the LMB sensitivity and the effect of RXR on the export of unliganded VDR and demonstrated that VDR export plays a role in the regulation of transcriptional activity. These insights into the subcellular trafficking of VDR and RXR suggest a new role for RXR heterodimerization in the regulatory control of constitutive VDR actions.

It is now gaining acceptance that many nuclear receptors and transcription factors shuttle between the nucleus and the cytoplasm and that this shuttling is important for the regulatory control of transcriptional activities. For decades, the generally accepted model divided nuclear receptors into two categories: one category for receptors that always reside in the nucleus and another for receptors that reside in the cytoplasm and translocate into the nucleus only after hormone binding. According to this model, both VDR and RXR belong to the group of receptors that reside in the nucleus. However, it is now clear that nuclear receptors are highly mobile. Shuttling of these receptors was studied with multiple techniques, including heterokaryon and microinjection experiments on living cells and immunocytology on fixed cells. Experiments with heterokaryons have suggested continuous shuttling between the nucleus and the cytoplasm for the TR (20), the liganded GR (26), the liganded and unliganded ER (29), and the progesterone receptor (38). Microinjection of recombinant receptors into Xenopus oocytes showed the export of TR{alpha} (28). Because mammalian nuclei are too small for microinjection, heterokaryon experiments are not suitable for determining transit kinetics, and because of our concerns about artifacts associated with fixation and with antibody cross-reactivity, we used photobleaching experiments (FRAP) in living cells that express fluorescent protein chimeras of VDR and RXR. Previously, we and others have used such FRAP techniques to demonstrate the fast intranuclear movements of GRs (39, 40) and estrogen receptors (41). In the present study, FRAP technique gave us reproducible results on receptor shuttling across the nuclear membrane, which were validated with multiple controls and are in agreement with the results of our permeabilization experiments. Our studies have shown that both RXR and VDR shuttle, that ligand-dependent differences in VDR transit time exist, and that both RXR overexpression and LMB treatment increase transit time of the unliganded VDR. The FRAP technique is limited in that only one cell can be monitored at any given time. Because there are differences between cells in the cell cycle and receptor expression levels, obtaining a sufficient number of experiments for statistical analysis is essential. The recognition of VDR and RXR shuttling with FRAP led us to explore the multiple mechanisms of receptor import and export and their role in the regulation of transcriptional activity in more detail.

Mutational analysis showed that the RXR has a mini-bipartite NLS in its DBD, similar to the NLS of the VDR (32). This NLS1 confers binding of RXR to import receptors. This is indicated by two observations. First, mutations in this region of the RXR resulted in a predominantly cytoplasmic localization even in the presence of an RXR ligand. Second, the absence of another basic amino acid-rich domain, such as the bipartite NLS in the hinge region of VDR (42) or the NLS2 in the ligand-binding domain of other nuclear receptors, indicate that the liganded and unliganded RXRs are both imported by the same mechanism. Previously, we found that the NLS mutant GFP-VDRs remain in the cytoplasm without the hormone and only partially translocate into the nucleus with hormone. This nlsGFP-VDR, however, accumulated in the nucleus when it was coexpressed with RXR-BFP, suggesting that RXRs facilitate nuclear import of VDR. The use of nlsRXR-BFP coexpression further corroborated that RXR has the capability to influence VDR import, as nlsRXR-BFP retained unliganded GFP-VDR in the cytoplasm. Taken together with the fact that liganded GFP-VDRs bring nlsRXR-BFP into the nucleus, our data suggest that RXR and VDR can dimerize in the cytoplasm and that RXR and VDR influence each other’s import. RXRs also increase nuclear accumulation of unliganded VDR by slowing their export. Nuclear accumulation of VDR is similarly influenced by LMB treatment. We tested whether these effects of RXR on VDR localization could play a role in the regulation of ligand-independent functions of VDR. We found that coexpression of RXR and treatment with LMB similarly increases baseline transcriptional activity of VDR, whereas coexpression of nlsRXR-BFP decreases baseline transcriptional activity of VDR. Dimerization with RXR may play a significant role in the constitutive activities of other dimerization partners, at least in part, by bringing them into the nucleus, as indicated by the ability of RXR to increase nuclear accumulation of TR (20). Experiments with nlsRXR-BFP could identify these functions in the future.

We found a number of ligand-dependent differences in the nuclear import and export mechanisms of VDR. Results of both FRAP and permeabilization experiments showed that calcitriol increases the speed of GFP-VDR import and export. Calcitriol treatment also induced nuclear accumulation of nlsRXR-BFP, thereby further supporting the notion that these receptors can dimerize in the cytoplasm. Other protein interactions also play a role in the ligand-dependent nuclear import of VDR. This is indicated by our earlier observation that the ligand-dependent VDR import requires an intact AF-2 region, unlike the ligand-independent import (34). Differences are also indicated in the export mechanisms. Our previous studies showed the differences in temperature sensitivity between liganded and unliganded VDR export (24). In this study we found that calcitriol treatment results in a loss of LMB sensitivity of VDR export. These differences can be explained by differences in receptor conformation and coactivator binding, which could alter VDR interactions with import and export receptors or could activate indirect import and export routes. Not all the export pathways are equally affected by ligand binding. We found that mutations within the calreticulin-binding region impaired export of VDR and RXR irrespective of ligand binding. The apparent LMB insensitivity of the unliganded calGFP-VDR and ex5GFP-VDR shows the complexity of the multiple export pathways. Understanding this regulation is important, as indicated by our transactivation assays demonstrating that both receptor import and export are necessary for ligand-dependent functions. Taken together, these results support the hypothesis that RXR is the dominant partner for the mobility and functions of the unliganded VDR, whereas liganded VDR regulates the mobility and calcitriol-dependent functions of RXR.

Our studies contribute to the emerging complex model of nuclear receptor functional states. Earlier, we believed that VDR and RXR are either inactive or active only after ligand binding. Now we understand that VDR/RXR dimers are active in the absence of the ligand. Similarly to membrane receptors (43), we may eventually find antagonists to counteract these activities. Studies with calcitriol analogs have already demonstrated that several active states of VDR must exist, because the calcemic activities can be separated from the antiproliferative and immunoregulatory functions (44). Differential binding of interacting proteins also revealed different activation states of VDR (4). With this in mind, we predict that the impact of protein-protein interactions and multiple agonists/antagonists on the import and export of VDR will better characterize these functional states.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells and Expression Constructs
Transcriptionally active GFP-VDR and YFP-RXR were generated as described earlier (24). GFP-VDR was stably expressed in human embryonic kidney (HEK-293) cells, and a high-expressing clone (GL48) was selected as described earlier (24). YFP-RXR was stably expressed in green monkey kidney (CV-1) cells, and a high-expressing clone (CYR) was selected. Stable expressing cell lines were cultured in DMEM containing 10% fetal bovine serum (HyClone Laboratories, Inc., Logan, UT), 2 mM glutamine, 0.1 µM insulin, and 2 mM geneticin (Invitrogen, Carlsbad, CA).

COS-7 and CV-1 cells were obtained from ATCC (Manassas, VA) and were grown as described previously (24, 34). Briefly, cell cultures were maintained in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 0.1 µM insulin, and 0.1 mg/ml gentamicin. ROS cells stably expressing p24OH/Luc (ROSA I cells) were a gift from H. F. DeLuca (University of Wisconsin, Madison, WI).

Mutational Analysis of Receptor Trafficking
Point mutations were introduced into the coding sequences of the YFP-RXR, the RXR-BFP, and the GFP-VDR using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). Up- and downstream oligonucleotides were designed according to the manufacturer’s instructions. Point mutations in the putative NLS of the RXR-BFP and the YFP-RXR (nlsRXR-BFP and nlsYFP-RXR) were introduced at amino acids 160, 161, 164, 165 (K160Q, lysine to glutamine; R161G, arginine to glycine; R164G, lysine to glycine, K165Q, lysine to glutamine). The hdRXR-BFP has been generated by introducing point mutations at amino acids 419 and 420 as described previously (17).

To create mutations in leucine-rich domains of GFP-VDR, we mutated leucines to alanines in leucine-rich sequences amino acids 219, 221, and 224 (ex1GFP-VDR), amino acids 221, 224, and 227 (ex2GFP-VDR), amino acids 224, 227, and 230 (ex3GFP-VDR), amino acids 227, 230, and 233 (ex4GFP-VDR), amino acids 320, 323, and 325 (ex5GFP-VDR), amino acid 333 (ex6GFP-VDR), amino acids 390 and 393 (ex7GFP-VDR), and amino acids 417 and 420 (cofGFP-VDR). To create mutations in the calreticulin-binding sites of GFP-VDR at amino acids 47 and 48 (calGFP-VDR) and YFP-RXR at amino acids 158 and 159 (calYFP-RXR), we mutated phenylalanines to alanines.

In all constructs, including the mutants, the coding sequences for the fusion proteins were confirmed using the ABIPrism sequencing kit (Perkin-Elmer Corp., Norwalk, CT).

Microscopy
COS-7 cells were plated on chambered cover slips (Nalge Nunc International, Naperville, IL) and transfected with either wild-type or mutant GFP-VDR and YFP-RXR (0.25 µg/slide), or combinations of wild-type GFP-VDR (0.25 µg/slide) and wild-type or mutant RXR-BFP (0.5 µg/slide) using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). After transient transfection, cells were used for microscopy within 48 h. Selected experiments were done in the presence of 10 µg/ml cycloheximide. Cells were treated with calcitriol as indicated (generous gift from Dr. M. Uskokovic, Hofmann-LaRoche, Nutley, NJ) or with vehicle (0.1% ethanol). Selected experiments were done either in the presence of 2–10 nM LMB (a gift from Dr. B. Wolff, Novartis Pharmaceuticals, Vienna, Austria) or vehicle (0.1% ethanol).

Images were collected from a Carl Zeiss Axiovert 100 fluorescent microscope equipped with a LSM 410 laser-scanning unit (Carl Zeiss, Thornwood, NY) using a 100x 1.4 NA or a 63x 1.2 NA objective. The 488-nm line of a krypton-argon laser with a band-pass 510–525 nm emission filter was used for GFP and YFP detection, and the 364-nm line of a UV laser with a 397-nm long-pass emission filter for BFP detection.

FRAP was recorded to explore the shuttling of receptors between the cytoplasm and nucleus. To minimize photodamage to the cells, we paid special attention to precise alignments of the laser and pinholes for these experiments. We took advantage of the feature that about 1% of COS-7 cells form syncytia (45). All FRAP experiments were performed on a temperature-controlled microscope stage. After equilibration to the desired temperature, a preexposure image was recorded from an area containing a multinucleated fluorescing cell. Then a 16-pixel (2 µm2) area was chosen within one of the nuclei and exposed at full power for 1 sec/pixel using the 488-nm line of the krypton-argon laser. Immediately after this photoexposure, sequential images were taken every 10–30 sec from the same microscope field as the preexposure image for up to 30 min. To minimize photobleaching during the recovery phase, images were taken with 10% of laser power and 30% attenuation. For quantitative analysis of digitized images, the area and brightness values were determined over the bleached and the unbleached nuclei. For control, brightness values of the background and neighboring cells were also measured.

Export Assays
The export assay was modified from Refs. 46 and 47 . COS-7, GL48, and CYR cells were plated on chamber slides (Nalge Nunc International). COS-7 cells were transfected with expression vectors encoding GFP-VDR, calGFP-VDR, YFP-RXR, calYFP-RXR, and GFP-VDR (0.25 µg/well of a two-chamber slide) together with either RXR-BFP or hdRXR-BFP (0.5 µg/well of a two-chamber slide). Lipofectamine 2000 was used for transfection according to manufacturer’s instructions. Cells were used for the export assay 24 h after transfection.

Cells were pretreated for 1 h with vehicle, 2 nM LMB, 10 nM calcitriol, or 100 nM 9-cRA. In a subset of experiments, GL48 cells were pretreated with 10 nM calcitriol for 1 h and then incubated with 2 nM LMB for another hour. Then, cells were washed with ice-cold TB [containing 20 mM HEPES, pH 7.3; 110 mM potassium acetate; 5 mM magnesium acetate; 1 mM EGTA; one protease inhibitor cocktail tablet per 50 ml (Roche Diagnostics, Mannheim, Germany); 15 µg/ml cycloheximide (Biomeda, Hayward, CA); 1% rabbit reticulocyte lysate (Sigma, St. Louis, MO)]. Subsequently, cells were incubated with TB or with TB supplemented with 50 µg/ml digitonin (Sigma) for 10 min on ice. Then, cells were washed twice with ice-cold TB and incubated in TB for 1 h at 37 C. In another set of experiments the nuclear membrane was permeabilized using 0.5% Triton X-100 in TB. Wheat germ agglutinin conjugated with Alexis 590 (Molecular Probes, Inc., Eugene, OR) in a concentration of 0.5 mg/ml was used to confirm integrity or disruption of the nuclear membrane in either experiment. Results were consistent with previous reports indicating that digitonin treatment does not perturb nuclear pore structure and functions (48). The same concentrations of calcitriol, 9-cRA, or LMB were maintained during both the permeabilization and the transport phases of the assay. After incubation, cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 10 min at room temperature, and then washed with Dulbecco’s PBS, and mounted. Residual brightness values from digitized images were determined over 50–150 cell nuclei from nonpermeabilized cells (control) and compared with values over nuclei of digitonin-permeabilized cells. Data are expressed as percentage, mean ± 1 SE.

Transactivation Assays
CV-1 or COS-7 cells were subcultured into 12-well plates (Corning, Inc., Corning, NY) and after 24 h, cells were transfected using Lipofectamine 2000 reagents. Transcriptional activities of VDR fusion proteins were tested in cotransfection experiments. Cells were transfected with the receptor expression plasmids (0.3 µg/well), the luciferase reporter plasmid p24OH/Luc (0.07 µg/well; gift from H. F. DeLuca, University of Wisconsin, Madison, WI), and the ß-galactosidase standardization plasmid pGL3 (0.7 µg/well; Promega Corp., Madison, WI). The p24OH/Luc plasmid encodes a luciferase gene that is under the control of the 25-hydroxyvitamin D3 24-hydroxylase promoter (49). Receptor expression plasmids were combinations of GFP-VDR with RXR-BFP or nlsRXR-BFP. Transcriptional activities of YFP-RXR and nlsYFP-RXR were tested by cotransfection experiments. Cells were transfected with expression plasmids (0.3 µg/well) together with DR1-Luc (0.07 µg/well; gift from Dr. S. Minucci, European Institute of Oncology, Milan, Italy) and the ß-galactosidase standardization plasmid pGL3 (0.7 µg/well) (24). Transfected cells were treated with 10 nM calcitriol, 100 nM 9-cRA (Sigma) or vehicle (0.1% ethanol) in culture media for 24 h. Then, cells were lysed on the plates, and luciferase activities of cell lysates were determined with reagents from Promega Corp. ß-Galactosidase activities were determined with reagents from Sigma, as described previously (34). Luminescence data were normalized to ß-galactosidase values and expressed as fold induction relative to vehicle-treated controls. Experiments were done with triplicate samples and repeated at least twice. Data are presented as mean ± 1 SD.

ROSA I cells were used to assess the effect of LMB on the transcriptional activity of endogenous VDR. Cells were plated into 12-well plates and on the next day treated with either vehicle (0.2% ethanol) or 10 nM calcitriol. In another set of experiments, cells were pretreated with 2 nM LMB or 0.2% ethanol for 3 h, and then each pretreatment group was treated with either vehicle or 10 nM calcitriol. A third set of experiments applied LMB and vehicle simultaneously with calcitriol and vehicle. A fourth set of experiments applied first 10 nM calcitriol or vehicle, and 3 h later 2 nM LMB or vehicle was added. Twenty-four hours after drug treatment, cells were lysed on the plates and luciferase activities were determined as described above.


    ACKNOWLEDGMENTS
 
We are grateful to Dr. S. Minucci (EIO, Milan, Italy) for the RXRE DR-1-Luc plasmid; to Dr. H. F. DeLuca (University of Wisconsin, Madison, WI) for the p24OH/Luc23 plasmid and the ROSA I cells; and to Dr. M. Uskokovic for the calcitriol. We thank Dr. B. Wolff (Novartis) for providing LMB.


    FOOTNOTES
 
Abbreviations: AR, Androgen receptor; BFP, blue fluorescent protein; 9-cRA, 9-cisretinoic acid; CYR, CV-1 cells stably expressing YFP-RXR; DR-1/Luc, DR-1 luciferase reporter; ER, estrogen receptors; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; GFP-VDR, chimera of green fluorescent protein with VDR; GL48, HEK-293 cells stably expressing GFP-VDR; GR, glucocorticoid receptors; LMB, leptomycin B; NES, nuclear export receptors that bind to specific export signal sequences; NLS, nuclear localization signal; 24-OH/Luc, 24-hydroxylase luciferase reporter; PR, progesterone receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; TB, transport buffer TR, thyroid hormone receptor; VDR, vitamin D receptor; YFP, yellow fluorescent protein; YFP-RXR, chimera of yellow fluorescent protein with RXR{alpha}.

Received for publication December 19, 2001. Accepted for publication April 30, 2002.


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 DISCUSSION
 MATERIALS AND METHODS
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