Department of Anatomy and Neurobiology Kyoto Prefectural University of Medicine Kawaramachi Hirokoji, Kamigyo-ku Kyoto 602-8566, Japan
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ABSTRACT |
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INTRODUCTION |
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Recent studies have used a chimera of green fluorescent protein (GFP), a 27-kDa protein from the jellyfish Aequorea victoria, and GR (GFP-GR) showing dynamic cytoplasmic-to-nuclear translocation of GR upon ligand treatment (11, 12). Furthermore, Nishi et al. (13) demonstrated that there is no significant difference in the patterns of nuclear translocation of GFP-GR between neural cells and nonneural cells. Although fusion proteins of GFP and MR (GFP-MR) were also used to examine the dynamic changes in the subcellular localization in response to ligands using CV-1, CHO, and MDCK cells (14, 15), MR dynamics have never been studied in living neural cells. In the present study, we examined the intracellular trafficking of MR, focusing on 1) the trafficking time course, 2) relationship with transcriptional sites in the nucleus, 3) ligand specificity, 4)association with the signal transduction system, 5) effects of microtubule disruption, and 6) comparison of these parameters between neurons and nonneural cells.
Another interesting unsettled question is how MR interacts with GR in the brain. In the central nervous system, MRs are localized mainly in the hippocampus, while GRs are distributed throughout the brain (16, 17, 18, 19). However, MR and GR are highly colocalized in the hippocampus, particularly in the pyramidal cells of the CA1 region (20, 21, 22). MR has a high affinity for corticosterone (CORT), a common endogenous ligand for MR and GR in rodents, and is extensively bound at low levels of circulating CORT, while GR has a lower affinity for CORT and is extensively bound at high CORT levels (1, 23, 24, 25, 26). The differential actions of MR and GR in the hippocampal regions at different concentrations of CORT are intriguing. To elucidate possible differential responses of MR and GR to CORT, and also possible interactions between MR and GR, we investigated the subcellular distribution of MR and GR in response to CORT in single living cells using fusion proteins labeled with two different spectral variants of GFP, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP). We report here, for the first time, the results of the visualization of trafficking of MR and GR simultaneously in single living neurons in comparison with nonneural cells.
Our findings clearly showed that the subcellular distribution of GFP-MR is dynamically changed in response to ligands, and that the trafficking patterns of GFP-MR in neurons and nonneural cells are nearly the same. We also demonstrated that CORT-induced nuclear accumulation of YFP-MR and CFP-GR exhibited differential time courses at different ligand concentrations and in different cell types.
Furthermore, to scrutinize the different response of MR and GR to CORT, we examined the role of receptor association with heat shock proteins (hsp), particularly hsp90, in the nuclear translocation of MR and GR. Since hsp90 affects the hormone binding activity of MR (27) and GR (28), hsp90 association may provide an explanation for differential actions of CORT on MR and GR. We used geldanamycin that binds to hsp90 and inhibits hormone binding affinity to steroid hormone receptors (29). Different mechanisms that might be involved in the ligand-induced nuclear translocation of MR and GR regulated by the chaperoning effect of hsp90 in cultured hippocampal neurons and nonneuronal cells were discussed.
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RESULTS |
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As a control, cells transfected with only GFP showed neither MR nor GR immunoreactivity, which demonstrates that COS-1 cells express no endogenous corticosteroid receptor proteins. GFP-MR- or YFP-MR-transfected cells showed MR immunoreactivity in both the cytoplasm and the nucleus. After treatment with CORT, MR immunoreactivity was detected within the nucleus. In the CFP-GR-transfected cells, GR immunoreactivity was found predominantly in the cytoplasm in the absence of ligand, whereas the treatment with CORT induced localization of GR immunoreactivity in the nucleus (data not shown).
An immunoblot of GFP-MR- or YFP-MR-transfected COS-1cells showed a
single band at 121 kDa labeled by anti-MR antibody (Fig. 1A, lane 2, and Fig. 1C
, lane 1,
respectively), which was at the expected size of GFP-MR and YFP-MR
fusion proteins, while no specific band was observed in cells
transfected with GFP alone (Fig. 1A
, lane 1). The expected 108-kDa
protein was detected in CFP-GR-transfected COS-1 cells using anti-GR
antibody (Fig. 1C
, lane 2).
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Dynamics of GFP-MR in the Transfected Cells
Time Course Study
We then introduced GFP-MR fusion protein into primary cultured
hippocampal neurons as well as COS-1 cells. In the absence of ligand,
GFP-MR was distributed almost homogeneously in both cytoplasm and
nucleus in the majority of transfected cells in both cell types (Fig. 2, A and B), although in about 10% of
transfected cells, GFP-MR was observed in either the cytoplasmic or the
nuclear region. There were no significant differences in the GFP-MR
nuclear translocation patterns among the different cell types used in
this study: COS-1 cells, which express no endogenous MR, and cultured
hippocampal neurons, which express endogenous MR. Exposure to CORT
caused a nuclear accumulation of GFP-MR in about 100% of the
transfected cells. GFP-MR was mostly accumulated in the nuclear region
within 60 min at 37 C in both COS-1 cells and hippocampal neurons (Fig. 2
, A and B). In each cell type, the nuclear accumulation started at
around 5 min after CORT addition, and nearly half-maximal accumulation
was observed at about 15 min. The images shown in Fig. 2
, A and B, were
treated with an image deconvolution procedure. The fluorescence
appearance of GFP-MR in COS-1 cells became more heterogeneous in
approximately 3060 min after CORT treatment and finally showed
heterogeneous dot-like distribution patterns in the nucleus, while
there was no fluorescence distribution in the nucleoli. The appearances
of the cells after recording images for 1 h were almost the same
as those before recording images, indicating that the cells did not
deteriorate during 1 h of time-lapse imaging.
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Ligand Specificity
In addition to CORT, we employed treatment with aldosterone (ALD),
dexamethasone (DEX), progesterone (PRG), or estradiol
(E2) to examine the ligand specificity of GFP-MR.
ALD is a specific ligand for MR with a very high affinity. DEX, a
typical synthetic agonist for GR, has a chemical structure similar to
that of ALD (32). Therefore, DEX shows binding affinity for MR,
although the dissociation constant (Kd) value of
DEX for MR is about 10-fold lower than that of ALD. It is also known
that PRG, which also has a chemical structure similar to that of ALD,
binds to MR with a Kd value mostly the same as
that of ALD (33, 34). We observed that 1 x 10-
7 M ALD, DEX, or PRG induced the nuclear
translocation of GFP-MR in essentially the same manner as observed with
CORT (Fig. 4, A, B, and C, respectively),
and hippocampal neurons (data not shown). In contrast,
E2 has a different structure at the ligand-
binding site and cannot bind to MR. We found that GFP-MR was not
translocated into the nuclear region in cells treated with 1 x
10-7 M E2
(Fig. 4D
), or in hippocampal neurons (data not shown). Taken together,
the present findings confirm that GFP-MR retains its native receptor
structure and exhibits high specificity for ligands.
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We next addressed the question of whether a certain pathway is involved
in the receptor trafficking between the cytoplasm and the nucleus. We
focused on microtubules in the present study. We pretreated cells with
10 µM colchicine or 10 µg/ml nocodazole for 3 h
before exposure to CORT. These doses of drugs were shown to induce an
almost complete depolymerization of microtubules within 1 h (7, 36). We confirmed the microtubule disruption by immunocytochemistry
using antibody against tyrosinated -tubulin (37), as shown in a
previous report (13). The pretreatment with colchicine or nocodazole
did not cause significant changes in the manner of CORT-induced nuclear
accumulation of GFP-MR in COS-1 cells and hippocampal neurons (Fig. 5
, A and B, respectively). In both types
of cells, GFP-MR was completely accumulated in the nuclear region
within 60 min after CORT addition.
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In COS-1 cells, the nuclear translocation of CFP-GR was started
around 10 min after addition of 10-6
M CORT and was completed within 30 min (Fig. 6A). YFP-MR was also
accumulated in the nuclear region within 30 min in the presence of
10-6 M CORT (Fig. 6B
). The average
nuclear/cytoplasmic ratios of YFP-MR and CFP-GR fluorescence
intensities for each time point in the presence of
10-6 M CORT are shown in Fig. 6E
(top). Each resulting curve reflected a relative increase in
the nuclear intensity of YFP-MR or CFP-GR. These quantitative data and
fluorescence images revealed that the rates of nuclear accumulation of
YFP-MR and CFP-GR in the presence of 10-6
M CORT were nearly equal. In contrast, in the
presence of 10-9 M CORT,
CFP-GR started to translocate into the nuclear region after about 15
min, and its fluorescence remained in the cytoplasm even after 60 min
(Fig. 6C
), while YFP-MR began to accumulate in the nuclear region
around 5 min after exposure to 10-9
M CORT, and was completely localized in the
nucleus within 30 min (Fig. 6D
). The results of quantitative analysis
(Fig. 6E
, bottom) and fluorescence images showed that the
nuclear accumulation rate of YFP-MR was faster than that of CFP-GR in
the presence of 10-9 M
CORT.
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In contrast, hippocampal neurons exhibited no significant differences
between the rates of nuclear accumulation of CFP-GR and YFP-MR in the
presence of 10-6 M or
10-9 M CORT (Fig. 7, AD). Both CFP-GR and YFP-MR began to
accumulate in the nuclear region about 10 min after the treatment and
mostly localized in the nucleus within about 30 min at both low and
high concentrations of CORT. These results were also confirmed by the
quantitative analyses shown in Fig. 7E
.
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Effect of Geldanamycin
Finally, we investigated the effects of hsp90 association on
receptor trafficking using geldanamycin. COS-1 cells cotransfected with
YFP-MR and CFP-GR were pretreated with 1 µM geldanamycin
for 3 h and then exposed to CORT. In the presence of both
10-6 M (Fig. 8, A and B) and
10-9 M CORT (data not
shown), most of the fluorescent intensities of
YFP-MR and CFP-GR remained in the cytoplasm even 60 min after CORT
treatment. There were no significant differences in the inhibitory
action of geldanamycin on the method of CORT-induced nuclear
translocation between YFP-MR and CFP-GR.
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DISCUSSION |
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Time-Lapse Study of GFP-MR Trafficking in Single Living Cells
The subcellular distribution of MR in the absence of ligands
remains quite controversial. Some studies have suggested mostly
cytoplasmic (9, 10), whereas others have suggested mostly nuclear (39),
localization. Our results observed in living hippocampal neurons and
COS-1 cells showed that GFP-MR was distributed both in the cytoplasm
and the nucleus in the absence of ligands in the majority of
transfected cells. These results are mostly consistent with those of a
previous study, which also employed GFP-MR fusion proteins in living
CHO cells and CV-1 cells (15). They indicated that MR could belong to a
third category within the steroid/thyroid receptor superfamily, in
which the unliganded receptor is not predominantly in the cytoplasm
like the GR (11, 12, 13) or in the nucleus as other members of this family
(40). A shuttling of unliganded receptors between the cytoplasm and the
nucleus has been reported for other steroid receptors (40, 41, 42, 43), which
is one of the possible mechanisms to explain the distribution pattern
of GFP-MR in the absence of ligand observed in the present study. It is
also possible that the subcellular localization of MR is altered
depending on the cell cycle, although hippocampal neurons, which are
postmitotic cells, are independent of cell cycles. Since COS-1 cells
used in the present study were maintained in the absence of serum for
24 h before observation, most of the cells might be synchronized
in the G1 phase. The cell cycle of COS-1 cells
used in the present study was evaluated by using BrdU incorporation for
S phase and lamin A/C or cyclin C1 antibody for
G1 markers. We observed that approximately
7080% of the cells were in G1 and about
2030% cells were in S (data not shown). These results suggest that
the heterogeneity of cell cycles observed in the present experiment
causes the varieties of subcellular distribution of GFP-MR in COS-1
cells.
Relationship with Transcriptional Sites
After GFP-MR entered the nucleus, the fluorescence appeared to be
accumulated in certain specific nuclear regions and was distributed in
heterogeneous dot-like patterns in the nuclear region, like GFP-GR (12, 13). Fejes-Toth et al. (15) reported that agonist-activated
MR accumulates in discrete clusters in the nucleus, and that this
phenomenon occurs only with transcriptionally active MR. In contrast,
van Steensel et al. (44, 45) demonstrated the spatial
distribution of GR and MR in clusters in specific nuclear domains using
an immunofluorescence technique with confocal microscopy. They
indicated that there was no correlation between the GR clusters and the
distribution of newly synthesized pre-mRNA, suggesting that the
clusters are not directly involved in active transcription.
We performed double immunofluorescence staining of GFP-MR and
hyperphosphorylated pol II to examine whether the heterogeneous
dot-like distribution of GFP-MR in the nucleus after CORT treatment is
associated with transcription sites or not. Since hyperphosphorylated
pol II is associated with sites of ongoing transcription (31), the
colocalization studies could indicate the relationship between GFP-MR
and active transcription sites in the nucleus. The heterogeneous
dot-like nuclear distribution of GFP-MR partially coincides with that
of hyperphosphorylated pol II both in COS-1 cells and cultured
hippocampal neurons transfected with GFP-MR. Our results suggest that
some of the receptors are associated with the transcription sites after
hormone treatment, while others are not directly correlated with
transcription sites. Furthermore, the effects of transcriptional
inhibition on the nuclear distribution of GFP-MR were investigated by
using -amanitin. The nuclear distribution patterns were not
significantly changed after treatment with transcription inhibitor.
However, we cannot determine whether the dot-like distributions of
GFP-MR induced after hormone treatment are involved in the active
transcription sites or not from the transcription inhibition
experiment. Because the interaction of transcription factor and active
transcription sites could be very dynamic, it is difficult to evaluate
whether heterogeneous dot-like patterns directly associate with active
transcriptional sites or not. Recent studies showed that various
nuclear proteins, such as transcription factors and splicing factors,
continuously and rapidly associate and dissociate with nuclear
compartments such as regulatory sites in living cells (46, 47, 48, 49). These
studies investigated the nuclear dynamics of GFP-labeled proteins in
living cells using FRAP (fluorescence recovery after photobleaching)
and FLIP (fluorescence loss in photobleaching) to determine how
fast these nuclear proteins move within the nucleus. The techniques of
FRAP and FLIP demonstrated the possibility that the nuclear proteins
are freely diffusing or constrained by structure, perhaps actively
recruited from one place to the next.
Another possible reason for the discrepancies in the relationship between receptors and active transcription sites is that the observed focal distribution of receptors represents a primary step leading to transcriptional activation. The exact nature and the function of the nuclear distribution patterns of GFP-MR remain to be determined.
Effect of PKA
A recent study demonstrated that PKA, a major mediator of signal
transduction pathways, modulates transcriptional activity of human MR
(35). That study used transient transfection assays in HepG2 cells to
show that PKA stimulates glucocorticoid response element
(GRE)-containing promoters in a ligand-independent manner. However, the
present findings indicate that activation of MR with FK alone cannot
induce nuclear accumulation of MR, the primary step in activation of
specific transcription sites by MR. Massaad et al. (35)
showed that cAMP alone induced a 3- to 4-fold activation of
transcription, but this was about 35% of the activation induced by ALD
alone. They also indicated that a synergistic activation was achieved
when cells were treated with both ALD and cAMP. Taken together, these
results suggest that cAMP alone cannot induce nuclear translocation of
GFP-MR, but can activate small amount of MR that are distributed in the
nucleus, even in the absence of ligand, and induce a lower
transcriptional activation than that induced by ALD alone. When cells
are treated with both ALD and cAMP, MR is fully translocated into the
nucleus by binding with ALD, showing synergistic activation.
Effects of Cytoskeletal Elements
Extensive studies have recently focused on the nuclear-cytoplasmic
transport of macromolecules including transcription factors (50, 51).
These studies investigated mainly nuclear pore complexes and shuttling
proteins, all of which are elements involved in transport in the region
of the nuclear membrane or perinuclear sites. In contrast, very little
is known about the pathway of transport of nuclear receptors from the
cytoplasmic region to the perinuclear site. Some studies suggested that
cytoskeletal elements are involved in nuclear translocation of
receptors (52, 53), while others reported that microtubule disruption
does not prevent nuclear translocation (54, 55, 56). The present study
examined the effects of microtubule disruption on the nuclear
translocation of GFP-MR in living cells. The GFP-MR was completely
translocated into the nuclear region even after treatment with
colchicine or nocodazole. These results suggest that the microtubules
are not essentially involved in transporting GFP-MR from the cytoplasm
to the nucleus. The same result was obtained in the case of GFP-GR
(13). However, we cannot entirely rule out the possible interactions of
microtubules and MR in the cells. Future studies will examine more
precise relations between MR and specific organelles, including
cytoskeletal elements, involved in the trafficking of nuclear
receptors.
Differential Responses of MR and GR to CORT
We investigated the trafficking patterns by which MR and GR
respond to a common natural ligand, CORT, in a single living cell using
dual-color labeling with two different GFP spectral variants. The
double labeling strategies with CFP and YFP have allowed simultaneous
time-lapse imaging of two different receptors in single living cells.
The dynamics of YFP-MR and CFP-GR were essentially the same as those of
GFP-MR and GFP-GR, respectively. The trafficking characteristics
of these fusion proteins in the cotransfected cells were basically the
same as those in the singly transfected cells.
In COS-1 cells, YFP-MR was accumulated in the nucleus faster than CFP-GR in the presence of 10-9 M CORT, a concentration between the Kd values of MR and GR. In contrast, no significant difference was observed in the accumulation rate in the presence of 10-6 M CORT, a concentration much higher than the Kd values of both receptors. Since COS-1 cells have no endogenous MR or GR, the difference in trafficking kinetics detected in the presence of 10-9 M CORT is considered to directly reflect the difference in affinities for CORT between MR and GR; more specifically, MR has about 10-fold higher affinity for CORT compared with GR. The findings suggest that both MR and GR are saturated in cells treated with 10-6 M CORT, causing the lack of difference in trafficking kinetics. These results are in agreement with the present finding that MR plays major roles at physiological concentrations of CORT, while GR is mainly effective at high concentrations of CORT (20, 23, 57, 58).
Hippocampal neurons did not show any obvious difference in the nuclear accumulation rates of MR and GR in the presence of either 10-9 M or 10-6 M CORT. Since hippocampal neurons express endogenous MR and GR (20, 21, 22), these endogenous receptors may affect the trafficking of YFP-MR and CFP-GR. Another possible explanation for the present results is that hippocampal neurons, in contrast to COS-1cells, may have a unique nuclear transporting system for accumulating MR and GR in the nucleus together. A recent study showed that vesicles containing NMDA receptor 2B are transported along microtubules by KIF (kinesin superfamily)17, a neuron-specific molecular motor (59). Although our present and previous data indicated that microtubules are not essentially involved in the nuclear import of GFP-MR or GFP-GR, the results of Setou et al. (59) suggest that some receptors expressed in neuronal cells are transported by a neuron-specific molecular motor. These results led us to speculate that MR and GR could be translocated into the nucleus at mostly the same speed using specific motor molecules in cultured hippocampal neurons.
To elucidate another possible mechanism of differential responses of MR and GR to CORT, we examined an effect of hsp90 association with the receptors. Since geldanamycin binds to hsp90 and inhibits hormone binding to GR and MR (29, 60), which inhibits ligand-dependent nuclear translocation of the receptors (54), it may be a good tool with which to analyze an effect of hsp90 on nuclear-cytoplasmic trafficking of corticosteroid receptors. The treatment with 1 µM of geldanamycin mostly inhibited CORT-induced nuclear translocation of both YFP-MR and CFP-GR in COS-1 cells. We observed no significant differences in the inhibitory action of geldanamycin on the CORTinduced nuclear translocation manner between YFP-MR and CFP-GR. These results indicate that both receptors are associated with hsp90 in the cytoplasm, and their ligand-binding affinities and ligand-induced nuclear translocations could be regulated by hsp90 in COS-1 cells in the same way. We also treated cultured hippocampal neurons with geldanamycin in the same way as described for COS-1 cells. To investigate whether the treatment with 1 µM geldanamycin actually causes accumulation of geldanamycin within the primary cultured hippocampal neurons, we examined geldanamycin-induced induction of hsp70 (61, 62). We confirmed that geldanamycin at the concentration used highly induced hsp70 expression, showing mostly the same induction level as that in COS-1 cells. These results indicated that geldanamycin was effective in the cultured hippocampal neurons. Our data showed that geldanamycin had no effects on CORT-induced nuclear translocation of MR and GR in cultured hippocampal neurons, suggesting that the manner in which these receptors associate with chaperone protein for regulating ligand binding in hippocampal neurons differs from that of COS-1 cells.
Taken together, these results suggest that neuronal cells have unique regulatory systems for nuclear translocation of GR and MR that differ from those of COS-1 cells for responding to diversified stress stimuli in the brain. Future studies are required to clarify whether hippocampal neurons utilize any specific nuclear import machinery as both receptors and the mechanism of hsp90 chaperoning in these receptors are different from nonneuronal cells.
In conclusion, the present study using a GFP-MR chimera system revealed dynamic changes in the subcellular localization of GFP-MR in response to ligands. Furthermore, this is the first report to demonstrate the differential actions of two different corticosteroid receptors in a single living cell by employing dual-color imaging with YFP and CFP. This system should be quite useful for clarifying dynamic changes in the subcellular localization and interactions of functional molecules that cannot be detected in fixed cells.
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MATERIALS AND METHODS |
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Cell Culture and Transfection
Dissociated hippocampal primary neuronal cultures were prepared
from 18-day-old Sprague Dawley rat fetuses according to the method of
Azmitia and Hou (63). Briefly, the rat fetuses were removed from the
placenta in a laminar flow hood and transferred to ice-cold dissecting
solution (0.8% NaCl, 0.04% KCl, 0.006%
Na2HPO4·12
H2O, 0.003% KH2PO4, 0.5% glucose, 0.00012%
phenol red, 0.0125% penicillin G, and 0.02% streptomycin). The
isolated hippocampus was mechanically dissociated by triturating
through a fire-polished glass pipette. The dissociated cells were
plated on 16-well glass slides precoated with 0.1 mg/ml
polyethylenimine (Sigma, St. Louis, MO) at an initial
plating density of 1 x 105 cells per well
by adding 200 µl of the cell suspension to each well (area of 0.28
cm2; Lab-Tek Chamber Slide, Nunc, Naperville,
IL). The cultures were maintained in complete neuronal medium (CNM),
consisting of 92.5% (vol/vol) Eagles minimum essential medium (MEM,
Sigma), 1% (wt/vol) nonessential amino acids (Life Technologies, Inc., Gaithersburg, MD), 0.16% (wt/vol) glucose,
and 5% (vol/vol) FCS (Sigma) in a
CO2 incubator at 37 C with 5%
CO2/95% air. COS-1 cells were maintained in DMEM
(Sigma), without phenol red, supplemented with 10% FCS
overnight in a four-well Multidish with 16-mm diameter (Nunc) at an
initial plating density of 2 x 104 cells
per well in 400 µl of medium.
Plasmid DNA was transiently transfected into cells by a liposome-mediated method using LipofectAMINE PLUS (Life Technologies, Inc.) according to the manufacturers instructions. Hippocampal cells were cultured in CNM for 48 h and then treated with 1 mM cytosine-ß-D-arabinofuranoside for 24 h to suppress the proliferation of glial cells. They were cultured in serum-free medium (SFM) without steroids (MEM with 0.16% of glucose, 1% nonessential amino acids, 20 mM putrescine, 15 nM sodium selenite, 5 µg/ml insulin, and 100 µg/ml transferrin) for 48 h before transfection. For COS-1 cells, the medium was replaced with SFM 2 h before transfection. Cells were transfected with 200 µl of OPTI MEM (Life Technologies, Inc.) containing 8 µl of LipofectAMINE solution and 200 ng of plasmid DNA per well of 1 x 105 cells for 5 h at 37 C.
For ligand stimulation, cells were washed in SFM and treated with 10-6 or 10-9 M CORT (Sigma), 10-7 M ALD (Sigma), DEX (Sigma), PRG (Sigma), or estradiol (E2) (Sigma) at 37 C. The effect of FK was examined using 10-6 M FK (Sigma). To analyze the role of microtubules in MR trafficking, cells were pretreated with 1 µM or 10 µM colchicine (Sigma) or 1 µg/ml or 10 µg/ml nocodazole (Sigma) for the indicated period of time and then treated with 10- 9 M CORT at 37 C. To investigate the interactions between hsp90 and MR or GR, the cultured COS-1 cells were treated with 1 µM or 10 nM geldanamycin (Sigma) for 3 h before CORT treatment.
Immunocytochemistry and Immunoblotting
The immunocytochemistry of the cultured cells was performed
according to a previously described method (64). Briefly, the cells
were fixed in 4% paraformaldehyde in PBS and then subjected to
blocking with 2% BSA in PBS including 0.2% Triton X-100 for 1 h
at room temperature. The fixed cells were incubated with rabbit
polyclonal anti-GR antibody [1:2,000 dilution, (19)], rabbit
polyclonal anti-MR antibody [1:500 dilution, (65)], rabbit polyclonal
anti-GFP antibody (1:1,000 dilution, CLONTECH Laboratories, Inc.), mouse monoclonal antihyperphosphorylated pol II antibody
[1:10, kindly donated by Dr. Bart Sefton, Salk Institute, La Jolla, CA
(30)] for 48 h at 4 C. After washing with PBS five times, cells
were reacted with biotinylated goat antirabbit antibody (1:250
dilution; Roche Molecular Biochemicals, Mannheim, Germany)
for 1 h at room temperature. The cultures were reacted with
avidin-biotin-peroxidase complex (Vector Laboratories, Inc. Burlingame, CA) for 1 h at room temperature. The cells
were visualized with 0.02% 3,3'-diaminobenzidine (Sigma)
and 0.006% H2O2 in Tris-HCl buffered
saline (pH 7.6). For dual immunofluorescence staining, primary
antibodies from different sources (mouse or rabbit) were used
simultaneously and detected with species-specific secondary antibodies
linked to Alexa 488, Alexa 546, or Alexa 568 (Molecular Probes, Inc., Eugene, OR). To confirm disruption of microtubules, we
used monoclonal antityrosinated -antibody (Sigma) at a
dilution of 1:800.
Immunoblotting analysis was performed using COS-1cells transfected with GFP-MR, YFP-MR, or CFP-GR as described previously (13), or COS-1 cells and cultured hippocampal neurons treated with 1 µM geldanamycin for hsp70 induction analysis. Proteins were separated by a 10% SDS-PAGE). Samples were electroblotted to polyvinylidene difluoride membranes (Immobilon-P, Millipore Corp., Bedford, MA) by using a semidry blotting apparatus (Transblot-SD, Bio-Rad Laboratories, Inc. Hercules, CA). The membranes were incubated with anti-GR (1:5,000 dilution), anti-MR (1:500 dilution), anti-GFP polyclonal antibody(1:1,000 dilution), or anti-hsp70 monoclonal antibody (1:1,000 dilution, StressGen Biotechnologies Corp., Victoria, British Columbia, Canada) overnight at 4 C. Secondary goat-antirabbit or mouse-horseradish peroxidase (HRP) (Bio-Rad Laboratories, Inc.) was added at 1:5,000 for 1 h at room temperature. Blots were visualized using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Buckinghamshire, UK). For a quantitative analysis of the effect of geldanamycin on hsp70 expression in COS-1 cells and cultured hippocampal neurons, cells were treated with 1 µM geldanamycin for 3 h and then harvested on ice-cold lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.6, 1% Triton X100) containing 1 µM leupeptin, 1 µM pepstatin A, and 1 mM phenylmethylsulfonyl fluoride PMSF at 4 C. The protein content of cell lysates was determined by using Bradford reagent (Bio-Rad Laboratories, Inc.) with BSA as standard. The following immunoblotting procedures were performed in the same way as described above except that 30 µg of proteins were separated by SDS-PAGE. Relative hsp70 levels were determined from densitometric scanning of ECL-exposed film using NIH image. To show that equivalent protein was loaded in each lane, the samples were probed with antiglyceraldehyde-3-phosphate dehydrogenase polyclonal antibody (1:3,000 dilution; Trevigen, Inc., Gaithersburg, MD).
Examination of Transcriptional Activity
COS-1 cells plated on 35-mm dishes were cotransfected with 1
µg of MMTV-Luc reporter (11) and 1 µg of GFP-MR, YFP-MR, or CFP-GR
by lipofection. One microgram of pCH110, a mammalian positive control
vector for the expression of ß-galactosidase (66), was also
cotransfected as an internal standard. Cells were transfected as
described previously (13). Cell lysates were centrifuged at 12,000 rpm
for 2 min at 4 C, and the luciferase activity of the resulting
supernatants was assayed at 25 C using the luciferase assay system Pica
Gene (Toyo Inki, Tokyo, Japan) according to the manufacturers
protocol, and normalized to ß-galactosidase activity. The maximum
induction obtained with 10-7 M CORT
for 12 h was taken as 100 after normalization by the
ß-galactosidase activity, and the relative reporter luciferase
activities were plotted.
Time-Lapse Image Acquisition and Analysis
For the living cell imaging experiments, the culture medium was
replaced with SFM buffered with 20 mM HEPES
(Sigma), and the image acquisition was performed in a room
temperature-controlled at 37 C. Images were acquired using a SenSys1400
high-resolution, cooled CCD camera (Photometrics, Tucson, AZ) attached
to a microscope (IXL70, Olympus Corp., Tokyo, Japan)
equipped with an epifluorescence attachment. For the observation of
neurons, a 60x objective lens was used, while COS-1 cells were
observed with a 40x objective lens. For the identification of the
nuclear position, the chromatin was stained with 100 ng/ml Hoechst
33342 (Sigma). GFP fluorescence was observed using a
filter set with 480-nm excitation and 515-nm emission, and a 505-nm
dichroic mirror (Olympus Corp.); YFP fluorescence was
viewed using a filter set with 500-nm excitation and 545-nm emission,
and a 525-nm dichroic mirror (Omega Optical, Inc., Brattleboro, VT);
and CFP fluorescence was observed using a filter set with 440-nm
excitation and 480-nm emission, and a 455-nm dichroic mirror (Omega
Optical Inc.). Data were evaluated with the image analysis software
program, IPLab Spectrum (Signal Analytical Corp., Vienna, VA) or Meta
Morph (Universal Imaging Corp., West Chester, PA). In the time-lapse
analysis, images were captured using the time-lapse program of IPLab
Spectrum or Meta Morph. For high-resolution analysis, an image
deconvolution procedure (Meta Morph) was applied to series of images. A
"nearest neighbor estimate" was calculated from the raw data. To
measure nuclear/cytoplasmic ratios of the fluorescence intensity, data
were collected and quantitated using a line intensity profile across
the cell. For each set of conditions, the average intensities of the
pixels were collected within the individual nuclei and cytoplasms of at
least five cells from three independent experiments.
Nuclear/cytoplasmic fluorescence ratios were calculated for each time
point. The results were normalized relative to the value at 0 min taken
as 1.
Confocal scanning laser microscopic images were collected with a Radiance 2000 confocal microscope equipped with a 488 argon ion laser and 543 green helium-neon laser (Bio-Rad Laboratories, Inc.). An oil-immersion objective (x60; NA = 1.4; Nikon, Tokyo, Japan) was used.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan (to M.N. and M.K.).
Received for publication June 26, 2000. Revision received February 1, 2001. Accepted for publication March 16, 2001.
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REFERENCES |
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