Nucleocytoplasmic Trafficking of Steroid-free Glucocorticoid Receptor*

Robert J. G. HachéDagger §, Raymond TseDagger , Terry ReichDagger , Joanne G. A. Savory, and Yvonne A. LefebvreDagger §parallel

From the Departments of Dagger  Medicine and § Biochemistry, Microbiology, and Immunology and the  Graduate Program in Biochemistry, University of Ottawa, The Loeb Health Research Institute at the Ottawa Hospital, Ottawa, Ontario K1Y 4E9, Canada

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Glucocorticoid receptor (GR) recycles between an inactive form complexed with heat shock proteins (hsps) and localized to the cytoplasm and a free liganded form that regulates specific gene transcription in the nucleus. We report here that, contrary to previous assumptions, association of GR into hsp-containing complexes is not sufficient to prevent the shuttling or trafficking of the GR across the nuclear membrane. Following the withdrawal of treatment with cortisol or the hormone antagonist RU486, GRs recycled rapidly into hsp-associated, hormone-responsive complexes. However, cortisol-withdrawn receptors redistributed to the cytoplasm very slowly (t1/2 = 8-9 h) and RU486-withdrawn receptors not at all. Persistent localization of these GRs to the nucleus was not due to a gross defect in export, since in both instances the complexed nuclear GRs transferred efficiently between heterokaryon nuclei. Moreover, the addition of a nuclear retention signal to the N terminus of GR induced the transfer of naive receptor to the nucleus in the absence of steroid. These results suggest that the localization of GR to the cytoplasm is determined by fine control of the rates of transfer of GR across the nuclear membrane and/or by active retention that occurs independently from the association of GR with hsps.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the absence of hormone, steroid receptors are packaged into similar multiprotein complexes that migrate at 8 S on sucrose gradients (1, 2). These complexes contain heat shock proteins (hsps),1 most particularly hsp90 (3). They also contain hsp90-associated proteins, such as hsp70, p23, p14, and high molecular weight immunophilins, including Cyp40 and FKBP56 (3). Hormone binding induces dissociation of the active receptors from the hsp complexes and promotes sequence-specific DNA binding and transcriptional regulation. Steroid binding is transient, and the loss of hormone from the receptor leads to a repackaging or recycling of the receptor into the hsp-containing complex (4). For the glucocorticoid receptor (GR), association with hsps is required for steroid binding (5, 6).

When liganded, steroid receptors are shuttling proteins that traffic continuously between nucleus and cytoplasm (7-9). The primary steroid receptor nuclear localization signal (NLS), NL1, overlaps with the C-terminal ends of the receptor DNA binding domains (10-13). Additionally, the receptor for glucocorticoids (GR) has a second, ligand-dependent NLS (NL2) in the ligand binding domain whose sequence has not been delimited (11). Receptor nuclear export signals, however, remain to be identified. For GR, nuclear localization also appears to be dependent in large part on nuclear retention mediated through the binding of the receptors to DNA (14, 15), the nuclear matrix (16, 17), and other nuclear components (18).

Despite similarities in nuclear-cytoplasmic trafficking in the liganded state, the similar nature of their NLSs, and a common association into hsp-containing complexes, steroid receptors are differentially localized in the cell in the absence of hormone. Unliganded estrogen (ER) (19) and progesterone receptors (PR) (20) are nuclear, while naive GR, mineralocorticoid receptor, and possibly androgen receptor are localized to the cytoplasm (11, 14, 21, 22). The molecular basis for these differences in localization is not understood. However, the nuclear localization of ER and PR does not appear to be due to a defect in nuclear export, since naive PR transfers rapidly between nuclei in heterokaryon fusion experiments (9).

The usual explanation offered for the cytoplasmic localization of naive GR is that its association into the hsp-containing complex acts to mask its NL1 in a way that prevents recognition by the nuclear import machinery (23-25). This proposal is supported by a study demonstrating that hsp association of GR prevents the binding of an NL1-specific antibody (26).

However, other results are inconsistent with this simple model. For example, the redistribution of GR to the cytoplasm upon hormone withdrawal occurs far more slowly than could be expected to be accounted for by the time required for hormone dissociation and hsp reassociation (14, 27-29). This is true even for GR DNA binding mutants that never become more than 75% nuclear in the presence of hormone (14). Second, overexpression of GR in many cell lines results in the nuclear localization of the hsp-associated receptor, without discernible change in other properties (30, 31). Third, treatment of cytoplasmic GRs expressed by transient transfection with the hormone antagonist RU486 results in a nuclear GR that reacquires steroid responsiveness following antagonist withdrawal but does not redistribute to the cytoplasm (14, 32).

In this study, we have directly examined the nucleocytoplasmic trafficking of hormone-free GR prior to hormone treatment and following hormone withdrawal. Our results demonstrate that hsp-associated GRs exchange continuously between nucleus and cytoplasm and suggest that the cytoplasmic localization of naive receptors occurs through a process that is more complex than has been previously appreciated.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Culture, Plasmids, and Transfections-- GrH2 cells (derived from a rat hepatoma cell line stably transfected with rat GR, expressing approximately 100,00 GR molecules/cell) (33), COS7 cells (ATCC CRL-1651), or NIH 3T3 (ATCC CRL-6474) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum.

For chloramphenicol acetyl transferase (CAT) assays, lipofectamine (15 µg) was used to transfect GrH2 cells with plasmids RSV-beta gal (1 µg), to monitor transfection efficiency, and pBLMMTVCAT (34) (MMTV -631/+105, 3 µg), as the reporter gene. Sixteen hours after transfection, the cells were switched to serum-free medium. After 16 h in serum-free medium, hormonal treatments were initiated. RU38486 (RU486) was generously provided by RousselUclaf. Cells were treated and harvested as indicated in individual experiments. CAT assays were carried out as we have previously described (14). beta -Galactosidase activity was used to correct transfection efficiency, and the results displayed are the average of three experiments performed in duplicate.

The DNA-binding domain of c-Abl, amino acids 862-959 (35), was cloned as a StuI-SalI fragment into the pTL-GR expression vector (36) and into a pTLmyc-GR vector with Lys-Asn mutations at amino acids 513-515.

Indirect Immunofluorescence (IIF)-- To monitor the subcellular distribution of GR by immunofluorescence, GrH2 or COS7 cells were plated onto poly-L-lysine (500 µg/ml)-coated glass coverslips and incubated overnight in 35-mm tissue culture plates in DMEM containing 10% charcoal-stripped fetal calf serum. Cells were synchronized to G0 by incubation in serum-free DMEM for a further 21 h prior to the initiation of treatment. Dexamethasone, cortisol, and RU486 were added to final concentrations of 10-6 M in serum-free medium. To monitor redistribution of GR to the cytoplasm following hormone withdrawal, cells pretreated with cortisol (10-6 M) or RU486 (10-6 M) for 1 h were washed three times with phosphate-buffered saline and twice in serum-free medium with 5% bovine serum albumin (BSA). Subsequent incubation was in serum-free supplemented with BSA to 5%.

For heterokaryon analyses, GrH2 cells were co-plated with NIH 3T3 cells onto poly-L-lysine-coated coverslips. After attachment, the cells were cultured in serum-free medium for 16 h, treated with RU486 (10-6 M) or cortisol (10-6 M) for 1 h, and washed as described above and then with serum-free medium for 2 h before fusion. Cell fusion was performed by adding 50% polyethylene glycol 4000 (Life Technologies, Inc.) for 2 min at 37 °C (37). The cells were then washed five times with calcium/magnesium-free Hanks' balanced salt solution and incubated for 4 h in serum-free DMEM before being processed for IIF. Nuclei from GrH2 cells and NIH 3T3 cells were identified by staining with the Hoechst dye 33258 at 0.5 µg/ml.

IIF was carried out exactly as described previously (14) with primary murine BUGR-2 antibody (Affinity BioReagents, Inc.). Quantification was performed using double-blind encryption. For each time point, at least 400 stained cells were counted in each experiment. Each experiment was repeated 3-8 times over a period of several months.

Biochemical Fractionation-- To prepare cytoplasmic and nuclear fractions, GrH2 cells (5 × 105 cells/sample) were collected, washed, and suspended in hypotonic buffer (10 mM HEPES, pH 7.9, at 4 °C, 1.5 mM MgCl2, 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol). The cells were lysed with a Dounce homogenizer (B pestle), and nuclei were collected by centrifugation (3000 × g) (38). For each experiment, we confirmed before fractionation that fewer than 5% of the homogenized cells excluded trypan blue. The supernatant was saved as the cytosolic fraction. Nuclei were suspended in a low salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 0.02 M KCl), and proteins were extracted with a high salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 1.2 M KCl) over 30 min. Prior to fractionation through 10% SDS-polyacrylamide gels, GRs were concentrated and desalted by immunoprecipitation from the cytosolic and nuclear fractions as described previously (14, 39). After electrophoresis, samples were electrophoretically transferred to Immobilon P (Millipore Corp., Bedford, MA), and GR was probed on Western blots with BuGR-2 and sheep anti-mouse IgG conjugated to horseradish peroxidase (Amersham Pharmacia Biotech). Detection of GR was by enhanced chemiluminescence (Amersham Pharmacia Biotech). Chemiluminescent signals were quantified using a CH imaging screen (Bio-Rad) on a Bio-Rad GS-525 Molecular Imager.

Analysis of GR by Sucrose Density Gradient-- Whole cell extracts of GrH2 cells prepared as described previously (14) were fractionated through 15-30% sucrose gradients for 16 h at 55,000 rpm at 4 °C (40). 300-µl fractions were collected, immunoprecipitated with BuGR-2, and fractionated through 10% SDS-polyacrylamide gels. Chemiluminescent signals were quantified using a CH screen on a Bio-Rad GS-525 Molecular Imager. Markers for the gradients were aldolase (7.3 S) and BSA (4.6 S).

Steroid Binding Assays-- GrH2 cells were cultured overnight in serum-free DMEM supplemented with 5% BSA prior to treatment with cortisol (10-6 M) or RU486 (10-6 M) for 1 h. Cells were withdrawn from ligand treatment in serum-free media and harvested at the time points indicated, and cell number was determined before resuspension in phosphate-buffered saline containing either 3H-cortisol or 3H-cortisol + 50 µM cold cortisol (1000-fold excess) as described previously (41). After a 30-min incubation, the cells were washed five times with phosphate-buffered saline and counted in a liquid scintillation counter. Experiments were performed three times in duplicate, and values were compared with the initial level of specific cortisol binding in untreated cells.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

RU486-withdrawn GR Fails to Redistribute to the Cytoplasm-- Maintenance of GR-expressing cells in culture in the absence of serum allows for the examination of the subcellular trafficking of a stable pool of GR without appreciable new synthesis or degradation of the receptor molecules for periods exceeding 48 h (14). Further, under these culture conditions, the cells mimic the usual G0-G1 state of differentiated glucocorticoid-responsive cells in the majority of mammalian target tissues (42). Previously, we have observed in transient transfection experiments in which GRs were overexpressed that redistribution of GR to the cytoplasm following hormone withdrawal occurred only over a period of several hours (14). Receptors in RU486-treated cells, however, failed to redistribute appreciably to the cytoplasm for at least 48 h following withdrawal of treatment.

To begin a detailed analysis of the exchange of unliganded GRs between nucleus and cytoplasm that underlies their localization, we sought to determine whether GRs expressed at more physiological levels redistributed similarly to overexpressed receptor in response to hormone agonists and antagonists. In untreated GrH2 cells, which stably express rat GR and have been used extensively to study glucocorticoid hormone action (7, 43), the receptor is entirely localized to the cytoplasm prior to hormone treatment (7). To monitor the time course of redistribution of GRs in these cells, we employed an indirect immunofluorescence assay that we and others have previously demonstrated to allow quantification of the changes in the subcellular localization of steroid hormone receptors over a period of time (13, 14).

Treatment of GrH2 cells with the nonmetabolizable hormone agonist dexamethasone induced a rapid and nearly complete nuclear uptake (t1/2 = 4-5 min, 90-95% nuclear) of GR (Fig. 1). This rate was identical to the one we previously observed for transiently transfected GRs (14) and the rate that others have noted for endogenous GR in rat thymus cells measured by biochemical fractionation (27). Similar uptake results were also obtained with the natural steroid cortisol.2 Nuclear transfer of GR upon RU486 treatment was similar but occurred more slowly (t1/2 = 9-10 min). This difference is believed to reflect a decreased rate of dissociation of RU486-bound GR from the heat shock protein complex rather than a difference in nuclear import (44). Thereafter, in the continuous presence of dexamethasone or RU486, greater than 90% of cells contained GR that remained exclusively nuclear for at least 48 h.


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Fig. 1.   Nuclear uptake of GR after treatment with 10-6 M dexamethasone or 10-6 M RU486. Dexamethasone (bullet ) or RU486 () treatment of GrH2 cells was initiated 16 h after serum withdrawal. Subcellular distribution was determined by semiquantitative IIF as has previously been described (14). Immunofluorescent cells were classified into five categories ranging from completely nuclear (N) to completely cytoplasmic. The percentage of cells with exclusively nuclear distribution is plotted as a function of time after hormone treatment. S.E. values were calculated from eight experiments performed in duplicate. Error bars are not visible, since in this presentation the error was for all samples smaller than the point plotted. Display of the data with a logarithmic time scale facilitates discrimination of localization at early times after treatment. Previous studies have shown that the results obtained with this technique accurately reflect the subcellular distribution of GR obtained by other methods (14).

Upon withdrawal of a 1-h treatment with cortisol, a process that included thorough washing of the cells to ensure complete washout of the steroid, GR redistributed to the cytoplasm only very slowly over an extended period of time (Fig. 2, t1/2 = 8-9 h). This contrasts with the rapid off rate (t1/2 = 10 min) of cortisol and RU486 from GR (27). More strikingly, upon withdrawal of RU486 treatment, GR remained fully nuclear and did not appreciably redistribute to the cytoplasm. Further, the persistence of GR in the nucleus upon ligand withdrawal is unlikely to reflect the low residual level of intracellular ligand, since we have previously demonstrated, in transient transfection experiments, that a GR mutant with a 10-fold lower affinity for ligand responded to the withdrawal of RU486 and cortisol identically to WT GR (14).


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Fig. 2.   Redistribution of GR from the nucleus of GrH2 cells after withdrawal of cortisol or RU486 from the serum-free tissue culture medium following a 1-h treatment. Withdrawal from 10-6 M RU486 () or 10-6 M cortisol (bullet ) was initiated by extensive washing, followed by incubation in ligand-free medium as described under "Materials and Methods." To facilitate comparison of the rates of redistribution of cortisol and RU486-treated GRs from the nucleus upon withdrawal, values for nuclear occupancy are expressed as a percentage of the equilibrium nuclear localizations at 1 h after the addition of ligand (t0). The percentages of immunofluorescent cells with exclusively nuclear distribution (N) are displayed. The values represent the average of three independent experiments performed in duplicate. Again, the S.E. values are not illustrated, since they were smaller than the size of the points. The addition of cycloheximide (50 µg/ml) to incubations did not alter kinetics of export.2

RU486 and Cortisol-withdrawn GRs Rapidly Reassemble into an 8 S Complex Able to Bind Ligand and Regulate Transcription-- Previously, it has been demonstrated that upon withdrawal of hormone agonist, the subnuclear localization of GR is altered prior to its redistribution to the cytoplasm (29), but the association of GR with heat shock proteins during this period has not been examined. However, this intermediate state is marked by an increased sensitivity of the GR in the agonist-withdrawn cells to biochemical extraction.

In GrH2 cells treated with cortisol or RU486 for 1 h, virtually 100% of the GRs remained nuclear upon hypotonic lysis of the cells at 4 °C (Fig. 3A). However, 2 h after withdrawal of cortisol, a time when over 90% of the GRs were nuclear according to immunofluorescence data, the receptor was completely lost into the cytosol upon hypotonic cellular lysis. We observed the same result with RU486-withdrawn cells. Within 2 h of withdrawal of RU486, essentially all of the receptor fractionated biochemically into the cytosol upon hypotonic lysis of the cells (Fig. 3A).


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Fig. 3.   RU486-withdrawn receptors rapidly reassociate into 8 S complexes that are lost from the nucleus upon hypotonic cellular lysis. A, biochemical fractionation of RU486-withdrawn receptors. GrH2 cells in serum-free medium were fractionated by hypotonic cellular lysis into nuclear (N) and cytoplasmic (C) fractions following treatment with either 10-6 M dexamethasone or 10-6 M RU486 for 1 h (withdrawal = t0) and after 2 h of withdrawal from the cortisol/RU486 treatment in serum-free medium (t = 2 h). GRs quantitatively immunoprecipitated from the extracts were fractionated on a 10% SDS-polyacrylamide gel and identified by Western blotting with GR antibody BuGR2 (14). The distribution of GRs between the two fractions was quantified by a phosphor imager. B, sucrose density analysis of GR withdrawn from RU486 treatment for 2 h. Following a 1-h treatment with 10-6 M RU486 and a 2-h withdrawal period, a whole cell extract of GrH2 cells was fractionated over linear (15-30%) sucrose density gradients with aldolase (7.3 S) and BSA (4.6 S) markers. GRs in individual fractions were immunoprecipitated, resolved on 10% SDS-polyacrylamide gels, and quantified by phosphor imager analysis of Western blots. Migration of the RU486-withdrawn GR (solid line) is compared with that of naive GR extracted from the cytoplasm of untreated GrH2 cells (dashed line) and dexamethasone-liganded GRs extracted from the nucleus of cells treated with 10-6 M dexamethasone for 1 h (dotted line).

The loss of nuclear occupancy upon hypotonic cellular lysis following the withdrawal of hormone was reminiscent of both the facile nuclear extraction of liganded GRs containing mutations that eliminate DNA binding (14) and that of untreated nuclear ERs and PRs associated with hsps (45). Association of GRs with heat shock proteins is reflected on sucrose gradients by a shift in sedimentation coefficient from 4 to 8 S (1). To determine whether GR in RU486-treated cells had recycled into an 8 S complex upon withdrawal of treatment, the cytosol from hypotonically lysed cells shown in Fig. 3A was fractionated over sucrose gradients (Fig. 3B). Within 2 h of the withdrawal of treatment, GRs from the RU486-treated cells appeared to be completely reassociated with hsps into 8 S complexes.

Association of GR into the hsp complex occurs through a highly ordered, ATP-dependent process that can be efficiently reconstituted in rabbit reticulocyte lysate at 30 °C (4, 46) but that is not known to occur in cellular extracts at 4 °C (3). Nonetheless, to discount the possibility of artifactual reassociation of GR with hsps in our cellular extracts, we carried out several control experiments. In the first instance, we assessed the ability of heat-transformed GRs to reassociate with hsps in cellular extracts at 4 °C. Following heat transformation in whole cell extract, incubation of the GR-containing extract for 30 min on ice in the presence of molybdate was completely ineffective in promoting the reassociation of GR into the 8 S complex.2 This was also true for heat-transformed in vitro translated GR mixed with whole cell extract that had been spared the heat treatment and for cellular GRs in extracts supplemented with untreated whole cell extracts from the GR- parental cell line of GrH2 cells.2 Therefore, we conclude that the rapid reassociation of GRs with heat shock proteins upon withdrawal of RU486 was not sufficient to initiate the redistribution of GR to the cytoplasm. Similarly, reassociation of cortisol-withdrawn GRs into 8 S complexes was complete at 2 h, when only 10% of the receptors had redistributed to the cytoplasm.2

Ligand binding by GR is strictly dependent upon an ordered assembly of the receptor with the heat shock proteins that fixes the receptor ligand binding domain in a conformation competent to bind ligand (4-6). To determine whether cortisol and RU486-withdrawn GRs also reacquired ligand binding ability more rapidly than they redistributed to the cytoplasm, we performed whole cell steroid binding assays on naive cells and cells withdrawn for increasing times from treatment with cortisol and RU486 (Fig. 4). The results indicate that over 75% of high affinity glucocorticoid binding sites were regenerated in GrH2 cells within 1 h of the withdrawal of the cells from either cortisol or RU486. Indeed, approximately 40% of the binding sites were regenerated within 30 min of withdrawal. Thus, upon withdrawal of steroid treatment, the GRs reassociated into 8 S complexes competent to bind steroid well in advance of their redistribution to the cytoplasm. Further, it suggests that the failure of GRs in RU486-treated cells to redistribute to the cytoplasm upon hormone withdrawal was not due to a defect in the recycling of the receptor into a hormone-responsive state.


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Fig. 4.   RU486 and cortisol-withdrawn GRs rapidly recover ligand binding activity. GrH2 cells in serum-free medium were treated with 10-6 M cortisol or 10-6 M RU486 for 1 h. Following withdrawal of the stimuli, the cells were withdrawn from ligand in serum-free media and harvested at the times indicated. Whole cell binding assays to measure specific GR binding sites were performed in duplicate in phosphate-buffered saline with 3H-cortisol for 30 min at 0 °C. Binding to the withdrawn cells is expressed as a percentage of the level of binding in untreated cells (un, stippled bar), which was approximately 100,000 sites/cell, similar to previous reports (33). Cortisol binding upon withdrawal from cortisol is shown by the solid bars, while cortisol binding upon withdrawal from RU486 is represented by the open bars. The data shown is the average ± S.E. of three experiments performed in duplicate.

To evaluate the effect of recycling of GR from RU486 treatment in the nucleus on its ability to activate transcription in response to subsequent hormonal stimuli, we monitored transcription from a mouse mammary tumor virus (MMTV) reporter gene containing a strong glucocorticoid-responsive promoter (Fig. 5). RU486 is known to be a partial glucocorticoid agonist that induces a weak response from the MMTV promoter. Treatment of GrH2 cells transiently transfected with an MMTV CAT reporter gene with RU486 for 1 h had only a small effect on CAT activity produced from the MMTV promoter (lanes 1 and 2). The CAT activity recorded thereafter was the same whether the cells had been withdrawn from RU486 for 1 h or 23 h prior to harvesting (lanes 2 and 4). This indicated that production of the low level of CAT protein in response to RU486 treatment was complete within 1 h of withdrawal of the antagonist. Therefore, subsequent hormone-dependent increases in CAT activity would directly reflect the consequences of secondary steroidal treatment on transcription of the reporter gene.


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Fig. 5.   Induction of MMTV transcription by steroid in RU486-withdrawn GrH2 cells. GrH2 cells transfected with an MMTVCAT reporter plasmid were placed in serum-free medium for 16 h prior to the initiation of hormonal treatments, which was taken as the starting time for experiments. Cells harvested for CAT assay were treated as follows. Lane 1, control, incubated 2 h in serum-free medium; lane 2, 1 h with 10-6 M RU486, followed by 1 h of withdrawal in serum free medium; lane 3, control incubated an additional 24 h in serum-free medium; lane 4, 1 h with 10-6 M RU486, followed by 23 h of withdrawal in serum-free medium; lane 5, 1 h with 10-6 M RU486 followed by withdrawal for 2 h in serum-free medium and secondary treatment with 10-6 M dexamethasone for a further 21 h; lane 6, 3-h initial incubation in serum-free medium followed by a 21-h treatment with 10-6 M dexamethasone. Numerical values for CAT activities are shown above the bars and represent the average ± S.E. from three experiments performed in duplicate.

This enabled us to compare the steroid hormone induction of MMTV transcription in naive cells with that in cells subjected to a cycle of a 1-h RU486 treatment and a 1-h withdrawal. Treatment of naive cells with dexamethasone for 21 h, at a time after transfection equal to the completion of the 1-h RU486 withdrawal, led to a strong induction of CAT activity (lane 6). Secondary treatment of cells with nuclear, RU486-withdrawn GRs resulted in a similar total accumulation of CAT activity 21 h after dexamethasone treatment (lane 5). When corrected by subtraction for the contributions to the total CAT activity made by the hormone-independent and RU486-dependent accumulation of CAT, the primary and secondary responses to dexamethasone were almost identical. A similar result was also obtained when the period of secondary treatment was initiated 23 h following withdrawal of RU486.2 Together, these results indicated that the nuclear RU486-withdrawn GRs were not discernibly different from the cytoplasmic GRs in untreated cells in their association into a high molecular weight complex, ability to bind steroid hormone, and ability to activate transcription in response to steroid treatment. However, they did appear to be permanently localized to the nucleus.

Persistent Nuclear Localization of RU486-treated GRs Is Reversible-- In order to determine whether the nuclear localization of RU486-withdrawn GR could be reversed by secondary steroid treatment, the localization of GR in GrH2 cells was monitored by immunofluorescence following the withdrawal of a secondary cortisol treatment from cells initially treated with RU486 (Fig. 6). For the first cycle, the cells were treated with RU486 for 1 h, washed extensively, and incubated for 2 h in hormone-free medium. Secondary stimulation was accomplished by treatment with cortisol for 1 h. The consequences of withdrawal from the cortisol were monitored over the subsequent 24 h.


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Fig. 6.   RU486-withdrawn GR recovers ability to redistribute to the cytoplasm after secondary treatment with steroid agonist. GrH2 cells treated with 10-6 M RU486 for 1 h and then withdrawn from RU486 for 2 h were treated a second time for 1 h with 10-6 M cortisol, followed by withdrawal from cortisol in serum-free medium in the presence (dotted line) or absence (solid line) of 10 µg/ml cycloheximide. Subcellular distribution of GR was monitored by IIF at the time points indicated over the course of the experiment beginning at 1 h following RU486 treatment. The percentage of cells with exclusively nuclear (N) GRs is shown. The data are the average of four separate experiments performed in duplicate ± S.E.

Notably, following withdrawal of this secondary treatment with cortisol, GRs redistributed to the cytoplasm in a manner that closely mimicked the redistribution of the receptor following withdrawal of a primary treatment with cortisol (compare with Fig. 2). These results indicate that the changes in GR, or elsewhere in the cell, that occurred in response to RU486 and led to the long term localization of GR in the nucleus following withdrawal of the antagonist, could be reversed by a subsequent cycle of binding to steroid agonist. In these experiments, it was also clear that the redistribution of GR was not due to the synthesis of new receptor, as maintenance of the cells in cycloheximide throughout the course of treatment resulted in a closely overlapping pattern of subcellular distribution of GR compared with untreated cells.

RU486- and Cortisol-withdrawn, hsp-associated GRs Shuttle between Nucleus and Cytoplasm-- While the export of GR and other steroid receptors from the nucleus is poorly understood, it is known that liganded 4 S GRs traffic continuously or shuttle between nucleus and cytoplasm (7). It has also been demonstrated that unliganded, hsp-associated PRs shuttle continuously between nucleus and cytoplasm despite its apparent nuclear localization (9, 47). One potential explanation for the block in redistribution of GRs to the cytoplasm upon withdrawal of RU486 and for the slow return of GRs in cells withdrawn from steroid treatment was that the export of GRs from the nucleus upon loss of steroid or RU486 was somehow compromised (7).

To investigate this possibility, we evaluated the ability of nuclear unliganded, 8 S GRs to be exchanged between heterologous nuclei in cell fusion experiments (Fig. 7). Following treatment with cortisol or RU486 for 1 h and withdrawal for 2 h (a time at which GRs treated by each ligand migrated at 8 S on sucrose gradients and were competent to bind hormone; Figs. 3B and 4), GrH2 cells were fused with NIH 3T3 cells, and the exchange of GRs between nuclei was assessed by immunofluorescence. Upon fusion to the NIH 3T3 cells, the unliganded, hsp-associated GRs in the nuclei of RU486-withdrawn cells transferred efficiently to the NIH 3T3 nuclei (Fig. 7, A and B). Similarly, the GRs in GrH2 cells withdrawn from cortisol also redistributed equally between the two nuclei of the heterokaryons at this early time following hormone withdrawal when the GRs were still localized to the nucleus (Fig. 7, C and D). Thus, these results indicated that unliganded, hsp-associated GRs in cells withdrawn from steroid or RU486 treatment shuttled continuously between nucleus and cytoplasm. For GRs withdrawn from cortisol, this continuous shuttling between nucleus and cytoplasm was separate from the slow relocalization of GRs to the cytoplasm observed above. Therefore, the failure of GRs to redistribute rapidly to the cytoplasm upon withdrawal of cortisol or RU486 could not be accounted for by loss of the ability of the GRs to be exported from the nucleus.


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Fig. 7.   GRs traffic between nuclei of heterokaryons 2 h after the withdrawal of RU486 and cortisol treatment. GrH2 and NIH 3T3 cells plated together at high density incubated for 16 h in serum-free medium and then were treated with 10-6 M RU486 (A and B) or 10- 6 M cortisol (C and D) for 1 h. The cells were then washed and incubated for a further 2 h in serum-free medium. Cellular fusion was promoted by treatment with polyethylene glycol. After fusion, the cells were incubated for a further 4 h in serum-free medium. The cells were then processed for detection of GR by IIF (A and C). Hoechst dye was used to distinguish GrH2 nuclei from NIH 3T3 nuclei (B and D). The arrows point to NIH 3T3 and GrH2 nuclei in the right panels. Immunofluorescence in both GrH2 and NIH 3T3 cells indicates the transfer of GR to the NIH 3T3 nuclei. The images displayed are examples of the results consistently observed in over 400 fused cells viewed over three independent cell fusion experiments. FITC, fluorescein isothiocyanate

Localization of Unliganded GR to the Nucleus by the Addition of a Nuclear Retention Signal-- Unliganded 8 S GR prior to exposure to hormone has been proposed to be localized to the cytoplasm through a masking of the nuclear localization signals by the heat shock protein complex (23-25). However, to date there has been no direct demonstration that unliganded cytoplasmic GR does not transiently enter the nucleus. By contrast, our results indicated that the packaging of GR into hormone-responsive nuclear 8 S complexes following hormone withdrawal was not a barrier to the nuclear import of GR. In order to determine whether the unliganded GRs localized to the cytoplasm of untreated cells might also possess the ability to transiently shuttle into the nucleus, we designed an experiment that would allow us to directly visualize the accumulation of untreated GRs in the nucleus. Specifically, we reasoned that if unliganded naive GRs shuttled transiently between nucleus and cytoplasm, then the addition to GR of a signal demonstrated to promote protein retention in the nucleus might be expected to shift the equilibrium distribution of GR in untreated cells toward the nucleus.

The creation of GR fusion proteins by adding sequences to the N terminus of GR is not known to force redistribution of GR to the nucleus in the absence of nuclear localization activity within the fragment. Indeed, fusion of heterologous proteins with the ligand binding domain of GR has long been used as a tool to regulate the nuclear entry of protein fragments (48-50).

We have previously reported that the ability to bind DNA promotes the nuclear retention of liganded, transformed GR, following the entry of GR into the nucleus (14). c-Abl is a nuclear tyrosine kinase that has the ability to bind DNA (51). Unlike most other DNA-binding proteins (10), the c-Abl DNA binding domain is not linked to an NLS-like sequence (35). Thus the c-Abl DNA binding domain alone is unable to promote the transport a cytoplasmic protein into the nucleus (35). Therefore, to test our hypothesis that the unliganded GRs in untreated cells can exchange transiently between nucleus and cytoplasm, we sought to determine whether the 97-amino acid-long c-Abl DNA binding domain expressed fused to the N terminus of GR would alter the localization of GR in the absence of ligand (Fig. 8).


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Fig. 8.   The addition of a nuclear retention sequence to GR directs naive, unliganded receptor to the nucleus. IIF of COS7 cells transiently transfected to express either c-Myc-GR fusion protein (A and B), GFP-GR fusion protein (C and D), c-Abl-GR fusion protein with three Lys-Asn substitutions in the GR NL1 (E and F), c-Abl-GR fusion protein (G and H). IIF was performed on cells incubated in serum-free medium, prior to (A, C, E, and G) and following a 2-h treatment with 10-6 M dexamethasone (B, D, F, and H).

This experiment was performed by expressing GR fusion proteins by transient transfection into COS7 cells as we have previously described (14) and monitoring the localization of the fusion proteins by indirect immunofluorescence. Initially, several controls were performed to ensure that any nuclear transfer of the Abl-GR fusion protein would correlate directly with the ability of the c-Abl DNA binding domain to interact with DNA. First, to reiterate that the fusion of peptides to the N terminus of GR do not affect the hormone responsiveness of GR or nonspecifically promote the transfer of GR to the nucleus by promoting the exposure of the constitutive GR NLS, we examined the localization of GRs with N-terminal fusions of a 6× c-Myc epitope tag or green fluorescent protein (Fig. 8, A-D)

Expression of GR with the 82-amino acid c-Myc epitope tag (Fig. 8, A and B) or the 240-amino acid GFP sequence (Fig. 8, C and D) resulted in the expression of GR fusion proteins that were fully cytoplasmic in the absence of hormone (<3 ± 2% nuclear; Fig. 8, A and C) and that transferred indistinguishably from WT GR to the nucleus in response to dexamethasone (94 ± 3% nuclear; Fig. 8, B and D).

Second, expression of the c-Abl DNA binding domain fused to GR containing three point mutations that inactivate the basic NL1 motif of the receptor3 was also completely localized to the cytoplasm in the absence of hormone (92 ± 3% cytoplasmic; Fig. 8E). This result confirmed that the c-Abl DNA binding domain has no inherent nuclear localization potential. Upon the addition of hormone, this chimeric receptor transferred partially to the nucleus (21 ± 3% nuclear + nuclear > cytoplasmic, 50 ± 6% nuclear = cytoplasmic; Fig. 8F). This behavior closely mimics that expected for WT GR whose entry into the nucleus is dependent solely upon the hormone-dependent NL2 activity in the receptor ligand binding domain (52, 53).

By contrast, the fusion protein with the c-Abl DNA binding domain fused to WT GR was localized mainly to the nucleus prior to any exposure of the cells to hormone (81 ± 3% nuclear + nuclear > cytoplasmic; Fig. 8G) and became fully nuclear in response to treatment of the cells with dexamethasone (96 ± 2% nuclear; Fig. 8H). The localization of c-Abl-GR to the nucleus was not due to inappropriate dissociation of the heat shock proteins, since transcriptional responses to the c-Abl-GR fusion protein to dexamethasone were the same as that observed for WT GR.2 Further, Western analysis showed that all of the GR constructs expressed in this experiment accumulated in the cells to the same level as wild type GR.2

These data indicate that the GR NL1 was constitutively accessible in c-Abl-GR fusion protein and mediates the hormone-independent import of the hsp-associated protein into the nucleus. They also provide the first evidence that unliganded, hsp-associated, cytoplasmic GRs may constitutively shuttle between nucleus and cytoplasm. Thus, in both the liganded and unliganded states, GR appears to exist in a dynamic equilibrium between the nucleus and cytoplasm.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The cytoplasmic localization of GR prior to the addition of hormone is one tangible difference in the molecular mechanism of action of GR compared with ER and PR for which an explanation remains elusive. In the present work, we have determined that unliganded, hsp-complexed, hormone-responsive GRs exist in a dynamic equilibrium between nucleus and cytoplasm and shuttle continuously across the nuclear membrane. These observations suggest that the nucleocytoplasmic trafficking of unliganded GR is fundamentally similar to the trafficking of ER and PR. Further, our results suggest that the localization of unliganded, hsp-complexed GR to the cytoplasm is accomplished through an active mechanism that can be differentially affected by the transient binding of hormone agonists and antagonists.

8 S, hsp-associated GRs were observed to traffic across the nuclear membrane similarly to the liganded, free receptors. First, following withdrawal from both RU486 and cortisol, GR recycled rapidly into hormone-responsive complexes but redistributed at best only very slowly to the cytoplasm. Nonetheless, despite their prolonged nuclear occupancy, these 8 S GRs transferred efficiently between nuclei in heterokaryon experiments. Thus, the persistence of these GRs in the nucleus following hsp association occurred in the absence of obvious effects on the trafficking of GR across the nuclear membrane.

Second, DNA binding is hypothesized to facilitate the concentration of liganded GR in the nucleus by decreasing the pool of GR available for export (14). That the addition of an ectopic DNA binding domain from c-Abl could stimulate a very strong, NL1-dependent, accumulation of naive GR in the nucleus is substantive evidence that cytoplasmic unliganded GRs are also in a dynamic equilibrium between nucleus and cytoplasm. However, for unliganded WT receptors expressed at physiological levels, this equilibrium is normally expected to strongly favor cytoplasmic localization of the receptor population.

Third, additional support for the nuclear-cytoplasmic trafficking of unliganded, 8 S GRs is found in the reports that overexpression of GR in some cells promotes the partial transfer of GR to the nucleus in the absence of hormonal stimulus. (30, 31). In other experiments, we have recently observed that this shift in the naive GR population toward the nucleus upon receptor overexpression is also dependent upon NL1.3

While our results strongly support the rapid transfer of unliganded GR between cellular compartments, they do not directly address the status of the GR molecules during transport through the nuclear pore. In principle, nuclear pores are large enough to accommodate the passage of molecular complexes considerably larger in size than hsp-complexed receptors (mRNA-protein complexes, for example) (54). Alternatively, steroid receptors have been shown to be in a dynamic equilibrium between hsp-complexed and free forms (55). Therefore, it is also possible that the transfer of unliganded, hsp-complexed steroid receptors across the nuclear membrane is dependent upon transient complex disassembly, followed by reassembly on the other side of the membrane. We note, however, that in our experiments, the loss of 8 S GRs from the nucleus upon hypotonic cellular lysis occurred under conditions that precluded reassociation of 8 S complexes in cytosol. This would favor the direct export of intact 8 S GRs from the nucleus.

Our results indicate that changes in the localization of the GR population between cytoplasm and nucleus are superimposed upon a background of individual receptors shuttling rapidly across the nuclear membrane. What then determines the localization of hsp-associated receptors to nucleus or cytoplasm? Two possibilities seem likely. First, active retention mechanisms may limit the accessibility of 8 S GR to the nuclear-cytoplasmic transport machinery. For example, it has recently been shown that following withdrawal of hormonal ligand GRs become concentrated in a discrete nuclear subcompartment prior to their relocalization to the cytoplasm (12). Targeting 8 S GR to this subcompartment could promote the maintenance of the GR population in the nucleus by decreasing its access to the nuclear export machinery in a manner similar to that discussed above for DNA binding. However, it is also possible that localization of naive GR to the cytoplasm requires active cytoplasmic retention. Active retention in the cytoplasm has been observed for other proteins (56, 57), and such a retention mechanism would be more compatible with the observation that overexpression of GR can lead to nuclear accumulation of the receptor in some cells (30, 31).

Alternatively, since nuclear import and export are now known to involve distinct transporters, is it also possible that changes in the localization of GR reflect changes in the relative activities of the nuclear localization and nuclear export signals on GR. Both import and export are complex processes, and knowledge of active mechanisms to regulate the rates of protein transport across the nuclear membrane through postranslational modification and association with modifying proteins are emerging (58-61).

In this respect, it would appear notable that GR is a phosphoprotein whose phosphorylation state is known to increase and decrease in response to the addition and withdrawal of hormone in actively growing cells (62, 63). Thus, it is tempting to speculate that postranslational modification of the receptor by phosphorylation plays a role in the persistent nuclear localization of 8 S GR following hormone withdrawal. However, several results suggest that the phosphorylation sites that have been identified to date for GR in actively growing cells, which are localized in the N terminus of the receptor, are unlikely to play a direct role in localizing 8 S GR in the cell. First, fusion of the GR N terminus with the ER DNA and ligand binding domains resulted in a protein that was constitutively localized to the nucleus (64). Similar analyses of GR-ER and GR-PR chimeras also suggested that cytoplasmic localization of unliganded GR is determined by its ligand binding domain (13). Second, substitution of individual and multiple phosphorylation sites in the N terminus of GR have been reported to have no effect on the localization of naive receptor to the cytoplasm of asynchronously growing COS1 cells (65). Last, the phosphorylation at these sites is highly dependent upon the cell cycle and mitogenic stimulli with the peak in phosphorylation occurring during G2 (62, 66-69).

Interestingly, however, GR is known to become differentially hyperphosphorylated in response to RU486 compared with glucocorticoid agonist (68, 70). Thus, the differential modification of GR remains an attractive possibility as a mediator of the prolonged but reversible nuclear localization of GR following the withdrawal of RU486 treatment.

Alternatively, it is also possible that the composition of the hsp-GR complex may vary in ways that rapidly lead to hormone-responsive complexes following the withdrawal of RU486 and cortisol but limit the relocalization of the receptor to the cytoplasm. Distinguishing between these possibilities will require a careful comparison of the composition and postranslational modification of cytoplasmic and nuclear 8 S GR complexes that are stably maintained during G0. Characterization of the molecular basis for the persistent nuclear localization of cortisol- and RU486-withdrawn GRs could be expected to contribute toward understanding the biological imperative that has dictated the cytoplasmic localization of unliganded GR but allows for the constitutive nuclear localization of the closely related receptors for estrogens and progestins.

    ACKNOWLEDGEMENTS

We thank our colleague M. Ekker for helpful discussions and comments on the manuscript. We are grateful to K. Yamamoto and D. DeFranco for providing cell lines and plasmids employed in this work and R. Van Etten for providing a c-Abl cDNA. We also appreciate the assistance provided by F. Sackey in establishing the indirect immunofluorescence technique.

    FOOTNOTES

* This work was funded by a grant from the Medical Research Council of Canada (to Y. A. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: The Ottawa Hospital Loeb Research Institute, 725 Parkdale Ave., Ottawa, Ontario K1Y 4E9, Canada. Tel.: 613-761-5142; Fax: 613-761-5036; E-mail: lefebvre{at}civich.ottawa.on.ca.

The abbreviations used are: hsp, heat shock protein; GR, glucocorticoid receptor; ER, estrogen receptor; PR, progesterone receptor; MMTV, mouse mammary tumor virus; IIF, indirect immunofluorescence; NLS, nuclear localization signal; CAT, chloramphenicol acetyl- transferase; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; WT, wild type.

2 R. J. G. Haché, R. Tse, T. Reich, J. G. A. Savory, and Y. A. Lefebvre, unpublished observation.

3 Savory, J. G. A., Hsu, B., Laquain, I. R., Giffin, W., Reich, T., Haché, R. J. G., and Lefebvre, Y. A. (1999) Mol. Cell. Biol., in press.

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