Dynamics of Intracellular Movement and Nucleocytoplasmic Recycling of the Ligand-Activated Androgen Receptor in Living Cells
Rakesh K. Tyagi,
Yan Lavrovsky,
Soon C. Ahn,
Chung S. Song,
Bandana Chatterjee and
Arun K. Roy
Department of Cellular and Structural Biology (R.K.T., Y.L.,
S.C.A., C.S.S., B.C., A.K.R.) The University of Texas Health
Science Center at San Antonio and Audie L. Murphy Memorial Veterans
Affairs Hospital (B.C.) San Antonio, Texas 78284
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ABSTRACT
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An expression construct containing the cDNA
encoding a modified aequorea green fluorescent protein (GFP) ligated to
the 5'-end of the rat androgen receptor (AR) cDNA (GFP-AR) was used to
study the intracellular dynamics of the receptor movement in living
cells. In three different cell lines, i.e. PC3, HeLa, and
COS1, unliganded GFP-AR was seen mostly in the cytoplasm and rapidly
(within 1560 min) moved to the nuclear compartment after androgen
treatment. Upon androgen withdrawal, the labeled AR migrated back to
the cytoplasmic compartment and maintained its ability to reenter the
nucleus on subsequent exposure to androgen. Under the condition of
inhibited protein synthesis by cycloheximide (50 µg/ml), at least
four rounds of receptor recycling after androgen treatment and
withdrawal were recorded. Two nonandrogenic hormones, 17ß-estradiol
and progesterone at higher concentrations
(10-7/10-6
M), were able to both transactivate the
AR-responsive promoter and translocate the GFP- AR into the nucleus.
Similarly, antiandrogenic ligands, cyproterone acetate and casodex,
were also capable of translocating the cytoplasmic AR into the nucleus
albeit at a slower rate than the androgen 5
-dihydrotestosterone
(DHT). All AR ligands with transactivation potential, including the
mixed agonist/antagonist cyproterone acetate, caused translocation of
the GFP-AR into a subnuclear compartment indicated by its punctate
intranuclear distribution. However, translocation caused by casodex, a
pure antagonist, resulted in a homogeneous nuclear distribution.
Subsequent exposure of the casodex-treated cell to DHT rapidly (1530
min) altered the homogeneous to punctate distribution of the already
translocated nuclear AR. When transported into the nucleus either by
casodex or by DHT, GFP-AR was resistant to 2 M
NaCl extraction, indicating that the homogeneously distributed AR is
also associated with the nuclear matrix. Taken together, these results
demonstrate that AR requires ligand activation for its nuclear
translocation where occupancy by only agonists and partial agonists can
direct it to a potentially functional subnuclear location and that one
receptor molecule can undertake multiple rounds of hormonal signaling;
this indicates that ligand dissociation/inactivation rather than
receptor degradation may play a critical role in terminating hormone
action.
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INTRODUCTION
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Steroid hormone receptors are ligand-activated transcription
factors. Initial identification of the estrogen receptor in the early
1960s and studies on its mechanism of action through subcellular
fractionation led to the development of a two-step model for steroid
hormone action (1, 2). In this model, unliganded receptor was thought
to be localized in the cytoplasm as a heteromeric complex with heat
shock proteins and, upon ligand binding, to undergo conformational
transition, dissociation from the heteromeric partners,
homodimerization, and nuclear translocation to initiate target gene
regulation. Subsequent studies with different approaches, such as
immunostaining, autoradiography with labeled steroids, and cytofusion
analysis, have generated conflicting pictures with respect to the
intracellular distribution of unliganded steroid hormone receptors
(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). In the case of androgen receptor, primary localization of the
unliganded receptor either in the cytoplasmic (9) or in the nuclear
compartment (10) of transfected cells has been reported. The prevalent
notion that, with the exception of the glucocorticoid receptor,
unliganded steroid receptors are nuclear proteins is largely based on
the assumption that observations of the cytoplasmic existence of other
unliganded receptors by cell fractionation may reflect nuclear leakage
of these proteins due to their loose association with the chromatin
(16, 17). In addition to introducing experimental artifacts, both
biochemical and histochemical approaches can provide only a static
picture of the otherwise dynamic state of a living cell. Recent
advances in imaging techniques allow monitoring the intracellular
molecular movements in real time (18). Chimeric gene constructs
expressing proteins of interest tagged with the modified and improved
version of aequorea green fluorescent protein (GFP) can be used to
follow movement of proteins in real time through wide-field water
immersion objectives and a high-resolution charge-coupled detection
device (19). We have used this approach to explore the intracellular
movement and subnuclear compartmentalization of the androgen receptor
(AR) after ligand treatment and ligand withdrawal. We report that most
of the unliganded AR under the steady-state condition resides in the
cytoplasm and, upon hormone exposure, rapidly migrates into the
nucleus. Furthermore, androgen withdrawal releases the receptor from
its chromatin association and exports it back into the cytoplasmic
compartment for recycling when the hormone is reintroduced.
Additionally, we show that ligands with both transactivation potential
and inhibitory action can cause translocation of the AR from cytoplasm
to nucleus albeit to a varying extent. However, only ligands with
agonist activity and not the pure antagonist, i.e. casodex,
are capable of translocating the AR into a distinct subnuclear
compartment. The role of ligands in nuclear translocation of GFP-AR
with a truncated AR and a weaker version of GFP has been reported
previously (20).
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RESULTS
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Ligand-Dependent Transactivational Activity of the Chimeric GFP-AR
and Kinetics of Its Nuclear Translocation
A fusion protein containing an amino-terminal extension of the
intact rat AR with GFP was used for this study. We first tested the
ability of this chimeric protein for androgen-dependent transactivation
of the mouse mammary tumor virus-chloramphenicol acetyltransferase
(MMTV-CAT) promoter-reporter in AR-negative PC3 cells. In the presence
of 10-9 M 5
-dihydrotestosterone
(DHT), cells transfected with the chimeric (GFP-AR) expression vector
showed about one third transactivation function as compared with the
nonchimeric AR (relative CAT activities of 183 ± 19 for GFP-AR
vs. 588 ± 11 for AR). The partial loss of
transactivational activity of the GFP-AR may be due to the GFP
interference with the amino-terminal TAF-1 function of the AR protein.
However, transactivation function of both of these proteins was almost
totally dependent on the presence of the androgen (relative CAT
activity without DHT of less than 3).
We then examined the localization of the GFP-AR in three different cell
lines, i.e. prostate-derived PC3, uterus-derived HeLa, and
kidney-derived COS1 cells. In the absence of hormone, GFP-AR was found
to be mostly cytoplasmic in all three cell types (Fig. 1
, AC). However, addition of DHT to the
culture medium resulted in a time-dependent translocation of the GFP-AR
into the nuclear compartment. Nuclear migration of the receptor was
rapid and clearly evident within 15 min after hormone treatment, and
the receptor became primarily or exclusively nuclear within 60 min. A
similar DHT-mediated nuclear translocation was also observed by
immunostaining of both the endogenous AR in AR-positive LNCaP cells and
COS1 cells transfected with the pCMV-AR expression vector (Fig. 2
, A and B).

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Figure 1. Androgen-Dependent Nuclear Translocation of GFP-AR
in Transfected Cells
A, PC3; B, HeLa; C, COS1. Images from the same cells were acquired at
0, 15, 30, and 60 min after treatment with 10-8
M DHT. Bar, 15 µm.
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Figure 2. Androgen-Dependent Nuclear Translocation of
Untagged AR Detected by Immunostaining
A, Untransfected LNCaP cells. B, COS1 cells transfected with pCMV-AR
expression vector. Cells in separate culture dishes were treated with
10-8 M DHT and fixed at different time
intervals (0, 15, 30, and 60 min) as indicated on the
top of the figure. Bar, 15 µm.
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We also observed that, at any particular hormone concentration, there
were cell-to-cell variations in the extent of nuclear translocation,
i.e. some of the transfected cells were more responsive than
others in the androgen-dependent nuclear import of the GFP-AR.
Therefore, to obtain a normalized rate of nuclear import, we scored 100
transfected cells within a field for the nucleocytoplasmic fluorescence
intensity. Cells with exclusively nuclear and predominantly nuclear
fluorescence (N>C) within the same field were counted at different
time points after hormone exposure. Similar quantitation was also
performed with immunostained COS1 cells transfected with the wild-type
rat AR expression plasmid. Results presented in Fig. 3
show the kinetics of hormone-dependent
nuclear translocation of the GFP-AR and immunodetected wild-type AR in
transfected COS1 cells. In both cases, a linear increase in nuclear
import after DHT treatment was observed, and the import was complete
between 60 to 90 min.

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Figure 3. Time course of Nuclear Translocation of GFP-AR and
Untagged AR in Transfected COS1 Cells
Cells in separate culture dishes were exposed to DHT (10-8
M) and fixed at different time intervals for either direct
observation (GFP-AR) or immunostaining (untagged AR). Each
point represents percent cells with predominantly
nuclear fluorescence. Open circles, GFP-AR;
closed circles, AR without the GFP tag.
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Nucleocytoplasmic Recycling of AR after Androgen Treatment and
Androgen Withdrawal
The dynamic nature of the nucleocytoplasmic movement of the AR and
its hormonal control was explored further by imaging of the GFP-AR
fluorescence after androgen treatment and withdrawal. For this set of
experiments, we used COS1 cells, where the expression plasmid is
subjected to intracellular amplification, yielding a higher level of
GFR-AR that is necessary for monitoring the decaying fluorescent
protein after prolonged cycloheximide inhibition of new protein
synthesis. Results presented in Fig. 4A
show a reversal of cytoplasmic to nuclear migration of the GFP-AR after
hormone withdrawal in the presence of 50 µg/ml cycloheximide. At
12 h after hormone withdrawal, no significant difference in the
intensity of fluorescence between nuclear and cytoplasmic compartments
can be distinguished. Since export of the receptor from the nuclear to
the cytoplasmic compartment only became evident between 6 to 12 h
after hormone withdrawal, it was of interest to compare the rate of
ligand dissociation with nuclear export. Data presented in Fig. 4B
show
about 50% ligand dissociation within the same time frame when nuclear
export became clearly evident. A discordant relationship between ligand
dissociation and nuclear export of GR has been reported earlier (21).
We speculate that a dynamic process involving capture of the
dissociated ligand from one receptor molecule by another unoccupied
receptor provides a threshold effect of nuclear retention after hormone
withdrawal. The fact that the concentration of cycloheximide used in
these experiments (50 µg/ml) caused a total inhibition of
immunodetectable GFP-AR synthesis is demonstrated by results presented
in Fig. 4C
. Even at 10 µg/ml, no immunodetectable GFP-AR can be seen
within 15 h of culture period while untreated cells produced a
high level of this protein. The GFP-AR exported from the nucleus
after 12 h of hormone withdrawal could again be transported into
the nuclear compartment after reexposure to its hormonal ligand (Fig. 5A
). Quantitative analysis of this
reutilization process for up to four cycles of import and three cycles
of export by sequential hormone treatment and withdrawal (Fig. 5B
)
showed a gradual decline of the recycling competency of the receptor
protein. We interpret this observation to indicate a time-dependent
structural damage (degradation) of the receptor protein. However, a
decreased level of importins or other proteins in nucleocytoplasmic
transport after inhibition of protein synthesis by cycloheximide may
also be a contributing factor. During the first two rounds of
recycling, the receptor was almost equally translocation competent.
Upon subsequent hormone treatment the same batch of cells at their
third and fourth import cycles showed a gradual decline of
ligand-dependent nuclear import. Thus, the same receptor may be able to
mediate multiple rounds of hormonal signaling, which suggests that
inactivation and/or dissociation of hormonal steroids, rather than
nuclear degradation of the receptor protein after its initial import,
may play a dominant role in the termination mechanism of hormone
action.

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Figure 4. Export of the AR into the Cytoplasmic Compartment
after Hormone Withdrawal
A, Time course of nuclear-to-cytoplasmic export of the AR after
androgen withdrawal. COS1 cells were first exposed to DHT for 4 h
to translocate the GFP-AR into the nucleus and then incubated in the
hormone-free medium containing cycloheximide (50 µg/ml) and imaged at
different time intervals (0, 2, 6, and 12 h). B, Intracellular
dissociation kinetics of 3H-DHT in the presence
of excess unlabeled DHT. C, Western blot showing the effect of 10 and
50 µg/ml cycloheximide on GFP-AR synthesis in transfected cells.
Cycloheximide was added 2 h after transfection, and cells were
harvested 15 h later for Western blotting. Lane 1 contains sample
derived from untransfected cells cultured in the presence of 50 µg/ml
cycloheximide; lanes 2, 3, and 4 contain samples derived from
transfected cells in the presence of 0, 10, and 50 µg/ml
cycloheximide, respectively. The location of the GFP-AR band is marked
with the arrowhead. The lower band marked with an
asterisk represents a nonspecific cellular protein that
cross-reacts with the antibody.
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Figure 5. Nucleocytoplasmic Recycling of GFP-AR after
Androgen Treatment and Withdrawal
A, Intracellular distribution of GFP-AR before hormone treatment (a),
4 h after treatment with 10-8 M DHT
(b), 12 h after hormone withdrawal in the presence of 50 µg/ml
cycloheximide (c), and 4 h after hormone treatment (in the
presence of cycloheximide) to hormone-withdrawn cells (d).
Bar, 15 µm. B, Quantitative analysis of cells
with exclusively nuclear fluorescence after four cycles of DHT-mediated
import and three cycles of export after hormone withdrawal. Each
histogram represents average values from four
independent experiments ± SD.
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The nuclear import of steroid receptors mediated by importins
interacting with the nuclear localization signal (NLS) has been fairly
well characterized (9, 22, 23, 24, 25, 26, 27). However, the corresponding nuclear
export signal (NES) of the steroid hormone receptors has not been
identified, and the mechanism of the nuclear export of steroid
receptors is not clearly understood (27). Leptomycin B (LMB) is a
specific inhibitor of exportins, a class of chaperone proteins that
bind to NES and facilitate the export of nuclear proteins into the
cytoplasmic compartment (28, 29). Recently the role of exportin in the
nucleocytoplasmic recycling of a cytoplasmically sequestered
signal-activated transcription factor, i.e. p65 subunit of
nuclear factor-
B (NF
B), has been described (30). Using p65-GFP as
our positive control, we examined the effect of LMB on the nuclear
export of the GFP-AR after androgen withdrawal. Although LMB failed to
prevent the export of GFP-AR from the nuclear to the cytoplasmic
compartment, the conditioned medium derived from the GFP-AR transfected
cells containing LMB was able to prevent the nuclear export of p65-GFP
with consequent accumulation of this protein in the nucleus (Fig. 6
). The latter observation shows that LMB
added to the culture medium of cells transfected with GFP-AR is still
biologically active and that the nuclear export of the
ligand-dissociated AR may involve an as-yet-uncharacterized
exportin-independent transport mechanism.

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Figure 6. LMB-Insensitive Nuclear Export of GFP-AR
The upper frames show that LMB is ineffective in
preventing the nuclear export of GFP-AR after androgen withdrawal. The
lower frames show that nucleocytoplasmic recycling of
p65-GFP is almost totally inhibited when the conditioned medium
containing LMB was added to cells transfected with the chimeric
expression vector producing p65 subunit of NF B tagged with GFP.
Frame a, COS1 cells treated with DHT for 4 h without LMB; frame b,
after 12 h of hormone withdrawal in the presence of LMB. Both
frames a and b show cells transfected with GFP-AR. Frames c and d,
p65-GFP transfected cells before and 60 min after incubation with LMB
containing conditioned medium retrieved from DHT-withdrawn cells used
for frame b. Bar, 15 µm.
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Nuclear Translocation and Differential Subnuclear
Compartmentalization of GFP-AR by Cross-Reactive AR Agonists and
Inhibitors of AR Function
The ligand-binding domain of the AR is known to cross-react with
17ß-estradiol (31). Activation of steroid hormone receptors by growth
factors such as epidermal growth factor (EGF) has also been reported
(32, 33, 34, 35, 36). We have examined relative translocation and transactivation
potentials of these and other potential AR modulators in the living
cell. Results presented in Fig. 7A
show
that both 17ß-estradiol and progesterone, especially at a higher
concentration (10-7 and
10-6 M), were effective in
translocating the GFP-AR into the nucleus. However, the gluococorticoid
dexamethasone and EGF were totally ineffective. Two antiandrogens,
cyproterone acetate (a mixed agonist/antagonist) (37) and casodex (a
pure antagonist) (38), also showed a relatively limited ability to
translocate GFP-AR into the nucleus. When these hormones and
antihormones were tested for their relative transactivation function
for the AR-dependent promoterreporter construct derived from the
rat probasin gene (i.e. ARR3-TK-Luc) (39), it showed that in
addition to DHT, 17ß-estradiol, progesterone, and cyproterone acetate
were transactivation competent (Fig. 7B
). However, the relative
abilities of these steroids for nuclear import and transactivation
function were different. Next to DHT, estradiol showed the best
translocation function, while cyproterone acetate was more competent in
transactivation than estradiol. Among the translocation-competent
ligands that we have tested, casodex, a pure antagonist, was found to
be the least effective. Again, some cells were more sensitive to
casodex-mediated translocation than others and after a prolonged
exposure (>20 h) to 10-6
M casodex, about 70% of the cells showed
primarily nuclear fluorescence (data not shown).

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Figure 7. Relative Effects of Hormones and Antihormones on
Nuclear Translocation and Transactivation Function of GFP-AR
A, Percent cells with exclusively nuclear fluorescence at 3 h
after hormone/antihormone treatment. Each histogram represent average
of two independent experiments. Sixty-seven percent of the transfected
cells treated with 10-8 M DHT showed
exclusively nuclear fluorescence, which was used as 100% for comparing
the potencies of other agents. B, Transactivation of ARR3-TK-Luc by
GFP-AR after treatment with different doses of hormones and
antihormones as labeled. E2, 17ß-estradiol; Prog,
progesterone; Dex, dexamethasone; EGF, epidermal growth factor (50,
100, and 200 ng/ml); CA, cyproterone acetate; Cdx, casodex. Each
histogram represents average values of six independent
experiments ± SD.
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Additionally, we noted that the pattern of nuclear distribution of the
GFP-AR was distinctly different for ligands with or without any
transactivation activity. DHT (a pure hormonal agonist),
17ß-estradiol (a cross-reactive hormonal agonist), and cyproterone
acetate (a mixed agonist/antagonist) showed a punctate distribution
pattern indicative of the association of the translocated receptor
within a subnuclear compartment. However, in the case of casodex (a
pure antagonist) the translocated receptor was evenly distributed (Fig. 8
). Since proteasome-dependent nuclear
degradation of the estrogen receptor has been observed (40), we wanted
to rule out the possibility of proteasomic inclusion of GFP-AR as the
cause of the punctate distribution. Treatment of GFP-AR transfected
cells with the proteasome inhibitor MG132 (3 µM) before
DHT treatment did not alter the intranuclear punctate distribution of
the GFP-AR (data not presented). This observation indicates that the
formation of the punctate foci is an intermediate step in the signaling
rather than degradation process. That ligand-mediated nuclear
translocation and subnuclear compartmentalization represent two
distinct steps in the process of hormonal signaling was further
indicated by a rapid conversion of the nucleoplasmic GFP-AR of the
casodex-treated cell by subsequent treatment with DHT (Fig. 9A
). In this case punctate appearance was
evident within 15 min, and formation of the punctate foci was complete
within 1 h after DHT treatment. Salt extraction of nuclear
proteins followed by immunoblot analysis showed that most of the GFP-AR
within the nuclei of either casodex-treated or DHT-treated cells is
present within the salt (2 M NaCl)-resistant nuclear matrix
fraction. Thus, the punctate foci may represent a distinct step in
steroid receptor function after its binding to the nuclear
matrix.

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Figure 8. Subnuclear Localization of GFP-AR after Nuclear
Translocation by Hormones and Antihormones
The pictures show the pattern of nuclear localization in transfected
COS1 cells after translocation induced by DHT (10-8
M), cyproterone acetate (CA, 10-6
M), casodex (10-6 M),
and 17ß-estradiol (E2, 10-6
M). Except casodex (a pure antagonist), all other
ligands produced a punctate nuclear distribution.
Bar, 10 µm.
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Figure 9. Conversion of Homogeneous to Punctate Nuclear
Distribution of Casodex-Treated Cells Subsequently Exposed to DHT
A, Fluorescence pattern of a transfected COS1 cell initially treated
with casodex (10-6 M) for 3 h and
subsequently treated with 10-8 M DHT. A
single cell showing exclusively nuclear fluorescence was imaged at 0,
15, 30, and 60 min after DHT treatment. B, Western blot of nuclear
extracts of cells treated with casodex and DHT. COS1 cells were
cultured for 15 h in the presence of 10-6
M casodex (CDX), for 4 h in the
presence of 10-8 M (DHT), and
19 h in the presence of 10-6 M
casodex followed by 4 h in the presence of 10-8
M DHT (CDX DHT). Monoclonal mouse
antibody to GFP was used to detect the GFP-AR. Three lanes for each of
the treatment condition represent as follows: F1, supernatant after
extraction with 0.25 M ammonium sulfate; F2,
supernatant after extraction with 2 M NaCl; F3, pellet
containing nuclear matrix.
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DISCUSSION
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Until recently, with few exceptions (14, 20, 41, 42), technical
limitations did not allow real time imaging of nucleocytoplasmic
movement of steroid hormone receptors within a single cell. Although
static pictures have generated a wealth of information, fixation
artifacts and nuclear leakage during the cell fractionation procedure
have often led to controversial conclusions. The general consensus,
based on these static approaches, suggests that, in the unliganded
state, certain members of the steroid hormone receptor family, such as
the glucocorticoid receptor, reside in the cytoplasmic compartment,
whereas another class typified by the estrogen receptor, irrespective
of the ligand-receptor interaction, is always located in the nucleus
(4, 7, 25, 43). However, the subcellular location of the unliganded AR
has still remained controversial (9, 10, 16). Recent improvements in
the imaging technology with wide-field water immersion objectives can
be used to examine the intracellular movement of GFP-labeled proteins
in the living cell with a high degree of resolution and in real time.
Additionally, the newly modified version of the jellyfish GFP is not
only stable at 37 C in mammalian cells but also emit stronger
fluorescence signal allowing its accurate localization within various
subcellular compartments. This avoids the need for disruption of the
cellular dynamics or lowering of the temperature for stabilizing the
chimeric protein. The GFP-AR chimera that we have used maintains its
ligand-dependent transactivation function at 37 C, albeit to a lesser
extent than that of the unmodified AR. Our unpublished results show
that introduction of a short histidine tag
(Met-Arg-Gly-Ser-His-His-His-His) at the N-terminal part of the AR also
reduces its transactivation potential to a similar extent. In both
cases, functional interference with the N- terminal TAF-1 domain
seems to be the likely possibility. In an earlier study a chimeric
GFP-AR was also used to examine its nuclear translocation (20). Due to
the lower fluorescence intensity of the earlier version of GFP, use of
an N-terminally truncated form of AR, whole-cell imaging, and culturing
of cells at 30 C to stabilize the GFP-AR, observations reported by
these authors are different from those presented in this article. The
newly modified GFP (i.e. EGFP) emits approximately 35-fold
more intense fluorescence than the wild-type GFP and the chimeric
protein is stable at 37 C. Additionally, optical sectioning allows
finer delineation and uniform imaging than the whole-cell images, which
blur the distribution pattern of the fluorescent protein due to
differences in the thickness of various parts of the cell. Because of a
combination of all of these experimental problems, these investigators
observed only very low transactivation function of GFP-AR, could not
detect complete nuclear translocation after hormone treatment, and
failed to observe any subnuclear sequestration of AR (20).
In all of the cell lines that we have examined, the unliganded GFP-AR
is primarily localized in the cytoplasmic compartment, and upon
androgen exposure, rapidly migrates into the nucleus. Translocation of
AR can be detected within a few minutes after hormone treatment and is
almost complete within 60 min. Translocation kinetics of the GFP-AR are
very similar to the pattern observed in immunostained COS1 cells
containing unmodified AR. We have also observed that both the
unliganded GFP-ER and GFP-peroxisome proliferator-activated receptor
(PPAR
) are primarily nuclear in COS1 cells (data not shown). From
all of these results and the existing literature it is reasonable to
conclude that members of the steroid hormone receptor family can be
classified into two distinct groups, one that requires ligand binding
for nuclear translocation [e.g. glucocorticoid receptor
(GR) and AR] and another that is nuclear, even without ligand binding
[e.g. estrogen receptor (ER), progesterone receptor (PR),
and PPAR
]. Structural features that differentiate these two groups
are presently unknown. Cytoplasmic proteins that are larger than 60 kDa
are transported into the nuclear compartment through a
chaperone-mediated transport process (44). In the case of unliganded GR
and AR, the NLS, which interacts with chaperone proteins (importins),
is likely to be masked by receptor-associated heat shock proteins and
immunophilins. This group of receptors may require ligand-mediated
conformational change to unmask the NLS site for its appropriate
interaction with importins, whereas in the case of ER and PPAR
, the
NLS may be easily accessible to importins, even in the absence of their
respective ligands.
Our observation of the nucleocytoplasmic recycling of the GFP-AR
in living cells has an important bearing on the termination mechanism
of the signaling process. We have observed multiple cycles of the
DHT-dependent import-export process of GFP-AR after inhibition of new
protein synthesis in the presence of cycloheximide. These results
suggest that termination of nuclear signaling may depend on
dissociation of the hormonal ligands due to ligand withdrawal or ligand
inactivation. However, the receptor protein, after its dissociation
from the ligand and chromatin, may be reutilized for another round of
the signaling process when the ligand is again made available. A recent
report has suggested an alternative mechanism for the termination of
hormonal signal by nuclear degradation of the specific receptor protein
through ubiquitination followed by proteasomal degradation (40).
However, the quantitative contribution of such a process of receptor
degradation in the termination of hormonal signaling has not been
established. Our results indicate that, upon ligand withdrawal, most of
the GFP-AR is exported back into the cytoplasmic compartment and is
still competent to undergo the ligand-dependent translocation process.
Multiple rounds of hormonal signaling by recycled receptors and
cross-reactive ligands under certain clinical conditions, such as
recurrent forms of AR-positive prostate cancer, may contribute to the
etiology of the disease. In a large number of these cases there are
abnormally high levels of AR, due to either AR gene amplification or
possibly to AR up-regulation after prolonged periods of androgen
deprivation and antiandrogen therapy (45, 46). These recurrent cancer
cells expressing high levels of AR may experience adequate androgenic
response caused by multiple rounds of receptor recycling through
intermittent exposure of the AR to cross-reactive partial agonists of
nongonadal origin.
The role of exportin/CRM1 chaperone proteins in the
nuclear-to-cytoplasmic export of a number of proteins has been well
established (27, 47, 48, 49). This mechanism is exquisitely sensitive to
inhibition by LMB. LMB sensitivity to nuclear export of PR and GR by
the immunostaining method has been examined earlier. Nuclear export of
PR is insensitive to LMB (50), and contradictory results have been
reported for GR (51, 52). Our results are consistent with the
conclusion that the nuclear export of AR is mediated through an
LBM-insensitive exportin-independent process, the nature of which has
yet to be established. We feel that the results presented in this
report concerning the LMB insensitivity of the export process are
especially convincing, because 1) our observations were made in the
living cell, without any additional intervention; and 2) inhibition of
the nuclear export of GFP-p65 by the LMB-containing conditioned medium
derived from the GFP-AR-transfected cells.
We have observed a general correlation between the dose-dependent
transactivational function of AR by two other natural steroid hormones
(i.e. 17ß-estradiol and progesterone, that are capable of
cross-reacting with AR), and their effectiveness for translocating the
receptor into the nucleus. Both dexamethasone and EGF were unable
either to cause translocation of GFP-AR into the nucleus or to
transactivate the ARR3-thymidine kinase (TK)-Luc in the cell
transfection assay. A number of studies have suggested the potential
role of EGF in enhancing steroid receptor function (32, 33, 34, 35, 36). The
results presented in this article indicate that such a modulating
influence of EGF may not be due to a direct influence on either the
nuclear translocation or transactivation function of the unliganded AR
and may be dependent on initial androgenic activation (32).
It is also of interest to note that both a mixed
agonist/antagonist (cyproterone acetate) and a pure antagonist
(casodex) were moderately effective in mediating the nuclear
translocation of GFP-AR as compared with DHT. However, the patterns of
nuclear distribution of the translocated receptor in these two cases
are distinctly different. Similar to other agonists, the cyproterone
acetate treatment led to a punctate nuclear distribution of GFP-AR,
and, in the case of casodex, the fluorescence was homogeneously
distributed within the nuclear compartment. However, upon subsequent
exposure to DHT, the receptor moved to a distinct subnuclear
compartment. A similar difference in the distribution pattern of GR and
ER after agonist and antagonist treatment has also been reported (41, 53, 54). Similar to ER (53), our results show that both homogeneous and
punctate forms of GFP-AR are associated with the nuclear matrix. Thus,
formation of the punctate foci appears to be a distinct step in the
mechanism of steroid hormone action, and this step lies beyond the
initial matrix association of the receptor protein. It is of interest
to note that only ligands with agonist or partial agonist activity can
carry the process up to this step and beyond to initiate
transcriptional activation.
 |
MATERIALS AND METHODS
|
---|
Plasmid Constructs
Chimeric GFP-AR was generated by inserting the rat AR
cDNA (provided by Dr. Shutsung Liao, University of Chicago, Chicago,
IL) into the BamHI site of the 3'- end of the coding region
of the modified aequorea GFP (pEGFP-C2, CLONTECH Laboratories, Inc. Palo Alto, CA). Chimeric GFP-ER
was
constructed similarly by inserting the human ER
cDNA into the
BamHI site of the pEGFP-C2 vector. For generating the
chimeric GFP construct of the p65 subunit of NF
B, the human p65
cDNA fragment was excised from the pCMV-p65 expression vector (provided
by Dr. John Cidlowski, NIEHS, Research Triangle Park, NC) and
inserted into the BamHI site of the GFP expression plasmid,
pEGFP-N3. Nucleotide sequences of all DNA constructs were
authenticated by manual sequencing. Construction of the expression
vectors pCMV-AR and ARR3-TK-Luc has been described previously (39). The
promoter-reporter construct pMMTV-CAT was a gift from Dr. Stephen
Harris (The University of Texas Health Science Center, San Antonio,
TX).
Cell Culture, Transfection, Cell Fractionation, and Western Blot
Analysis
PC3, HeLa, LNCaP, and COS1 cells were obtained from
American Type Culture Collection (Manassas, VA) and
cultured in serum-containing media as recommended by the supplier.
Cells were plated in six-well culture flasks at 150 x
103 cells per well in the growth medium (MEM
supplemented with 5% charcoal-stripped FBS), grown overnight, and
cotransfected with either pARR3-TK-Luc or pMMTV-CAT promoter-reporter
(1 µg) along with either wild-type steroid receptor-expression
plasmid (pCMV-rAR) or the chimeric GFP plasmids (pCMV-GFP-rAR,
pCMV-GFP-ER), using FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, IN) reagent, 6 µl/well, or
LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD),
8 µl/ml.
Media were changed at 24 h after transfection and were replaced
with fresh media with or without the hormonal ligands. Cells were
incubated for an additional 24 h before harvesting and extraction
of proteins. Cell extracts were assayed for protein concentration
(Bradford protocol), luciferase activity (assay kit, Promega Corp., Madison, WI), and CAT by enzyme-linked immunoassay (CAT
ELISA kit, Roche Molecular Biochemicals). For CAT activity
results were expressed as optical density (x
103) per µg protein.
For experiments involving imaging of GFP fluorescence, after
transfection, cells were allowed to express the appropriate chimeric
protein for 30 h before any hormonal treatment. For import/export
experiments after hormone treatment and withdrawal, hormone-treated
cells were rinsed at 0, 30, 60, and 120 min (twice at each time point
with 3 ml of the culture medium containing the charcoal-stripped serum)
before the next round of the translocation process was initiated. To
prevent de novo protein synthesis, the replacement medium
was supplemented with cycloheximide (50 µg/ml).
Cycloheximide-mediated inhibition of GFP-AR synthesis in transfected
cells was monitored by Western blot analysis with monoclonal antibody
to GFP (primary antibody) and horseradish peroxidase-conjugated
antimouse IgG. Peroxidase signal was visualized with ECL plus
reagent according to the manufacturers recommendation (Amersham Pharmacia Biotech, Arlington, IL). Leptomycin B (a gift from Dr.
M. Yoshida, University of Tokyo, Tokyo, Japan) was used at a final
concentration of 15 ng/ml.
Isolation of cell nuclei and nuclear fractionation were performed
according to the modified procedure described by Htun et al.
(53). Briefly, freshly harvested cells were suspended in TNM buffer (10
mM Tris, pH 8.0, 300 mM
sucrose, 100 mM NaCl, 2 mM
MgCl2, 1% dithioglycol, and 1
mM phenylmethylsulfonyl fluoride) and after
homogenization cells were treated with 0.5% Triton X-100. Isolated
nuclei were washed in TNM buffer and resuspended in DIG buffer (TNM
buffer with 50 mM NaCl and 3
mM MgCl2) and digested with
168 µ/ml DNase I. Digested nuclei were first fractionated with 0.25
M ammonium sulfate
(fraction 1), and the pellet was subjected to two sequential
extractions with 2 M NaCl (combined supernatants
were used as fraction 2), and the remaining pellet containing the
nuclear matrix was used as the fraction 3. After freeze-drying, all
three fractions were dissolved in 100 µl Laemlis SDS buffer for
Western blot analysis according to the procedure described above.
Fluorescence Imaging
Fluorescence imaging of live cells was performed through a E400
Eclipse epifluorescence microscope and water immersion objectives
(Nikon, Melville, NY) connected to a video monitor through
a cooled charge-coupled device camera (Princeton Instruments, Princeton, NJ; Roper Scientific Inc., Trenton, NJ).
The microscope contains a temperaturecontrolled stage, a stepper
motor for optical sectioning, and an automatic filter wheel for
appropriate filter selection. Optical sectioning was performed at
1-µm steps and images were reconstructed through Metamorph
software (Universal Imaging Corp., West Chester, PA).
For immunodetection, cells were cultured in two chambered glass slides
and fixed with 100% methanol at -20 C for 10 min. Fixed cells were
rinsed twice with PBS (10 min each wash) and air dried. Before antibody
treatment, slides were incubated in a humid chamber for 30 min,
followed by overnight incubation (at 4 C) with polyclonal anti-AR
antibody (affinity-purified IgG) produced in the rabbit. The antibody
was generated by conjugating the first 20 N-terminal amino acids of AR
to keyhole hemocyanin. After removal of the anti-AR antibody by rinsing
three times with PBS, cells were treated for 1 h with sheep
antirabbit IgG antibody conjugated with the fluorescent dye CY3. This
step was followed by rinsing and mounting with coverslips and
observation under the fluorescence microscope.
Dissociation Rate of AR
Binding of 3H-DHT and its rate of
dissociation were assayed according to Zhou et al. (55).
Briefly, COS1 cells transfected with GFP-AR expression plasmid for
48 h were subsequently placed in a serum-free medium containing 5
nM 3H-DHT in the presence
or absence of a 100-fold molar excess of unlabeled DHT and incubated
for 2 additional hours. Cells were then washed twice in PBS and
incubated further in the serum-free medium containing 50
µM unlabeled DHT for various time periods. Cell
samples collected at different time intervals were washed twice with
PBS, dissolved in Tris-SDS-glycerol buffer, and counted for
radioactivity.
 |
ACKNOWLEDGMENTS
|
---|
We thank Gilbert Torralva for dedicated technical assistance and
Lita Chambers for secretarial help.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Arun K. Roy, Ph.D., Department of Cellular & Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7762. E-mail:
roy{at}uthscsa.edu
B.C. is a career scientist with the Department of Veterans Affairs.
S.C.A. was partially supported by a fellowship from the Korean Science
and Engineering Foundation. This work was supported by NIH Grants
DK-14744 and AG-10486.
Received for publication December 31, 1999.
Revision received April 4, 2000.
Accepted for publication April 24, 2000.
 |
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