Protracted Nuclear Export of Glucocorticoid Receptor Limits Its Turnover and Does Not Require the Exportin 1/CRM1-Directed Nuclear Export Pathway

Jimin Liu and Donald B. DeFranco

Departments of Biological Sciences (J.L., D.B.D.), Neuroscience (D.B.D.), and Pharmacology (D.B.D.) University of Pittsburgh Pittsburgh, Pennsylvania 15260


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoid receptors (GRs) are shuttling proteins, yet they preferentially accumulate within either the cytoplasmic or nuclear compartment when overall rates of nuclear import or export, respectively, are limiting. Hormone binding releases receptors from stable heteromeric complexes that restrict their interactions with soluble nuclear import factors and contribute to their cytoplasmic retention. Although hormone dissociation leads to the rapid release of GRs from chromatin, unliganded nuclear receptors are delayed in their export. We have used a chimeric GR that contains a heterologous, leucine-rich nuclear export signal sequence (NES) to assess the consequences of accelerated receptor nuclear export. Leucine-rich NESs utilize the exportin 1/CRM1-dependent nuclear export pathway, which can be blocked by leptomycin B (LMB). The fact that rapid nuclear export of the NES-GR chimera, but not the protracted export of wild-type GR, is sensitive to LMB, suggests that GR does not require the exportin 1/CRM1 pathway to exit the nucleus. Despite its more rapid export, the NES-GR chimera appears indistinguishable from wild-type GR in its transactivation activity in transiently transfected cells. However, accelerated nuclear export of the NES-GR chimera is associated with an increased rate of hormone-dependent down-regulation. The increase in NES-GR down-regulation is overcome by LMB treatment, thereby confirming the connection between receptor nuclear export and down-regulation. Given the presence of a nuclear recycling pathway for GR, the protracted rate of receptor nuclear export may increase the efficiency of biological responses to secondary hormone challenges by limiting receptor down-regulation and hormone desensitization.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The glucocorticoid receptor (GR) is a shuttling protein (1 ) that, like other steroid receptors (2 3 4 ), has the capacity to bidirectionally traverse nuclear pore complexes (NPCs). Even in the context of this reversible trafficking, GRs accumulate within distinct subcellular compartments due to limitations in either overall rates of nuclear import or export (5 ). In most cells, unliganded GRs reside predominantly within the cytoplasm (6 7 8 9 ) as a stable association with heat shock proteins and small immunophilins restricts their access to soluble nuclear import factors (5 10 11 12 ). Upon hormone binding, GRs are unleashed from heteromeric complexes that serve to retain them in the cytoplasm and rapidly import into nuclei (5 13 ). Hormone-bound GRs that enter the nucleus rapidly locate and bind to high- affinity target sites within native chromatin and alter the transcriptional activity of linked promoters (14 15 ). As the overall rate of GR nuclear import exceeds the rate of receptor nuclear export (1 ), hormone-bound GRs predominantly reside within nuclei (5 ).

GR binding to chromatin is hormone-dependent (16 ) and may be reversible as the withdrawal of hormone leads to the apparent release of receptors from high-affinity chromatin-binding sites (17 18 ). While the kinetics of GR chromatin release is closely related to the rate of hormone dissociation (19 20 ), unliganded receptors remain nuclear for a considerable period of time after hormone withdrawal (20 ). The limitation of GR nuclear export does not appear to be mediated by a slow passage through the NPC, but more likely is influenced by retention of the receptor within distinct subnuclear compartments (20 21 ).

Specific nuclear export signal sequences (NESs) have been identified in various proteins that are capable of exporting from nuclei such as the human immunodeficiency virus (HIV) Rev protein (22 ), heterogeneous nuclear RNA-binding protein (hnRNP) A1 (23 ), hnRNP K (24 ), protein kinase inhibitor of protein kinase A (PKI) (25 ), and inhibitor of nuclear factor {kappa}B (I{kappa}B) (26 ). One class of NESs that direct rapid, energy-dependent nuclear export are hydrophobic, leucine-enriched sequences (27 ). Exportin 1/CRM1 has been identified as a receptor for leucine-rich NESs and mediates the targeting of exporting substrates to the NPC (28 29 ). CRM1 was originally identified as a 115-kDa protein in Schizosaccharomyces pombe, whose mutation causes abnormal chromosome morphology and renders S. pombe resistant to the cytotoxin leptomycin B (LMB) (30 ). LMB was subsequently found to bind directly to exportin 1/CRM1 (29 ) and to inhibit the nuclear export of Rev and U snRNAs in mammalian cells (31 ). The binding of LMB to exportin 1/CRM1 prevents the assembly of an export-competent trimeric complex containing exportin 1/CRM1, RanGTP, and an NES-containing protein (29 ).

Recent studies suggest that exportin 1/CRM1-independent nuclear export pathways may also exist. For example, HeLa cell cytosol treated with N-ethylmaleimide or phenyl-Sepharose to inactivate or deplete exportin 1/CRM1 remains competent to support the in vitro nuclear export of PKI (32 ). Furthermore, the export of importin {alpha}, a nuclear import factor that recycles from the nucleus to the cytoplasm, is mediated by a distinct nuclear export factor, CAS (33 ). In the presence of RanGTP, CAS binds strongly to importin {alpha} that has released its import cargo, forming an importin {alpha}/CAS/RanGTP complex. CAS also binds to the NPC and rapidly enters the nuclear envelope, where it uses similar sites on the NPC as importin ß (33 ). Nuclear export of importin ß appears to be mediated by a novel pathway that has yet to be defined (34 ).

The signal sequence directing GR import into nuclei has been well characterized (13 35 36 ) and is related to a prototypical bipartite, basic amino acid-enriched nuclear localization signal sequence (NLS) common to many other nuclear proteins (37 ). In contrast, the identity of NESs on steroid receptor proteins remains controversial. While the results of one study implicated a role for the progesterone receptor (PR) NLS in nuclear export (38 ), NLSs have generally not been found to participate in nuclear export (23 39 40 ). Furthermore, the recycling of NLS-binding importin {alpha} protein from the nucleus to the cytoplasm occurs when importin {alpha} is not associated with its NLS-containing substrates (33 ). It therefore seems unlikely that NLSs would direct protein export from nuclei using the same transport proteins (i.e. the importins) that function in NLS-mediated nuclear import. Although steroid receptors may contain some sequences with limited homology to leucine-rich NESs, these sequences do not appear to be functional, as the addition of LMB does not inhibit nuclear export of PR (41 ). However, conflicting results have been generated showing an effect of LMB on nuclear export of GR (12 ). Since very different conditions were used to assay PR vs. GR nuclear export (12 41 ), it is unclear whether the differential effects of LMB are accounted for by unknown experimental variables or due to the existence of distinct mechanisms for nuclear export of highly related steroid receptors.

In addition to unresolved questions concerning the mechanism of steroid receptor nuclear export, the physiological significance of receptor nucleocytoplasmic shuttling is unknown. The transcriptional regulatory capacity of steroid receptors is rapidly terminated upon hormone withdrawal (18 ), obviating the use of nuclear export to control the duration of transcriptional responses, as is the case for other regulated transcription factors (26 42 ). In this report we have used a novel approach to examine both the mechanism of GR nuclear export and the impact of nuclear export on various properties of the receptor. Our results suggest that GR does not utilize the exportin-1/CRM1 pathway for nuclear export. Furthermore, while nuclear export kinetics does not appear to impact the transcriptional regulatory capacity of GR, it exerts a dramatic effect on receptor down-regulation. Given the presence of a nuclear recycling pathway for GR (43 ), the protracted rate of receptor nuclear export may increase the efficiency of biological responses to secondary hormone challenges by limiting receptor down-regulation and hormone desensitization.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A Leucine-Rich NES Linked to the Amino Terminus of GR Accelerates Its Nuclear Export
If the relatively slow kinetics of GR nuclear export is biologically relevant for some receptor function, altering the kinetics of receptor export might provide a means for assessing those receptor activities that are dependent on, or sensitive to, receptor export kinetics. The leucine-rich NES from I{kappa}B is responsible for its rapid nuclear export, which proceeds via the exportin 1/CRM1 pathway (26 44 ). Although this NES can function when linked to heterologous proteins (26 ), its function has not been tested in the context of a protein that potentially utilizes a distinct nuclear export pathway. Therefore, we constructed a rat GR chimera (NES-GR) in which 17 amino acids of I{kappa}B (IQQQLGQLTLENLQMLP) were linked to amino acid 4 at the rat GR amino terminus (Fig. 1Go). The resulting construct encodes a chimeric protein designated NES-GR.



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Figure 1. Schematic Maps of GR and NES-GR

An NES originating from I{kappa}B (IQQQLGQLTL E NLQMLP) was inserted at the N terminus of GR (preceding the fourth amino acid of GR) to generate the NES-GR chimera.

 
Wild-type GR or NES-GR expressing plasmids were transiently transfected into Cos-1 cells and GR subcellular localization was examined by indirect immunofluorescence (IIF). As shown in Fig. 2Go (panels A and F), both unliganded GR and NES-GR reside predominantly within the cytoplasm. Upon hormone treatment, both GR and NES-GR translocated into the nucleus (Fig. 2Go, B and G). Although detailed analyses were not performed, it appears that the kinetics of GR and NES-GR nuclear import are indistinguishable with nearly complete import (as detected by IIF analysis) apparent within 30 min of hormone treatment.



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Figure 2. Kinetics of GR and an NES-GR Nuclear Export

Cos-1 cells, transfected with either GR (panels A–E) or NES-GR (panels F–J) expression plasmids, were pretreated with 1 µM cycloheximide for 1 h, then treated with 10-7 M corticosterone for 1 h. Cells were then withdrawn from hormone (indicated by +/-H) for indicated periods of time. Cycloheximide was also included in hormone-free medium. Cells were fixed either before hormone treatment (panels A and F), 1 h after hormone treatment (panels B and G), or after various periods of hormone withdrawal (panels C, D, E, H, I, and J), and processed for IIF to detect GR and NES-GR.

 
To reveal nuclear export of GR and NES-GR, we used a hormone withdrawal regimen that eventually leads to the redistribution of nuclear GRs to the cytoplasm (8 20 ). Transfected Cos-1 cells, pretreated with 1 µM cycloheximide for 1 h, were exposed to 10- 7 M corticosterone for 1 h and then subjected to various periods of hormone withdrawal. Cycloheximide was readded during the withdrawal period to insure that preexisting, rather than de novo synthesized, receptors were observed during the withdrawal periods. As expected from previous studies (8 ), GR exported slowly from nuclei of hormone-withdrawn cells, taking about 4–8 h to attain maximum cytoplasmic accumulation in most cells (Fig. 2Go, D and E). Note that some cells exhibit predominant nuclear GR staining even after an 8-h withdrawal period (Fig. 2EGo). In contrast, export of NES-GR was apparent even after only 30 min of hormone withdrawal (data not shown) and nearly complete within 1 h of withdrawal (Fig. 2HGo). Nearly all cells expressing NES-GR exhibited predominant cytoplasmic localization 4 h after hormone withdrawal (Fig. 2Go, I and J). A quantitative assessment of the IIF data presented throughout the text is shown in Fig. 4Go. Furthermore, a comparison of fluorescent staining intensity using the NIH Image program (see Materials and Methods) revealed that the ratio of nuclear/cytoplasmic GR was 16-fold greater than nuclear/cytoplasmic NES-GR after a 1-h withdrawal. Within 8 h of hormone withdrawal, the GR nuclear/cytoplasmic ratio exceeds that of NES-GR by only 3-fold. Thus, the presence of a heterologous leucine-rich NES on GR does not appear to limit GR import, but rather accelerates its rate and efficiency of nuclear export. Furthermore, the exportin 1/CRM1-dependent NES overrides whatever signal is present on GR that limits its nuclear export rate.



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Figure 4. Analysis of the Effects of LMB on GR and NES-GR Nuclear Export

Transfected Cos-1 cells were treated with 10-7 M corticosterone for 10 min and then with 1 µM cycloheximide for 1 h, in the presence or absence of 100 nM LMB. Hormone was then withdrawn for the indicated periods (+/-H) in the presence or absence of LMB. The relative nuclear and cytoplasmic staining of GR or NES-GR in about 100 transfected cells per condition was classified into five groups (N: exclusive nuclear staining; N > C: nuclear exceeds cytoplasmic staining; N = C: equal nuclear and cytoplasmic staining; N < C: cytoplasmic exceeds nuclear staining; C: cytoplasmic staining). The results are summarized for GR (A) and NES-GR (B). Similar patterns were obtained when the same experiment was repeated.

 
Differential Effects of LMB on GR and NES-GR Nuclear Export
Nuclear export that proceeds via the exportin 1/CRM1 pathway, including that driven by the I{kappa}B NES (26 ), can be specifically inhibited by LMB (29 44 ). If the linked I{kappa}B NES in fact drives the rapid export of NES-GR, its contribution to this rapid export should be negated by LMB. Since conflicting results have been generated regarding the effects of LMB on steroid receptor nuclear export (12 41 ), we also thought it important to assess LMB effects in our assays of GR nuclear export. Therefore, we examined the nuclear export of both GR and NES-GR during hormone withdrawal in the presence and absence of LMB.

Cos-1 cells transfected with GR or NES-GR were pretreated with 1 µM cycloheximide for 1 h, followed by a 10- 7 M corticosterone treatment for 30 min. Hormone-treated cells were then incubated with 100 nM LMB for 1 h and then withdrawn from hormone for up to 8 h. Cycloheximide was readded and LMB was either included in, or excluded from, the withdrawal medium. Cells were fixed at various time points and processed by IIF to observe the subcellular localization of GR (Fig. 3Go, A and C) or NES-GR (Fig. 3Go, D–F). As shown in Fig. 3Go (panels A–C), the apparent rate of GR nuclear export was not changed in the presence of LMB, suggesting that GR does not utilize the exportin 1/CRM1 nuclear export pathway.



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Figure 3. Leptomycin B Delays Nuclear Export of NES-GR but Not GR

Cos-1 cells transfected with GR (panels A–C) or NES-GR (panels D–F) were treated with 10-7 M corticosterone for 10 min and then with 1 µM cycloheximide for 1 h. LMB (100 nM) was added along with cycloheximide. Hormone was then withdrawn for the indicated periods (+/-H). Cycloheximide and LMB were replenished in the hormone-free medium after withdrawal. Cells were fixed and processed for IIF to detect GR (A–C) or NES-GR (D–F).

 
In contrast to the lack of effects on GR, the relatively rapid nuclear export of NES-GR was significantly dampened by LMB. As shown in Fig. 3DGo, after 1 h of hormone withdrawal in the presence of LMB, NES-GR export was completely blocked and NES-GR remained predominantly within nuclei. As shown earlier, significant export of NES-GR occurred after 1 h of hormone withdrawal (Fig. 2HGo). Even after 4 h of hormone withdrawal in the presence of LMB, NES-GR export was not apparent (Fig. 3EGo). This extent of hormone withdrawal (i.e. 4 h) in the absence of LMB led to nearly complete nuclear export of NES-GR (Fig. 2IGo). Interestingly, NES-GR export in LMB-treated cells appeared to be more restricted than GR (compare Fig. 3BGo and 3EGo). A potential explanation for this phenomenon is presented in the Discussion.

LMB treatment does not lead to the complete inhibition of NES-GR export, as the extent of GR and NES-GR nuclear export appeared indistinguishable in cells withdrawn from hormone for 8 h in the presence of LMB (Fig. 3Go, C and F). To exclude the possibility that NES-GR nuclear export after an 8 h withdrawal in the presence of LMB was due to an instability of LMB, additional LMB was added every 2 h during withdrawal. NES-GR was still found to be exported within 8 h of hormone withdrawal upon repeated additions of LMB (data not shown). It therefore seems likely that if prevented from using the exportin 1/CRM1 pathway, NES-GR will eventually utilize an exportin 1/CRM1-independent pathway for nuclear export. Our experiments do not reveal whether NES-GR nuclear export eventually proceeds via the same pathway used by GR, but this is consistent with the relatively slow rate of NES-GR export observed under these conditions.

To better illustrate the effects of LMB on GR and NES-GR export, about 100 cells from each time point shown in Figs. 2Go and 3Go were counted and classified into five groups [N, N > C, N = C, N < C, C; (11 )]. As shown in Fig. 4AGo, the subcellular distribution of wild-type GR was very similar throughout the time course of hormone withdrawal, irrespective of the presence or absence of LMB. As apparent from a comparison of panels A and B (Fig. 4Go), nuclear export of NES-GR (most easily visualized by loss of exclusive nuclear staining; filled bar) is much more rapid than GR in the absence of LMB. However, panel B in Fig. 4Go also illustrates the dramatic effects of LMB on the rate and extent of NES-GR nuclear export. Therefore, our results provide strong support for the notion that GR uses an export pathway that is unique and distinct from the exportin 1/CRM1 pathway.

Transactivation Activity of NES-GR Is Hormone Dependent and Apparently Not Distinguishable from GR in Transiently Transfected Cells
To evaluate the functional consequences of altered nuclear export kinetics of GR, detailed analyses of NES-GR transactivation activity were performed. In this case, a luciferase reporter gene was used to provide a sensitive assay of NES-GR transactivation function in transiently transfected Cos-1 cells. A plasmid containing the luciferase gene linked to the gluco-corticoid-responsive mouse mammary tumor virus (MMTV) promoter (i.e. MMTV-Luc) was cotransfected with either a wild-type GR or NES-GR expression plasmid. In the absence of hormone, luciferase expression was low in Cos-1 cells transfected with these GR constructs (Fig. 5Go, lanes 1 and 3). When transfected Cos-1 cells were treated with hormone for only 4.5 h, luciferase activity driven by the MMTV-luciferase reporter gene was increased to nearly the same extent in cells expressing either GR or NES-GR (Fig. 5Go, lanes 2 and 4). This brief hormone treatment was chosen to minimize GR down-regulation (see below). Western blot analysis confirmed that GR and NES-GR expression were comparable under these transfection conditions (data not shown). Furthermore, identical results were obtained when input GR cDNA concentration was decreased 1000-fold (i.e. to 1 ng/plate), indicating that the transactivation of NES-GR measured in these assays was not merely reflective of receptor overexpression (data not shown). Thus, in a transiently transfected cell, NES-GR maintains its capacity to locate a transiently transfected, glucocorticoid-responsive template.



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Figure 5. NES-GR Functions as a Hormone-Dependent Transactivator in Transiently Transfected Cos-1 Cells

Cos-1 cells were cotransfected with 0.5 µg of a GRE-linked luciferase reporter (MMTV-Luciferase), and 1 µg of either a GR (columns 1 and 2) or NES-GR (columns 3 and 4) expression plasmid. Cells were then collected after a 4.5-h treatment with (columns 2 and 4) or without (columns 1 and 3) 10-7 M dexamethasone and luciferase activity measured in equivalent amounts of total lysate protein.

 
To more fully evaluate the transactivation properties of NES-GR, we performed a detailed dose-response and time course analysis of hormone induction in transiently transfected Cos-1 cells. A hormone dose-response experiment showed that the NES-GR and GR exhibited nearly identical responses to hormone in their induction of reporter gene expression (data not shown). Induction of luciferase activity driven from the MMTV-Luc plasmid was detected at 10-11 M dexamethasone (Dex), both with GR and NES-GR (data not shown). Maximal induction for both receptors was attained at 10-9 M Dex, a physiologically relevant concentration of glucocorticoid hormone. A time course analysis of NES-GR and GR transactivation function was also performed and showed no obvious difference between the effectiveness of NES-GR and GR on transactivation from the transiently transfected MMTV-Luc template (data not shown). Thus, the hormone-dependent transactivation activity of NES-GR does not appear to be distinguished from wild-type GR in transiently transfected Cos-1 cells either by its hormone dose response or kinetics.

Accelerated Hormone-Dependent Down-Regulation of NES-GR
After its nuclear import, GR is subjected to alternative processing fates including nuclear recycling, nuclear export, and degradation (45 46 ). While GR nucleocytoplasmic shuttling occurs in hormone-treated cells (1 ), the reutilization of GR is counterbalanced by degradation of the receptor that is stimulated in hormone-treated cells (45 46 ). Does nuclear export or nucleocytoplasmic shuttling contribute to hormone-dependent down-regulation of GR? The NES-GR chimera with its accelerated rate of nuclear export provides a useful reagent to assess the possible link between GR nuclear export and degradation.

Cos-1 cells transfected with GR or NES-GR expression plasmids were pretreated with 1 µM cycloheximide for 1 h, and then incubated with 10-7 Dex for various lengths of time. Cells were then collected and subjected to Western blot analysis to assess steady state levels of wild-type GR or NES-GR. A typical Western blot for each GR construct is shown in Fig. 6Go, A and B. Two to three independent experiments were repeated for each GR construct and the results were quantified in Fig. 6CGo. Rather than illustrate absolute rates of GR degradation, which may be difficult to compare in different transfected cells, we compared GR levels in hormone-treated vs. untreated cells. The data shown in Figs. 6AGo and 6CGo confirm the hormone-dependent down-regulation of GR, which has been observed in transfected Cos-1 cells (46 ) and many other cell lines and tissues (45 47 ). It is quite apparent from the data shown in Fig. 6Go, B and C, that hormone-dependent down-regulation of NES-GR is dramatically accelerated relative to GR. Within 12 h of Dex treatment, steady state levels of NES-GR were reduced nearly 80% while minimal effects of hormone were noted on GR after a 12-h treatment of transfected cells (Fig. 6CGo). Thus, an increased capacity for GR nuclear export, while exerting no noticeable effects on GR transactivation after relatively short periods of hormone treatment, dramatically stimulates hormone-dependent down-regulation of the receptor.



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Figure 6. Accelerated Hormone-Dependent Down-Regulation of NES-GR

GR- or NES-GR-transfected Cos-1 cells were pretreated with 1 µM cycloheximide for 1 h and then untreated or treated with Dex. Both untreated (-H) and treated (+H) cells were collected at various times and processed for Western blot analysis to detect GR (A) or NES-GR (B) protein (normalized to lamin B protein detected on the same blot). The ratio of GR or NES-GR remaining in hormone-treated (+H) vs. untreated (-H) cells is calculated for each time point and then graphed in panel C, which is representative of at least three experiments.

 
To further confirm that accelerated down-regulation of NES-GR is due to a more rapid transit of NES-GR to the cytoplasm, LMB was included during hormone treatment. As seen in Fig. 7Go, the accelerated down-regulation of NES-GR observed after a 12-h hormone treatment was reduced by LMB. LMB did not affect hormone-dependent down-regulation of GR that was examined after a 24-h period of hormone withdrawal (data not shown). Taken together, our results suggest that the extent or kinetics of hormone-dependent down-regulation of GR is influenced by the rate of receptor nuclear export.



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Figure 7. Accelerated Hormone-Dependent Down-Regulation of NES-GR Is Blocked by LMB

NES-GR transfected Cos-1 cells were pretreated with 1 µM cycloheximide for 1 h and then untreated or treated with Dex and LMB as indicated. Both untreated and treated cells were collected after 12 h and processed for Western blot analysis to detect the NES-GR protein with lamin B detected on the same blot (A). The results were quantified with NES-GR levels normalized to lamin B protein, and displayed in panel B.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The regulation of nuclear import is widely used as a mechanism to restrict transcription factor access to target sites that are activated in response to specific extracellular stimuli (5 48 ) or at unique developmental stages (49 50 51 ). In recent years, it has become apparent that nuclear export of transcription factors often provides a mechanism to terminate an induced gene regulatory event (26 52 ). Although the nuclear export capacity of steroid receptor proteins has been recognized for a number of years (1 2 3 4 ), the physiological significance of this trafficking has not been established. Furthermore, opposing views have emerged recently regarding the mechanism of steroid receptor nuclear export (12 41 ).

In this report, we show that nuclear export of rat GR in asynchronous cells is unlikely to be mediated by the exportin 1/CRM1 nuclear export pathway. We have no explanation for the discrepancy between our results and those reported by Savory et al. (12 ), but it may be related to the cytotoxicity associated with LMB treatment of G0-synchronized cells in their studies (12 ). LMB was also found not to affect the nuclear export of PR in asynchronous cells (41 ), suggesting that exportin 1/CRM1-independent nuclear export may be a property that is shared by all steroid receptors.

Steroid receptors are not unique in their apparent exportin 1/CRM1-independent nuclear export. Human RNA helicase A also utilizes an exportin 1/CRM1-independent pathway for nuclear export, but its mechanism of export remains completely undefined (53 ). Likewise, importin ß does not use the exportin 1/CRM1 pathway and may export from nuclei via a direct interaction with distinct nucleoporins (34 ). It is difficult to judge whether GR exports by a similar mechanism as interactions between GR and nucleoporins have never been tested. Furthermore, we have not directly tested whether nuclear export of GR requires CAS, the factor that has been found to mediate importin {alpha} nuclear export (33 ). Both the amino- and carboxyl-terminal domains of rat GR are dispensible for its nuclear export (1 ), suggesting that if a novel NES exists within the receptor, it may be localized within its DNA-binding domain. The NLS of PR has been postulated to function as an NES (41 ), but this hypothesis remains controversial and has not been supported by other studies of related NLSs (23 39 40 53 ). The nucleotide requirements for steroid receptor nuclear export also remain undefined although in permeabilized cells, ATP and not GTP was found to be required for GR export (20 ). In independent studies, an ATP requirement for in vitro nuclear export was also noted (54 ).

Irrespective of the mechanism used by GR to exit the nucleus, its nuclear export is not involved in the termination of receptor transactivation, as hormone dissociation leads to the rapid release of GR from chromatin and cessation of receptor-mediated transcriptional responses (18 ). GRs that release from chromatin remain within the nucleus for a considerable period of time, held within an undefined subnuclear compartment that restricts receptor access to the nuclear export machinery (20 ). In contrast to the relative insensitivity of GR transactivation to receptor nucleocytoplasmic shuttling, nuclear export of other transcription factors can be involved in limiting the duration of transcriptional responses (26 42 ). Transactivation by the NF-AT transcription factor is limited by its NES, even in the absence of nuclear export (52 ). Calcium-dependent activation of NF-AT is brought about when calcineurin effectively competes with exportin 1/CRM1 for binding to the NF-AT NES (52 ). Although calcineurin has also been found to potentiate GR transactivation (55 ), it seems unlikely that it does so via the same mechanism responsible for calcineurin-dependent activation of NF-AT. Nuclear export does not limit GR transactivation to the same extent as NF-AT, nor does the receptor utilize the exportin 1/CRM1 nuclear export pathway.

Even if GR utilizes a distinct pathway for nuclear export, it is not precluded from using the exportin 1/CRM1-mediated nuclear export pathway. Thus, the rapid nuclear export of a GR chimera that possesses a heterologous leucine-rich NES (i.e. NES-GR) is likely to be exportin 1/CRM1 dependent given its sensitivity to LMB. In this case, the machinery that accounts for nuclear retention and relatively slow nuclear export kinetics of GR is overridden as the chimeric receptor is diverted to an exportin 1/CRM1-dependent pathway. NES-GR is not restricted to using the exportin 1/CRM1-dependent nuclear export pathway, as it will exit the nucleus, although quite slowly, in the presence of LMB. In fact, NES-GR nuclear export in the presence of LMB appears to be even less efficient than GR, suggesting that there may be some delay before the chimeric receptor can switch nuclear export pathways.

If nuclear export exerts minimal effects on GR transactivation, what other function(s) of the receptor might be sensitive to the rate or extent of GR nuclear export? Based upon the accentuated hormone-dependent down-regulation of NES-GR, we hypothesize that nuclear retention of the receptor, brought about by its relatively inefficient export, is responsible for limiting the extent of receptor turnover. Since the concentration of GR protein is an important parameter influencing cellular responsiveness to glucocorticoids (56 ), it may be critical to maintain some threshold level of nuclear GR. Even in hormone-withdrawn cells, nuclear GRs have the capacity to recycle to chromatin in response to a secondary hormone challenge (43 ). Thus, it may be advantageous to retain receptors within nuclei, particularly since the termination of their transactivation activity does not require their exit from the nucleus.

The exact mechanism of hormone-dependent GR down-regulation is not known. Since proteosome components can be found both in the cytoplasm and nucleus (57 58 ), GR degradation could occur in either compartment. However, a study of the intracellular distribution of GRs in response to hormone treatment suggested that the hormone-induced down-regulation of GR may occur in the cytoplasm (45 ). Although the accentuated down-regulation of NES-GR would be most consistent with a predominantly cytoplasmic localization of GR degradation, our results do not rule out the contribution of nuclear proteosomes to GR degradation.

If we presume that the accelerated turnover of GR in hormone-treated cells occurs within the cytoplasm, how are cytoplasmic receptors in hormone-treated cells distinguished from naïve receptors in untreated cells and more efficiently targeted for degradation? Although the nuclear reentry rate of recycling receptors has never been accurately measured, perhaps a delay in cytoplasmic, recycled receptor association with nuclear import factors provides an opportunity for these receptors to be diverted to the cytoplasmic degradation machinery. It is also possible that recycled cytoplasmic GRs are distinguished from naïve receptors by differences in either associated proteins (59 ) or posttranslational modifications (60 ). Interestingly, GRs possessing mutations at their multiple phosphorylation sites have a diminished rate of hormone-dependent down-regulation (46 ). Since the GR dephosphorylation rate is much slower than the rate of receptor (61 62 63 ), recycled cytoplasmic receptors would be hyperphoshorylated relative to naïve receptors (60 ). As examined most extensively with I{kappa}B (48 64 65 ), phosphorylation is a well established mechanism used to target cytoplasmic proteins for proteosome-mediated degradation. Phosphorylation of recycled cytoplasmic receptors could play a role in its preferential selection for degradation.

A relationship between nuclear protein export and degradation has been observed in numerous cases. For example, nuclear export is required for the degradation of endogenous p53, as the exposure of various cells to LMB results in increased p53 levels in the nucleus (42 ). The rate of I{kappa}B{alpha} degradation is also governed by its nuclear export as LMB treatment, which blocks exportin 1/CRM1-dependent nuclear export of I{kappa}B{alpha}, diminishes its stimulus-induced turnover (44 ). In each of these cases, the link between nuclear export and degradation is used as a mechanism to terminate the transcriptional responses regulated by these proteins. Steroid receptors do not require such drastic measures to terminate their transactivation activity as hormone release from the receptors terminates their action at target genes.

In summary, we hypothesize that nuclear export of GRs does indeed serve a physiological function, i.e. to increase the efficiency of receptor turnover. Steroid receptors are limited in their ability to interact with the nuclear export machinery due either to their lack of a bona fide NES, or to their retention within a unique subnuclear compartment. In either event, the receptors remain competent to respond to secondary hormone challenges as long as they remain nuclear. As a result, hormone dissociation from the receptors, and their corresponding release from chromatin, does not signify the termination of receptor function.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
Cos-1 cells were maintained at 37 C in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FBS (Irvine Scientific, Santa Ana, CA).

Plasmids
The pSTC-TK-GR3–795 expression plasmid was generously provided by Dr. Keith Yamamoto (University of California, San Francisco) and encodes amino acids 4–795 of rat GR. A plasmid containing the NES of I{kappa}B was obtained from Dr. Catherine Dargemont (Curie Institute, Paris). To construct the NES-GR chimera, a double-stranded oligonucleotide (DNA Synthesis Facility, University of Pittsburgh, Pittsburgh, PA) containing 51 bp of the I{kappa}B NES sequence (encoding IQQQLGQLTLENLQMLP) with an additional BamHI site at each terminus was inserted preceding the fourth amino acid of GR in pSTC-TK-GR3–795. A bacterial luciferase plasmid containing the MMTV long terminal repeat (i.e. MMTV-Luc) was used as a reporter of GR transactivation function (66 ).

Antibodies and Chemicals
The BuGR2 monoclonal antibody (67 ) was used to detect both GR and NES-GR. Monoclonal antilamin B antibody was purchased from Oncogene Science, Inc. (Cambridge, MA) while cycloheximide was purchased from Sigma (St. Louis, MO). Dr. Teruhiko Beppu (University of Tokyo, Tokyo) generously provided LMB.

Transient Transfections
Cos-1 cells were plated 18–24 h before transfection using a calcium phosphate precipitation method as described in Current Protocols in Molecular and Cellular Biology. Two micrograms of DNA/35-mm diameter plate of cells were used for each transfection. Cells were incubated with DNA precipitates for 8–16 h before DNA was removed and replaced with fresh growth medium. Cells were then grown for 16–24 h before harvesting.

In Vivo Hormone Treatment and Withdrawal
For hormone treatments, transfected cells were incubated with 10-7 M corticosterone (Sigma) for 1 h. For hormone withdrawal, hormone-treated cells were briefly rinsed three times with phenol red-free DMEM plus 5% charcoal-stripped FBS and then incubated with this medium for the times indicated. One hour before, and for indicated periods during hormone withdrawal, cells were treated with 1 µM cycloheximide; 100 nM LMB was included during hormone withdrawal where indicated.

IIF
Cells were fixed with -20 C methanol for 5 min at room temperature and then incubated with the BuGR2 anti-GR antibody (8 ). A fluorescein isothiocyanate-coupled antimouse IgG antibody (Roche Molecular Biochemicals, Indianapolis, IN) was used as secondary antibody to detect GR. 4,6-Diamidino-2-phenylindole (DAPI) (Sigma) was also included to detect DNA and to indicate position of nuclei. Stained cells were observed by fluorescence microscopy through an Optiphot-2 microscope (Nikon, Garden City, NY) and photographed with T-Max 400 film (Eastman Kodak Co., Rochester, NY).

Nuclear Export Characterization
The relative nuclear and cytoplasmic immunofluorescence in 100 cells per condition was classified into five groups: 1) exclusive nuclear staining (N), 2) nuclear staining exceeding cytoplasmic staining (N > C), 3) equivalent nuclear and cytoplasmic staining (N = C), 4) cytoplasmic staining exceeding nuclear staining (N < C), and 5) exclusive cytoplasmic staining (C) (11 ). In addition, the nuclear-cytoplasmic ratio of GR and/or NES-GR was determined from multiple cells in a representative field with scanned photographic negatives using the NIH Image program. Mean intensity measurements were used for these estimates so that the area of the sampling field was irrelevant. In addition, expression of these results as a nuclear-cytoplasmic ratio allowed measurements from different photographic negatives to be compared without regard to length of film development time.

Luciferase Assay
For Luciferase assays performed with Cos-1 cell extracts, calcium phosphate transfection was performed in 35-mm tissue culture plates as described above. Each plate was transfected with 0.5 µg of MMTV-Luc plasmid, 0.5 µg herring sperm DNA (Sigma), along with 1 µg of either wild-type GR or NES-GR expression plasmids. Sixteen to 24 h after transfection, Cos-1 cells were treated with or without 10-7 Dex for 4–8 h. Cells were then collected with PBS containing 2 mM EDTA. Cell pellets were resuspended in 200 µl Luciferase cell lysis buffer (Promega Corp., Madison, WI) and kept on ice for 5 min. After centrifugation at 14,000 x g for 5 min, supernatants were collected. Ten microliters of each lysate were used to measure total protein concentration (Bio-Rad Laboratories, Inc. Hercules, CA), and 40 µl for Luciferase assays (Promega Luciferase kit, Promega Corp.). Luciferase activity was normalized to total protein content. The experiments were performed in duplicate.

Western Blot Analysis of GR Down-Regulation
Transfected Cos-1 cells were pretreated with 1 µM cycloheximide for 1 h, and then untreated or treated with 10-7 M Dex for the lengths of time indicated. Both untreated (-H) and hormone-treated (+H) cells were collected at various times and processed for Western blot analysis to detect the GR protein using the BuGR2 antibody (68 ). To provide an internal control for gel loading and transfer efficiency, lamin B was also detected on the same blots using an antilamin B antibody. GR or lamin B on Western blots was visualized using the enhanced chemiluminescence (ECL) detection system (Amersham International, Little Chalfont, Buckinghamshire, UK). Relative amounts of GR and lamin B protein on the same blot were quantified using NIH Image software. The ratio of GR remaining in hormone-treated (+H) vs. untreated (-H) cells was calculated for each time point.


    ACKNOWLEDGMENTS
 
We thank Dr. Catherine Dargemont for providing the IkB plasmid, Drs. Jorge Iniguez-Lluhli and Keith Yamamoto for GR expression plasmids and luciferase reporter plasmids, and Dr. Teruhiko Beppu for LMB.


    FOOTNOTES
 
Address requests for reprints to: Donald B. DeFranco, Departments of Biological Sciences, Neuroscience, and Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260,

This work was supported by NIH Grant CA-43037.

Received for publication May 26, 1999. Revision received September 8, 1999. Accepted for publication September 10, 1999.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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