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Departments of Pharmacology (M.D.G., W.B.P.), Physiology
(J.H., C.C.-S.), and Anatomy and Cell Biology (M.J.W.) The
University of Michigan Medical School Ann Arbor, Michigan 48109
Department of Pharmacology (J.L.S., P.R.H.) University of
South Carolina School of Medicine Columbia, South Carolina
29208
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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5 min, and the
t1/2 in the presence of geldanamycin is 45
min. In cells treated for 1 h with the cytoskeletal disrupting
agents colcemid, cytochalasin D, and ß,ß'-iminodipropionitrile to
completely disrupt the microtubule, microfilament, and intermediate
filament networks, respectively, the GFP-GR still translocates rapidly
to the nucleus in a strictly dexamethasone-dependent manner but
translocation is no longer affected by geldanamycin. After withdrawal
of the cytoskeletal disrupting agents for 3 h, normal cytoskeletal
architecture is restored, and geldanamycin inhibition of
dexamethasone-dependent GFP-GR translocation is restored. We suggest
that in cells without an intact cytoskeletal system, the GFP-GR moves
through the cytoplasm by diffusion. However, under physiological
conditions in which the cytoskeleton is intact, diffusion is limited,
and the GFP-GR utilizes a movement machinery that is dependent upon
hsp90 chaperone activity. In contrast to the GR, GFP-STAT5B, a
signaling protein that is not complexed with hsp90, undergoes
GH-dependent translocation to the nucleus in a manner that is not
dependent upon hsp90 chaperone activity. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Observations of fluorescence recovery after photobleaching of
microinjected fluorescein isothiocyanate-dextrans indicate that
macromolecular solutes up to 500 kDa freely and rapidly diffuse in
the cytoplasm and within the nucleus (2). Yet, there is evidence that
protein solutes containing a nuclear localization signal (NLS) utilize
a cytoskeleton-linked machinery for targeted movement through axoplasm.
For example, when rhodamine-labeled human serum albumin coupled to a
peptide containing the NLS of the SV40 large T antigen was injected
into the axoplasm of Aplysia californica neurons, it was
rapidly transported in the retrograde direction to the cell body and
then into the nucleus (3). There was little movement in the anterograde
direction, and retrograde movement depended upon intact microtubules.
Inasmuch as rhodamine-albumin without the NLS was not transported, but
accumulated in organelles near the axonal injection site, it was
concluded that the NLS provided access to the retrograde movement
system as well as to the nuclear import apparatus.
Steroid receptors are ligand-regulated transcription factors that must move through the cytoplasm, traverse the nuclear pores, and subsequently move within the nucleus to arrive at their sites of action. Their nuclear localization is determined by NLS sequences in the receptors themselves (4), and shuttling of receptors into and out of the nucleus occurs constantly (5–8; for review, see Ref. 9). In hormone-free cells, two patterns of shuttling are seen under steady-state conditions. For example, the progesterone receptor (PR) is predominantly localized in the nucleus (10), whereas the glucocorticoid receptor (GR) is predominantly localized in the cytoplasm of most cells (4, 11). Because its transfer from the cytoplasm to the nucleus is entirely steroid dependent, the GR is an excellent model for studying targeted protein movement.
In their hormone-free state, the steroid receptors are recovered from cells in multiprotein heterocomplexes containing the protein chaperones heat shock protein (hsp)90, hsp70, p23, and one of several high mol wt immunophilins, such as FK506 binding protein (FKBP52) and cyclosporin A binding protein-40 (for review, see Ref. 12). Although no specific GR movement machinery has been identified, there is indirect evidence in support of the notion that chaperone proteins are somehow involved in GR movement from cytoplasm to nucleus (13, 14, 15, 16). For example, Yang and DeFranco (15) showed that molybdate, which binds to hsp90 and stabilizes receptor-hsp90 complexes in vivo (17), trapped both the GR and the PR in the cytoplasm of cells chronically exposed to hormone, suggesting that the receptors can export from nuclei but cannot be reimported into nuclei in the presence of molybdate. Smith (18) has shown that receptor-hsp90 complexes are in a dynamic state, in that they are constantly dissociating and being reformed under physiological conditions in the cell. Dynamic interaction with the chaperone may be a component of the protein movement mechanism, and molybdate stabilization of complexes hinders the dynamic process. Consistent with the notion that hsp90 plays a role in receptor trafficking through the cytoplasm, we have shown (16) that geldanamycin, an antibiotic that binds to hsp90 and disrupts its function (19), impedes steroid-dependent movement of the GR from cytoplasm to nucleus. Also, consistent with the notion that immunophilin components of the receptor-hsp90 heterocomplexes are involved in GR movement, microinjection of an antibody against FKBP52 into L cells was shown to impede steroid-mediated shift of the GR from cytoplasm to nucleus (14).
In considering the possibility that GR movement through the cytoplasm utilizes an, as yet undefined, movement machinery, it is a reasonable notion that some sort of cytoskeletal network must serve as a scaffold for such facilitated movement (20, 21). Immunolocalization studies have shown both diffuse dispersion of the GR throughout the cytoplasm (e.g. Refs. 4, 11, 14) and localization to microtubules (22–24; for review, see Ref. 25). Cytoplasmic vitamin D receptors have also been localized to microtubules (26, 27). Consistent with the possibility that steroid receptor-associated chaperones play a role in movement along cytoskeletal tracks, at least a portion of the chaperones hsp90 (28, 29, 30), hsp70 (31), and FKBP52 (32, 33) colocalizes with microtubules.
The quandary regarding models of GR movement by random diffusion vs. movement by an organized machinery along cytoskeletal tracts is still unresolved. There is clearly a bias toward the random diffusion model because microtubule-disrupting agents do not inhibit cytoplasmic-nuclear translocation of PR (34) or GR (14), and they do not affect hormone-mediated transcriptional activation by the GR (35). In contrast to the steroid receptors, microtubule- disrupting agents did inhibit both nuclear (27) and mitochondrial (36) accumulation of the vitamin D receptor, and they inhibited 1,25-dihydroxyvitamin D3-dependent modulation of gene transcription (36).
In this work, we utilize a fusion protein of murine GR with Aequorea green fluorescent protein (GFP-GR) to determine whether there is any linkage between hsp90-dependent movement and cytoskeleton. Two patterns of GFP-GR localization in the cytoplasm of living cells have been reported previously: Ogawa et al. (37) observed a general, diffuse distribution throughout the cytoplasm of COS-1 cells, whereas Htun et al. (38) observed that the GFP-GR accumulated along fibrillar structures in murine adenocarcinoma cells. In both cases, the GFP-GR moved to the nucleus rapidly in a strictly steroid-dependent manner. Here, we find that GFP-GR fluorescence is randomly distributed throughout the cytoplasm of hormone-free 3T3 fibroblasts, and we provide evidence that rapid hormone-dependent movement of the GFP-GR through the cytoplasm utilizes a movement machinery that is dependent upon both hsp90 chaperone activity and cytoskeleton.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, E82.A3 cells were transfected with expression plasmids for GFP or the
GFP-GR chimera, and fluorescence was examined in living cells. In cells
expressing GFP, fluorescence is located in both the cytoplasm and the
nucleus, with the fluorescence being more intense in the nucleus (Fig. 1A), and this distribution is unaffected by dexamethasone (Fig. 1B).
The GFP-GR chimera, however, is retained predominantly in the cytoplasm
in the absence of hormone (Fig. 1C), and it moves to the nucleus when
cells are treated with dexamethasone (Fig. 1D). Figure 1E shows that
the GFP-GR activates transcription from a reporter plasmid in a
dexamethasone-dependent manner to nearly the same extent as the
transfected wild-type GR.
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.
In the protocol used in these experiments, cells are first placed on
ice and incubated for 1 h with 1 µM dexamethasone to
permit steroid binding to receptors. Geldanamycin is added for the last
30 min at 0 C to permit its equilibration with the cells. The
temperature is then increased to 37 C for 20 min to permit the
steroid-bound receptors to undergo translocation from cytoplasm to
nucleus. The microtubules depolymerize when 3T3 cells are placed on
ice, but they repolymerize within 2 min of warming at 37 C (data not
shown). Geldanamycin interacts with hsp90 (19) and blocks the formation
of mature receptor-hsp90 heterocomplexes (40), but it does not cause
steroid to dissociate from prebound GR or inhibit the transformation of
steroid-bound GR (16). If dynamic interaction of hsp90 with the
steroid-bound and transformed GFP-GR is required for its rapid movement
through the cytoplasm, then geldanamycin should inhibit
cytoplasmic-nuclear translocation.
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), and dexamethasone causes it to
move to the nucleus (Fig. 2B). Treatment with geldanamycin alone does
not affect the cytoplasmic localization of GFP-GR in hormone-free cells
(Fig. 2C), but geldanamycin does inhibit nuclear transfer of the GFP-GR
in dexamethasone-treated cells (c.f. Fig. 2, D–F, with Fig. 2B). By 20 min at 37 C, essentially all cells treated with
dexamethasone alone show a concentrated nuclear fluorescence like that
shown in Fig. 2B, but cells treated with both dexamethasone and
geldanamycin show the range of fluorescence patterns illustrated in
Fig. 2, D–F. A scoring of cytoplasmic vs. nuclear
fluorescence was established from 0, which represents cytoplasmic
fluorescence >> nuclear, to 4, which represents nuclear >>
cytoplasmic, as illustrated in Fig. 2. The bar graphs in
Fig. 2G show the nuclear translocation scores from 0 to 4 for GFP-GR in
cells treated with dexamethasone alone (lane 3) and the inhibition of
dexamethasone-mediated translocation by geldanamycin (lane 4), as
determined for several hundred cells per condition. Geldampicin, an
inactive analog of geldanamycin that does not bind to hsp90 (19), does
not affect dexamethasone-mediated GFP-GR translocation (data not
shown).
Geldanamycin Does Not Inhibit GFP-GR Translocation When
Cytoskeleton Is Disrupted
Figure 3 presents the effects of a
1-h treatment with cytoskeletal disrupting agents (each at the
concentration used throughout this paper) on three cytoskeletal systems
in 3T3 cells. The top row (panel A) shows microtubules,
microfilaments, and intermediate filaments in untreated cells. The
second row (panel B) shows disruption of microtubule (left),
microfilament (middle), and intermediate filament
(right) networks by colcemid, cytochalasin D, or
ß,ß'-iminodipropionitrile (IDPN), respectively. Cells treated
simultaneously with all disrupting agents (panel C) have lost all three
cytoskeletal networks. When 3T3 cells that have been treated for 1
h with all disrupting agents are washed and incubated for 1 h in
normal medium without drug, the cytoskeletal networks are restored
(panel D). We have shown previously that in cells that are long-term (6
h) fixed with formaldehyde, hsp90 that is detected by indirect
immunofluorescence with the monoclonal AC88 antibody is colocalized
with microtubules (28, 30). The localization of this
microtubule-associated fraction of hsp90 is disrupted by colcemid (28),
but it is not affected by treatment of 3T3 cells with cytochalasin D,
IDPN, or geldanamycin (data not shown).
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shows the effect of geldanamycin
on dexamethasone-induced GFP-GR movement in control cells with normal
cytoskeleton (top row) and in cells treated simultaneously
with three cytoskeletal disrupting agents (bottom row). Both
in the presence and absence of cytoskeletal networks, the GFP-GR is
localized in the cytoplasm of hormone-free cells (left
panels, -Dex) and moves to the nucleus with dexamethasone treatment
(middle panels, +Dex). In untreated cells with normal
cytoskeleton, geldanamycin inhibits GFP-GR translocation, whereas
geldanamycin has no effect on cytoplasmic-nuclear transfer when all
three cytoskeletal networks are disrupted (right panels and
bar graphs). Treatment of 3T3 cells with all three
cytoskeletal drugs distorts cell shape, and vesicles from which the
GFP-GR is excluded can be seen in the cytoplasm of some of the cells
(Fig. 4, bottom panels).
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, 3T3 cells were treated with colcemid
(A), or cytochalasin D (B), or IDPN (C), or all three drugs
simultaneously (D). As shown by the open bars, in all cases
GFP-GR translocation was no longer affected by geldanamycin. As shown
by the black bars in Fig. 5, movement of the GFP-GR in
drug-treated cells that were then washed and incubated in drug-free
medium to allow restoration of the cytoskeletal networks (as shown in
Fig. 3D) has been returned to a geldanamycin-inhibited mode.
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Effects of Geldanamycin and Cytoskeletal Disruption on the Rate of
GFP-GR Translocation
As shown in Fig. 6A, dexamethasone-mediated GFP-GR cytoplasmic-nuclear translocation occurs
at a similar rate in cells lacking the three cytoskeletal networks
(solid squares) as it does in cells with intact cytoskeletal
networks (open squares). In both control cells and cells
treated for 1 h with all three cytoskeletal disrupting agents, the
t1/2 for translocation is 5 min (Fig. 6A), which is
consistent with previously published rates for GR translocation in COS7
cells (4) and L cells (14). The fact that the overall rate of
cytoplasmic-nuclear translocation is the same with and without
cytoskeleton suggests that movement of the GFP-GR through the cytoplasm
to the nucleus is not rate-limiting in either case, leaving nuclear
import and possibly subsequent movement within the nucleus as
rate-limiting. When geldanamycin is present, the cytoplasmic phase of
GFP-GR movement becomes rate limiting in cells with intact cytoskeletal
networks. As shown in Fig. 6B, geldanamycin slows the overall process
of translocation when the cytoskeleton is intact by nearly 1 order of
magnitude (t1/2 45 min). In cells treated with
cytoskeletal disrupting agents, the GFP-GR moves at the same rate in
the presence and absence of geldanamycin (Fig. 6C).
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, we show that in the presence of cytoskeletal disrupting agents,
geldanamycin has no effect on GFP-GR translocation rate even when it is
present at 500 µM concentration (
). Thus, in the event
that the cytoskeletal disruptors were to inactivate geldanamycin, they
would have to do so in a way that is not overcome by a 50-fold
elevation in geldanamycin concentration. The proposal that it is
disruption of the cytoskeleton that renders GFP-GR translocation
insensitive to geldanamycin is strongly supported by the data of Fig. 6D. In this case, 3T3 cells were treated either with colchicine
(black bars) or with 
-lumicolchicine (hatched
bars), a biologically inactive isomer of colchicine that does not
interact with tubulin (41). 3T3 cells treated with colchicine lose
their microtubules, but cells treated with -lumicolchicine do not
(data not shown). As shown in Fig. 6D, geldanamycin did not inhibit
GFP-GR translocation in colchicine-treated cells, but translocation is
inhibited in -lumicolchicine-treated cells.
Geldanamycin Does Not Inhibit GH-Dependent Translocation of
GFP-STAT5B
In contrast to steroid receptors, STAT (signal transducers and
activators of transcription) proteins are not bound to hsp90 (42), but
they translocate to the nucleus in response to a variety of cytokines,
hormones, and growth factors (43), much like the GR translocates in
response to steroid. When 3T3 cells are transfected with cDNAs encoding
the GH receptor (GHR) and GFP-STAT5B, the chimeric STAT5B translocates
to the nucleus in a GH-dependent manner (J. Herrington, L. Rui, G.
Luo, L. Yu-Lee, and C. Carter-Su, submitted). In the experiments shown
in Fig. 7, the effects of geldanamycin
and colcemid on GH-dependent GFP-STAT5B movement were assessed. As with
the GR and the GFP-GR, GFP-STAT5B translocated to the nucleus in a
hormone-dependent manner in both control cells (upper row
and black bars on the left) and in
colcemid-treated cells with disrupted microtubular structure
(lower row and hatched bars on the
right). Geldanamycin, however, did not affect nuclear
translocation under either condition.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Translocation of GFP-GR is
inhibited in cells with intact cytoskeleton by the inhibitor of hsp90
chaperone function, geldanamycin (Fig. 2). However, when 3T3 cells are
treated with cytoskeletal disrupting agents, translocation of GFP-GR
(Figs. 4 and 5) or endogenous GR is no longer geldanamycin sensitive.
The effect of cytoskeletal disruption is reversible in that withdrawal
of the disrupting agents results in restoration of cytoskeletal
networks (Fig. 3) and geldanamycin sensitivity (Fig. 5). Surprisingly,
no specificity for cytoskeletal systems was found in that any of the
three disruptive agents made GFP-GR translocation no longer
geldanamycin sensitive (Fig. 5). That this loss of geldanamycin effect
results from cytoskeletal disruption and is not a toxic effect of the
cytoskeletal disrupting agents is supported by the failure of
-lumicolchicine, the biologically inactive isomer of colchicine, to
affect geldanamycin sensitivity of GFP-GR movement (Fig. 6D). These
observations are consistent with a model in which the GR normally moves
along cytoskeletal tracts, and in this normal movement mode,
steroid-transformed receptors require dynamic interaction with the
hsp90-based chaperone system for rapid movement.
Because geldanamycin sensitivity was lost when 3T3 cells were treated
with any one of the disrupting agents alone (Fig. 5), it is not clear
which cytoskeletal networks are required for what we will call
hsp90-dependent (i.e. geldanamycin-inhibited) GFP-GR
movement. Much as rapid organelle movement in axoplasm proceeds along
both microtubules and actin filaments (e.g. Ref. 44),
hsp90-dependent movement of the GFP-GR could involve its interaction
with multiple cytoskeletal systems. Indeed, hsp90 has been localized in
various reports to multiple cytoskeletal networks, including actin in
membrane ruffles (45), microtubules (28, 29, 30), and intermediate
filaments (29, 30). However, the fluorescence methods we have used to
observe selective disruption of different cytoskeletal networks by
individual disrupting agents are crude, and we have no indication
whether or not the remaining networks are functionally intact. It is
entirely possible that disruption of one system compromises other
cytoskeletal systems as well, and at this time, we can only say that
hsp90-dependent movement requires intact cytoskeleton.
The fact that rapid-dexamethasone-dependent GFP-GR translocation is
hsp90 dependent under the physiologically normal condition in which
cytoskeletal networks are intact but is hsp90-independent
(i.e. geldanamycin-insensitive) when cytoskeleton is
disrupted suggests that there are two mechanisms of movement.
Perrot-Applanat et al. (34) shifted the PR from the nucleus
into the cytoplasm by administration of energy-depleting drugs and
observed reaccumulation of the receptor in the nucleus upon removal of
the drugs, regardless of whether cytoskeleton was intact or disrupted.
They concluded that cytoskeleton is not involved and that
"karyophilic signals and interactions with the nuclear pore seem to
be the primary determinants of the cellular traffic of the progesterone
receptor." Because we see the same rate of GFP-GR nuclear
translocation when cytoskeleton is intact as when it is totally
disrupted (Fig. 6A), it may be that nuclear import and not receptor
movement through the cytoplasm is rate limiting for nuclear
accumulation under both conditions. We suggest that in the presence of
a normal cytoskeletal network, receptor movement occurs on a
cytoskeleton-based movement machinery that requires dynamic interaction
of the receptor with hsp90. When cytoskeleton is disrupted, the GR may
move through the cytoplasm by diffusion.
Although movement of receptor through the cytoplasm is not the
rate-limiting step in cytoplasmic-nuclear translocation under
physiological conditions in cells with intact cytoskeleton, it becomes
rate limiting when geldanamycin is present. As shown in Fig. 6B, GFP-GR
that is bound by dexamethasone still translocates, but very slowly. If
movement through the cytoplasm was normal and nuclear import was
inhibited in the presence of geldanamycin, then the GFP-GR fluorescence
should accumulate at the nuclear periphery. This is not what we see.
Rather, the fluorescence remains diffusely distributed in the
cytoplasm, consistent with impairment of movement through the
cytoplasmic space. Thus, we suggest that the presence of an intact
cytoskeletal system may limit receptor diffusion, and rapid receptor
movement utilizes a movement machinery that is hsp90 dependent. In
contrast, hormone-dependent movement of GFP-STAT5B, a signaling protein
that is not recovered from cells in complexes with hsp90 (data not
shown), is not inhibited by geldanamycin (Fig. 7). At this time, it is
not known whether GFP-STAT5B moves solely by diffusion or utilizes a
movement machinery in a manner that does not require hsp90
chaperoning.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-lumicolchicine, and
charcoal-stripped, delipidated calf serum were from Sigma (St. Louis,
MO). IDPN was from Fisher (Pittsburgh, PA). Recombinant human GH was a
gift from Eli Lilly (Indianapolis, IN). Phenol red-free DMEM was from
BioWhittaker (Walkersville, MD). Opti-MEM medium and Lipofectamine were
from GIBCO BRL (Gaithersburg, MD). Geldanamycin was obtained from the
Drug Synthesis and Chemistry Branch of the Developmental Therapeutics
Program, National Cancer Institute, and geldampicin was generously
provided by Dr. Kenneth Rinehart (University of Illinois, Urbana). The
aP1 rabbit antiserum raised against amino acids 440–795 of the rat GR
(46) was a kind gift from Dr. Bernd Groner (Institute for Experimental
Cancer Research, Freiburg, Germany). Rat GHR cDNA (47) was kindly
provided by Dr. Gunnar Norstedt (Karolinska Institute, Stockholm,
Sweden).
Plasmids
The mouse wild-type GR cDNA vector SV2Wrec and the murine
mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT)
reporter plasmid have been described (48). CMVßgal and pEGFP-C3 were
from CLONTECH (Palo Alto, CA). The stop codon in the 5'-untranslated
region of the GR cDNA was converted to a leucine codon using
oligonucleotide-directed mutagenesis of SV2Wrec as described (49). The
BglII-XbaI fragment containing the GR cDNA was
then excised and cloned into the corresponding sites of pEGFP-C3 to
give an expression plasmid with the C terminus of GFP fused in frame to
the N terminus of the GR. Rat STAT5B (50) was inserted into the
BgIII site of EGFP-C1 (CLONTECH) as described (J.
Herrington, L. Rui, G. Luo, L. Yu-Lee, and C. Carter-Su,
submitted).
Cell Culture and Transfection
The E82.A3 subline of L929 mouse fibroblasts (39) and NIH-3T3
cells were grown on 11 x 22-mm coverslips in DMEM supplemented
with 10% bovine calf serum in 35-mm tissue culture dishes. When cells
were 60% confluent, they were rinsed three times with serum-free
medium and then incubated an additional hour in fresh medium. For each
transfection of GFP-GR cDNA, a 2-ml solution containing 2 µg DNA, 10
µl Lipofectamine, and 0.8 ml Opti-MEM medium was added to the culture
dish and mixed gently to assure uniform distribution. For transfection
of GFP-STAT5B, a 2 ml solution containing 2 µg GFP-STAT5B cDNA and 4
µg of GHR cDNA were added and incubated under the same conditions.
Cells were incubated with the transfection mixture for 5–6 h at 37 C,
and the medium was then replaced by complete growth medium for an
additional 18 h of incubation. At the end of this incubation, the
coverslips were washed extensively with and then incubated for 24
h in phenol red-free DMEM supplemented with 10% charcoal-stripped,
delipidated bovine calf serum. After the 18-h incubation, cells
expressing GFP-STAT5B and GHR were incubated for an additional 12
h in serum-free medium before treatment with colcemid, GH, and
geldanamycin as described in the legend to Fig. 7.
Assay of GFP-GR Fluorescence in Living Cells
To test the ability of the chimeric protein to move from
cytoplasm to nucleus, cells expressing GFP-GR were incubated for 20 min
at 37 C with 1 µM dexamethasone added to the medium in
the culture dish. Coverslips were then inverted onto a microslide with
a concavity (18 mm in diameter x 0.5 mm deep) that contained the
same medium. Cells were photographed with a Leitz Aristoplan
epiillumination fluorescence microscope (E. Leitz, Inc.,
Rockleigh, NJ) and a Leitz Vario-Orthomat camera using T-Max 3200 film.
The bars in the figures represent 10 µm.
To assay the effect of geldanamycin on GFP-GR movement, dexamethasone (1 µM) or vehicle (0.1% ethanol) was added to cells that had been precooled for 10 min on ice. Cells were maintained on ice for 1 h to allow steroid occupation of all receptors, with 10 µM geldanamycin or vehicle (0.1% dimethylsulfoxide) being added at 30 min. At the end of the 1-h preincubation on ice, cells were shifted to 37 C for 20 min to allow the steroid-bound receptors to translocate to the nucleus. Coverslips were then inverted onto microslides for fluorescence imaging.
To assay the effect of cytoskeletal disruption on GFP-GR nuclear translocation, cells were incubated for 1 h at 37 C with 0.6 µg/ml colcemid, 1 µg/ml cytochalasin D, and/or 1% IDPN before cooling them on ice and adding dexa-methasone.
Cells were scored for GFP-GR translocation using a score of 4 for nuclear fluorescence much greater than cytoplasmic fluorescence, 3 for nuclear fluorescence greater than cytoplasmic fluorescence, 2 for nuclear fluorescence equal to cytoplasmic fluorescence, 1 for nuclear fluorescence less than cytoplasmic fluorescence, and 0 for nuclear fluorescence much less than cytoplasmic fluorescence. The translocation scores represent the mean ± SEM from three experiments in which >100 cells per condition per experiment were scored.
Immunofluorescence Visualization
3T3 cells were grown on coverslips in DMEM with 10% calf-serum
for 24 h and then incubated for 1 h with the indicated
cytoskeletal disrupting agent. The coverslips were then rinsed with PBS
at room temperature and simultaneously fixed and permeabilized by
immersion in cold methanol (-25 C) for at least 15 min. Cells were
rinsed again with PBS, and the coverslips were inverted onto a 30 µl
drop of blocking solution (20 mM Tris, pH 8.0, 130
mM NaCl, 0.2% saponin, 0.05% Tween 20, 1% BSA)
containing 1 µl TUB 2.1 antibody against tubulin, 1 µl V9 antibody
against vimentin, 1 µl rhodamine-phalloidin (200 U/ml) to label
F-actin, or 0.3 µl of aP1 anti-GR serum. After overnight incubation
with antibody at 4 C and subsequent washing with PBS, coverslips were
inverted again on 30 µl drops of blocking solution containing 1 µl
of rhodamine-conjugated antimouse or antirabbit IgG and incubated for
2 h at room temperature. Incubations with rhodamine-phalloidin
were performed at room temperature for 45 min only. Cells on coverslips
were photographed as described above.
Transcriptional Activation
E82.A3 cells were incubated with DMEM containing DEAE-Dextran
(0.2 mg/ml), chloroquine (30 µM), MMTV-CAT (3 µg/ml),
CMVßgal (2 µg/ml), and the GFP expression plasmid, the GFP-GR
expression plasmid, or the wild-type GR expression plasmid (3 µg/ml)
for 2 h, and then shocked for 1 min with 15%
dimethylsulfoxide/HEPES-buffered saline. After 40 h of incubation in
phenol red-free DMEM and charcoal-stripped calf serum, 1.0
µM dexamethasone was added and cells were incubated an
additional 20 h to allow induction of CAT activity. Cell extracts
were assayed for CAT as described by Nordeen et al. (51),
and values were normalized to ß-galactosidase activity assayed in the
same cell samples.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by NIH Grants DK-34171 (to C.C.-S.), DK-47951 (to P.R.H.), CA-28010 (to W.B.P.), and ES-06265 and ES-07006 (to M.J.W.).
Received for publication May 27, 1998. Revision received August 3, 1998. Accepted for publication September 2, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, 25-dihydroxyvitamin
D3-dependent rapid and estrogen-dependent slow
reorganization of vitamin D receptors. J Cell Biol 111:2385–2395This article has been cited by other articles:
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 N. Kumar, J. Robidoux, K. W. Daniel, G. Guzman, L. M. Floering, and S. Collins Requirement of Vimentin Filament Assembly for beta3-Adrenergic Receptor Activation of ERK MAP Kinase and Lipolysis J. Biol. Chem., March 23, 2007; 282(12): 9244 - 9250. [Abstract] [Full Text] [PDF]
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 M. T. W. Handley, L. P. Haynes, and R. D. Burgoyne Differential dynamics of Rab3A and Rab27A on secretory granules J. Cell Sci., March 15, 2007; 120(6): 973 - 984. [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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 X. Zhang, A. F. Clark, and T. Yorio Heat Shock Protein 90 Is an Essential Molecular Chaperone for Nuclear Transport of Glucocorticoid Receptor {beta} Invest. Ophthalmol. Vis. Sci., February 1, 2006; 47(2): 700 - 708. [Abstract] [Full Text] [PDF]
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 H.-J. Kim, Y. I. Park, and M.-S. Dong Effects of 2,4-D and DCP on the DHT-Induced Androgenic Action in Human Prostate Cancer Cells Toxicol. Sci., November 1, 2005; 88(1): 52 - 59. [Abstract] [Full Text] [PDF]
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 J. Rosen and J. N. Miner The Search for Safer Glucocorticoid Receptor Ligands Endocr. Rev., May 1, 2005; 26(3): 452 - 464. [Abstract] [Full Text] [PDF]
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G. M. Wochnik, J. Ruegg, G. A. Abel, U. Schmidt, F. Holsboer, and T. Rein FK506-binding Proteins 51 and 52 Differentially Regulate Dynein Interaction and Nuclear Translocation of the Glucocorticoid Receptor in Mammalian Cells J. Biol. Chem., February 11, 2005; 280(6): 4609 - 4616. [Abstract] [Full Text] [PDF]
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J. M. Harrell, P. J. M. Murphy, Y. Morishima, H. Chen, J. F. Mansfield, M. D. Galigniana, and W. B. Pratt Evidence for Glucocorticoid Receptor Transport on Microtubules by Dynein J. Biol. Chem., December 24, 2004; 279(52): 54647 - 54654. [Abstract] [Full Text] [PDF]
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D. Walerych, G. Kudla, M. Gutkowska, B. Wawrzynow, L. Muller, F. W. King, A. Helwak, J. Boros, A. Zylicz, and M. Zylicz Hsp90 Chaperones Wild-type p53 Tumor Suppressor Protein J. Biol. Chem., November 19, 2004; 279(47): 48836 - 48845. [Abstract] [Full Text] [PDF]
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 J. Ruegg, F. Holsboer, C. Turck, and T. Rein Cofilin 1 Is Revealed as an Inhibitor of Glucocorticoid Receptor by Analysis of Hormone-Resistant Cells Mol. Cell. Biol., November 1, 2004; 24(21): 9371 - 9382. [Abstract] [Full Text] [PDF]
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P. J. M. Murphy, M. D. Galigniana, Y. Morishima, J. M. Harrell, R. P. S. Kwok, M. Ljungman, and W. B. Pratt Pifithrin-{alpha} Inhibits p53 Signaling after Interaction of the Tumor Suppressor Protein with hsp90 and Its Nuclear Translocation J. Biol. Chem., July 16, 2004; 279(29): 30195 - 30201. [Abstract] [Full Text] [PDF]
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M. D. Galigniana, J. M. Harrell, H. M. O'Hagen, M. Ljungman, and W. B. Pratt Hsp90-binding Immunophilins Link p53 to Dynein During p53 Transport to the Nucleus J. Biol. Chem., May 21, 2004; 279(21): 22483 - 22489. [Abstract] [Full Text] [PDF]
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 N. Z. Gerges, I. C. Tran, D. S. Backos, J. M. Harrell, M. Chinkers, W. B. Pratt, and J. A. Esteban Independent Functions of hsp90 in Neurotransmitter Release and in the Continuous Synaptic Cycling of AMPA Receptors J. Neurosci., May 19, 2004; 24(20): 4758 - 4766. [Abstract] [Full Text] [PDF]
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Y. Zhang, Y. Yang, S. Yeh, and C. Chang ARA67/PAT1 Functions as a Repressor To Suppress Androgen Receptor Transactivation Mol. Cell. Biol., February 1, 2004; 24(3): 1044 - 1057. [Abstract] [Full Text] [PDF]
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A. S. Sreedhar, K. Mihaly, B. Pato, T. Schnaider, A. Stetak, K. Kis-Petik, J. Fidy, T. Simonics, A. Maraz, and P. Csermely Hsp90 Inhibition Accelerates Cell Lysis: ANTI-Hsp90 RIBOZYME REVEALS A COMPLEX MECHANISM OF Hsp90 INHIBITORS INVOLVING BOTH SUPEROXIDE- AND Hsp90-DEPENDENT EVENTS J. Biol. Chem., September 12, 2003; 278(37): 35231 - 35240. [Abstract] [Full Text] [PDF]
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N. Saydam, F. Steiner, O. Georgiev, and W. Schaffner Heat and Heavy Metal Stress Synergize to Mediate Transcriptional Hyperactivation by Metal-responsive Transcription Factor MTF-1 J. Biol. Chem., August 22, 2003; 278(34): 31879 - 31883. [Abstract] [Full Text] [PDF]
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G. Nelson, G. J. C. Wilde, D. G. Spiller, S. M. Kennedy, D. W. Ray, E. Sullivan, J. F. Unitt, and M. R. H. White NF-{kappa}B signalling is inhibited by glucocorticoid receptor and STAT6 via distinct mechanisms J. Cell Sci., June 15, 2003; 116(12): 2495 - 2503. [Abstract] [Full Text] [PDF]
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 J. Y. Ji, H. Jing, and S. L. Diamond Shear Stress Causes Nuclear Localization of Endothelial Glucocorticoid Receptor and Expression From the GRE Promoter Circ. Res., February 21, 2003; 92(3): 279 - 285. [Abstract] [Full Text] [PDF]
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 W. B. Pratt and D. O. Toft Regulation of Signaling Protein Function and Trafficking by the hsp90/hsp70-Based Chaperone Machinery Experimental Biology and Medicine, February 1, 2003; 228(2): 111 - 133. [Abstract] [Full Text] [PDF]
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J. R. Petrulis, A. Kusnadi, P. Ramadoss, B. Hollingshead, and G. H. Perdew The hsp90 Co-chaperone XAP2 Alters Importin beta Recognition of the Bipartite Nuclear Localization Signal of the Ah Receptor and Represses Transcriptional Activity J. Biol. Chem., January 17, 2003; 278(4): 2677 - 2685. [Abstract] [Full Text] [PDF]
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P. Berg and I. Pongratz Two Parallel Pathways Mediate Cytoplasmic Localization of the Dioxin (Aryl Hydrocarbon) Receptor J. Biol. Chem., August 23, 2002; 277(35): 32310 - 32319. [Abstract] [Full Text] [PDF]
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 R. S. Edinger, S. C. Watkins, D. Pearce, and J. P. Johnson Effect of immunosuppressive agents on glucocorticoid receptor function in A6 cells Am J Physiol Renal Physiol, August 1, 2002; 283(2): F254 - F261. [Abstract] [Full Text] [PDF]
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G. A. Abel, G. M. Wochnik, J. Ruegg, A. Rouyer, F. Holsboer, and T. Rein Activity of the GR in G2 and Mitosis Mol. Endocrinol., June 1, 2002; 16(6): 1352 - 1366. [Abstract] [Full Text] [PDF]
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G. Piwien-Pilipuk, A. Ayala, A. Machado, and M. D. Galigniana Impairment of Mineralocorticoid Receptor (MR)-dependent Biological Response by Oxidative Stress and Aging. CORRELATION WITH POST-TRANSLATIONAL MODIFICATION OF MR AND DECREASED ADP-RIBOSYLATABLE LEVEL OF ELONGATION FACTOR 2 IN KIDNEY CELLS J. Biol. Chem., March 29, 2002; 277(14): 11896 - 11903. [Abstract] [Full Text] [PDF]
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T. H. Davies, Y.-M. Ning, and E. R. Sanchez A New First Step in Activation of Steroid Receptors. HORMONE-INDUCED SWITCHING OF FKBP51 AND FKBP52 IMMUNOPHILINS J. Biol. Chem., February 8, 2002; 277(7): 4597 - 4600. [Abstract] [Full Text] [PDF]
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T. Miura, R. Ouchida, N. Yoshikawa, K. Okamoto, Y. Makino, T. Nakamura, C. Morimoto, I. Makino, and H. Tanaka Functional Modulation of the Glucocorticoid Receptor and Suppression of NF-kappa B-dependent Transcription by Ursodeoxycholic Acid J. Biol. Chem., December 7, 2001; 276(50): 47371 - 47378. [Abstract] [Full Text] [PDF]
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 M. Wiesgigl and J. Clos Heat Shock Protein 90 Homeostasis Controls Stage Differentiation in Leishmania donovani Mol. Biol. Cell, November 1, 2001; 12(11): 3307 - 3316. [Abstract] [Full Text] [PDF]
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M. Nishi, H. Ogawa, T. Ito, K.-I. Matsuda, and M. Kawata Dynamic Changes in Subcellular Localization of Mineralocorticoid Receptor in Living Cells: In Comparison with Glucocorticoid Receptor using Dual-Color Labeling with Green Fluorescent Protein Spectral Variants Mol. Endocrinol., July 1, 2001; 15(7): 1077 - 1092. [Abstract] [Full Text] [PDF]
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A. Kazlauskas, S. Sundström, L. Poellinger, and I. Pongratz The hsp90 Chaperone Complex Regulates Intracellular Localization of the Dioxin Receptor Mol. Cell. Biol., April 1, 2001; 21(7): 2594 - 2607. [Abstract] [Full Text]
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F.-X. Beck, W. Neuhofer, and E. Muller Molecular chaperones in the kidney: distribution, putative roles, and regulation Am J Physiol Renal Physiol, August 1, 2000; 279(2): F203 - F215. [Abstract] [Full Text] [PDF]
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J. Cheung and D. F. Smith Molecular Chaperone Interactions with Steroid Receptors: an Update Mol. Endocrinol., July 1, 2000; 14(7): 939 - 946. [Full Text]
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 M. D. Galigniana, G. P. Vicent, G. Piwien-Pilipuk, G. Burton, and C. P. Lantos Mechanism of Action of the Potent Sodium-Retaining Steroid 11,19-Oxidoprogesterone Mol. Pharmacol., July 1, 2000; 58(1): 58 - 70. [Abstract] [Full Text]
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Y Shafrir, D ben-Avraham, and G Forgacs Trafficking and signaling through the cytoskeleton: a specific mechanism J. Cell Sci., January 8, 2000; 113(15): 2747 - 2757. [Abstract] [PDF]
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K. C. Kanelakis, Y. Morishima, K. D. Dittmar, M. D. Galigniana, S. Takayama, J. C. Reed, and W. B. Pratt Differential Effects of the hsp70-binding Protein BAG-1 on Glucocorticoid Receptor Folding by the hsp90-based Chaperone Machinery J. Biol. Chem., November 26, 1999; 274(48): 34134 - 34140. [Abstract] [Full Text] [PDF]
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A. Racz and J. Barsony Hormone-dependent Translocation of Vitamin D Receptors Is Linked to Transactivation J. Biol. Chem., July 2, 1999; 274(27): 19352 - 19360. [Abstract] [Full Text] [PDF]
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M. D. Galigniana, P. R. Housley, D. B. DeFranco, and W. B. Pratt Inhibition of Glucocorticoid Receptor Nucleocytoplasmic Shuttling by Okadaic Acid Requires Intact Cytoskeleton J. Biol. Chem., June 4, 1999; 274(23): 16222 - 16227. [Abstract] [Full Text] [PDF]
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M. D. Galigniana, C. Radanyi, J.-M. Renoir, P. R. Housley, and W. B. Pratt Evidence That the Peptidylprolyl Isomerase Domain of the hsp90-binding Immunophilin FKBP52 Is Involved in Both Dynein Interaction and Glucocorticoid Receptor Movement to the Nucleus J. Biol. Chem., April 27, 2001; 276(18): 14884 - 14889. [Abstract] [Full Text] [PDF]
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O. J. Shah, J. A. Iniguez-Lluhi, A. Romanelli, S. R. Kimball, and L. S. Jefferson The Activated Glucocorticoid Receptor Modulates Presumptive Autoregulation of Ribosomal Protein S6 Protein Kinase, p70 S6K J. Biol. Chem., January 18, 2002; 277(4): 2525 - 2533. [Abstract] [Full Text] [PDF]
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) all three cytoskeletal disrupting agents (colcemid,
cytochalasin D, and IDPN). Dexamethasone-mediated movement of GFP-GR at
37 C was then assayed by fixing the cells in cold methanol at the
indicated times and determining the nuclear translocation score on the
fixed cells. B, 3T3 cells expressing GFP-GR were preincubated at 0 C
with dexamethasone in the absence (
) or treatment with 10 µM
(•) or 500 µM (