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
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ABSTRACT
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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.
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INTRODUCTION
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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
B (I
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
, 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
that has released its import cargo, forming an importin
/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
protein from the nucleus to the cytoplasm
occurs when importin
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.
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RESULTS
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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
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
B (IQQQLGQLTLENLQMLP) were linked to amino acid 4 at the
rat GR amino terminus (Fig. 1
). 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 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.
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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. 2
(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. 2
, 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 AE) or NES-GR (panels
FJ) 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.
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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 48 h to attain maximum
cytoplasmic accumulation in most cells (Fig. 2
, D and E). Note that
some cells exhibit predominant nuclear GR staining even after an 8-h
withdrawal period (Fig. 2E
). 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. 2H
). Nearly all
cells expressing NES-GR exhibited predominant cytoplasmic localization
4 h after hormone withdrawal (Fig. 2
, I and J). A quantitative
assessment of the IIF data presented throughout the text is shown in
Fig. 4
. 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.
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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
B NES (26 ), can be specifically
inhibited by LMB (29 44 ). If the linked I
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. 3
, A and C) or
NES-GR (Fig. 3
, DF). As shown in Fig. 3
(panels AC), 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 AC) or NES-GR (panels DF)
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 (AC) or NES-GR (DF).
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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. 3D
, 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. 2H
). Even after 4 h of
hormone withdrawal in the presence of LMB, NES-GR export was not
apparent (Fig. 3E
). This extent of hormone withdrawal (i.e.
4 h) in the absence of LMB led to nearly complete nuclear export
of NES-GR (Fig. 2I
). Interestingly, NES-GR export in LMB-treated cells
appeared to be more restricted than GR (compare Fig. 3B
and 3E
). 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. 3
, 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. 2
and 3
were counted and
classified into five groups [N, N > C, N = C, N < C,
C; (11 )]. As shown in Fig. 4A
, 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. 4
), 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. 4
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. 5
, 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. 5
, 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.
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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. 6
, A and B. Two to three independent experiments were repeated for each GR
construct and the results were quantified in Fig. 6C
. 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. 6A
and 6C
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. 6
, 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. 6C
). 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.
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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. 7
, 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.
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DISCUSSION
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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
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
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
B
degradation is also governed by its nuclear export as LMB
treatment, which blocks exportin 1/CRM1-dependent nuclear export of
I
B
, 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
|
---|
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-GR3795 expression plasmid was generously provided
by Dr. Keith Yamamoto (University of California, San Francisco) and
encodes amino acids 4795 of rat GR. A plasmid containing the NES of
I
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
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-GR3795. 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 1824 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 816 h before DNA was
removed and replaced with fresh growth medium. Cells were then grown
for 1624 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 48 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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