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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 (IB) (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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 IB (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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(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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, 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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B NES (26 ), can be specifically
inhibited by LMB (29 44 ). If the linked IB 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, D–F). As shown in Fig. 3 (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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, 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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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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, 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 IB (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
IB degradation is also governed by its nuclear export as LMB
treatment, which blocks exportin 1/CRM1-dependent nuclear export of
IB, 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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
IB 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 IB 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B
promotes active transport of NF-B from the nucleus to the cytoplasm.
J Cell Sci 110:369–378[Abstract]
B protects it from signal-induced degradation and
inhibits nuclear factor B transcriptional activation. J Biol
Chem 274:9108–9115B/rel
transcription factor and IB inhibitor system. Curr Opin Cell Biol 5:477–487[CrossRef][Medline]
B by phosphorylation of its inhibitor IB. Nature 344:678–682[CrossRef][Medline]
B
requires proteolysis of the inhibitor IB-: signal-induced
phosphorylation of IB- alone does not release active NF-B.
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 P. Dewint, V. Gossye, K. De Bosscher, W. Vanden Berghe, K. Van Beneden, D. Deforce, S. Van Calenbergh, U. Muller-Ladner, B. Vander Cruyssen, G. Verbruggen, et al. A Plant-Derived Ligand Favoring Monomeric Glucocorticoid Receptor Conformation with Impaired Transactivation Potential Attenuates Collagen-Induced Arthritis J. Immunol., February 15, 2008; 180(4): 2608 - 2615. [Abstract] [Full Text] [PDF]
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 B. L. Conway-Campbell, M. A. McKenna, C. C. Wiles, H. C. Atkinson, E. R. de Kloet, and S. L. Lightman Proteasome-Dependent Down-Regulation of Activated Nuclear Hippocampal Glucocorticoid Receptors Determines Dynamic Responses to Corticosterone Endocrinology, November 1, 2007; 148(11): 5470 - 5477. [Abstract] [Full Text] [PDF]
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 N. L. Weigel and N. L. Moore Steroid Receptor Phosphorylation: A Key Modulator of Multiple Receptor Functions Mol. Endocrinol., October 1, 2007; 21(10): 2311 - 2319. [Abstract] [Full Text] [PDF]
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C. T. Kesler, D. Gioeli, M. R. Conaway, M. J. Weber, and B. M. Paschal Subcellular Localization Modulates Activation Function 1 Domain Phosphorylation in the Androgen Receptor Mol. Endocrinol., September 1, 2007; 21(9): 2071 - 2084. [Abstract] [Full Text] [PDF]
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 D. Nonclercq, F. Journe, I. Laios, C. Chaboteaux, R.-A. Toillon, G. Leclercq, and G. Laurent Effect of nuclear export inhibition on estrogen receptor regulation in breast cancer cells J. Mol. Endocrinol., August 1, 2007; 39(2): 105 - 118. [Abstract] [Full Text] [PDF]
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A. Carrigan, R. F. Walther, H. A. Salem, D. Wu, E. Atlas, Y. A. Lefebvre, and R. J. G. Hache An Active Nuclear Retention Signal in the Glucocorticoid Receptor Functions as a Strong Inducer of Transcriptional Activation J. Biol. Chem., April 13, 2007; 282(15): 10963 - 10971. [Abstract] [Full Text] [PDF]
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 M. Nishi and M. Kawata Brain Corticosteroid Receptor Dynamics and Trafficking: Implications from Live Cell Imaging Neuroscientist, April 1, 2006; 12(2): 119 - 133. [Abstract] [PDF]
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D. Gioeli, B. E. Black, V. Gordon, A. Spencer, C. T. Kesler, S. T. Eblen, B. M. Paschal, and M. J. Weber Stress Kinase Signaling Regulates Androgen Receptor Phosphorylation, Transcription, and Localization Mol. Endocrinol., March 1, 2006; 20(3): 503 - 515. [Abstract] [Full Text] [PDF]
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 I. M. Adcock and K. Ito Glucocorticoid Pathways in Chronic Obstructive Pulmonary Disease Therapy Proceedings of the ATS, November 1, 2005; 2(4): 313 - 319. [Abstract] [Full Text] [PDF]
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R. F. Walther, E. Atlas, A. Carrigan, Y. Rouleau, A. Edgecombe, L. Visentin, C. Lamprecht, G. C. Addicks, R. J. G. Hache, and Y. A. Lefebvre A Serine/Threonine-rich Motif Is One of Three Nuclear Localization Signals That Determine Unidirectional Transport of the Mineralocorticoid Receptor to the Nucleus J. Biol. Chem., April 29, 2005; 280(17): 17549 - 17561. [Abstract] [Full Text] [PDF]
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 N. D. Freedman and K. R. Yamamoto Importin 7 and Importin {alpha}/Importin {beta} Are Nuclear Import Receptors for the Glucocorticoid Receptor Mol. Biol. Cell, May 1, 2004; 15(5): 2276 - 2286. [Abstract] [Full Text] [PDF]
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B. E. Black, M. J. Vitto, D. Gioeli, A. Spencer, N. Afshar, M. R. Conaway, M. J. Weber, and B. M. Paschal Transient, Ligand-Dependent Arrest of the Androgen Receptor in Subnuclear Foci Alters Phosphorylation and Coactivator Interactions Mol. Endocrinol., April 1, 2004; 18(4): 834 - 850. [Abstract] [Full Text] [PDF]
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A. J. Saporita, Q. Zhang, N. Navai, Z. Dincer, J. Hahn, X. Cai, and Z. Wang Identification and Characterization of a Ligand-regulated Nuclear Export Signal in Androgen Receptor J. Biol. Chem., October 24, 2003; 278(43): 41998 - 42005. [Abstract] [Full Text] [PDF]
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R. F. Walther, C. Lamprecht, A. Ridsdale, I. Groulx, S. Lee, Y. A. Lefebvre, and R. J. G. Hache Nuclear Export of the Glucocorticoid Receptor Is Accelerated by Cell Fusion-dependent Release of Calreticulin J. Biol. Chem., September 26, 2003; 278(39): 37858 - 37864. [Abstract] [Full Text] [PDF]
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L. Amazit, Y. Alj, R. K. Tyagi, A. Chauchereau, H. Loosfelt, C. Pichon, J. Pantel, E. Foulon-Guinchard, P. Leclerc, E. Milgrom, et al. Subcellular Localization and Mechanisms of Nucleocytoplasmic Trafficking of Steroid Receptor Coactivator-1 J. Biol. Chem., August 22, 2003; 278(34): 32195 - 32203. [Abstract] [Full Text] [PDF]
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M. Qiu, A. Olsen, E. Faivre, K. B. Horwitz, and C. A. Lange Mitogen-Activated Protein Kinase Regulates Nuclear Association of Human Progesterone Receptors Mol. Endocrinol., April 1, 2003; 17(4): 628 - 642. [Abstract] [Full Text] [PDF]
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P. Maruvada, C. T. Baumann, G. L. Hager, and P. M. Yen Dynamic Shuttling and Intranuclear Mobility of Nuclear Hormone Receptors J. Biol. Chem., March 28, 2003; 278(14): 12425 - 12432. [Abstract] [Full Text] [PDF]
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M. Itoh, M. Adachi, H. Yasui, M. Takekawa, H. Tanaka, and K. Imai Nuclear Export of Glucocorticoid Receptor is Enhanced by c-Jun N-Terminal Kinase-Mediated Phosphorylation Mol. Endocrinol., October 1, 2002; 16(10): 2382 - 2392. [Abstract] [Full Text] [PDF]
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 J. M. Holaska, B. E. Black, F. Rastinejad, and B. M. Paschal Ca2+-Dependent Nuclear Export Mediated by Calreticulin Mol. Cell. Biol., September 1, 2002; 22(17): 6286 - 6297. [Abstract] [Full Text] [PDF]
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X. Wang, J. L. Pongrac, and D. B. DeFranco Glucocorticoid Receptors in Hippocampal Neurons that Do Not Engage Proteasomes Escape from Hormone-Dependent Down-Regulation but Maintain Transactivation Activity Mol. Endocrinol., September 1, 2002; 16(9): 1987 - 1998. [Abstract] [Full Text] [PDF]
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Z. Wang, J. Frederick, and M. J. Garabedian Deciphering the Phosphorylation "Code" of the Glucocorticoid Receptor in Vivo J. Biol. Chem., July 12, 2002; 277(29): 26573 - 26580. [Abstract] [Full Text] [PDF]
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D. B. DeFranco Navigating Steroid Hormone Receptors through the Nuclear Compartment Mol. Endocrinol., July 1, 2002; 16(7): 1449 - 1455. [Abstract] [Full Text] [PDF]
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 I. G. Macara Transport into and out of the Nucleus Microbiol. Mol. Biol. Rev., December 1, 2001; 65(4): 570 - 594. [Abstract] [Full Text] [PDF]
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P. Berg and I. Pongratz Differential Usage of Nuclear Export Sequences Regulates Intracellular Localization of the Dioxin (Aryl Hydrocarbon) Receptor J. Biol. Chem., November 9, 2001; 276(46): 43231 - 43238. [Abstract] [Full Text] [PDF]
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T. Kino, R. H. Stauber, J. H. Resau, G. N. Pavlakis, and G. P. Chrousos Pathologic Human GR Mutant Has a Transdominant Negative Effect on the Wild-Type GR by Inhibiting Its Translocation into the Nucleus: Importance of the Ligand-Binding Domain for Intracellular GR Trafficking J. Clin. Endocrinol. Metab., November 1, 2001; 86(11): 5600 - 5608. [Abstract] [Full Text] [PDF]
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C. F. Bunn, J. A. Neidig, K. E. Freidinger, T. A. Stankiewicz, B. S. Weaver, J. McGrew, and L. A. Allison Nucleocytoplasmic Shuttling of the Thyroid Hormone Receptor {{alpha}} Mol. Endocrinol., April 1, 2001; 15(4): 512 - 533. [Abstract] [Full Text]
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J. M. Holaska, B. E. Black, D. C. Love, J. A. Hanover, J. Leszyk, and B. M. Paschal Calreticulin Is a Receptor for Nuclear Export J. Cell Biol., January 8, 2001; 152(1): 127 - 140. [Abstract] [Full Text] [PDF]
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Y. Wan, K. K. Coxe, V. G. Thackray, P. R. Housley, and S. K. Nordeen Separable Features of the Ligand-Binding Domain Determine the Differential Subcellular Localization and Ligand-Binding Specificity of Glucocorticoid Receptor and Progesterone Receptor Mol. Endocrinol., January 1, 2001; 15(1): 17 - 31. [Abstract] [Full Text]
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R. K. Tyagi, Y. Lavrovsky, S. C. Ahn, C. S. Song, B. Chatterjee, and A. K. Roy Dynamics of Intracellular Movement and Nucleocytoplasmic Recycling of the Ligand-Activated Androgen Receptor in Living Cells Mol. Endocrinol., August 1, 2000; 14(8): 1162 - 1174. [Abstract] [Full Text]
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M. Prigent, I. Barlat, H. Langen, and C. Dargemont Ikappa Balpha and Ikappa Balpha /NF-kappa B Complexes Are Retained in the Cytoplasm through Interaction with a Novel Partner, RasGAP SH3-binding Protein 2 J. Biol. Chem., November 10, 2000; 275(46): 36441 - 36449. [Abstract] [Full Text] [PDF]
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