Heat Shock Protein 90-Dependent (Geldanamycin-Inhibited) Movement of the Glucocorticoid Receptor through the Cytoplasm to the Nucleus Requires Intact Cytoskeleton

Mario D. Galigniana, Jennifer L. Scruggs, James Herrington, Michael J. Welsh, Christin Carter-Su, Paul R. Housley and William B. Pratt

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We use here a chimera of the green fluorescent protein (GFP) and the glucocorticoid receptor (GR) to test the notion that the protein chaperone heat shock protein-90 (hsp90) is required for steroid-dependent translocation of the receptor through the cytoplasm along cytoskeletal tracks. The GFP-GR fusion protein undergoes steroid-mediated translocation from the cytoplasm to the nucleus, where it is transcriptionally active. Treatment of 3T3 cells containing steroid-bound GFP-GR with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, inhibits dexamethasone-dependent translocation from the cytoplasm to the nucleus. The t1/2 for translocation in the absence of geldanamycin is ~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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although there is considerable understanding of how organelles move through the cytoplasm and axoplasm (1), very little is known about how nonvesicle-associated proteins, such as steroid receptors and other signaling proteins, move through the cytoplasm to arrive at their sites of action in the nucleus. It could be that these protein solutes move through the cytoplasm by diffusion and then become trapped at their sites of action by protein-protein interactions, such as by the binding of a localization signal by a signal recognition protein. Alternatively, protein solutes may utilize a movement machinery to traverse the cytoplasm, in which case, movement would likely occur along cytoskeletal tracts. Evidence exists in support of both movement by diffusion and a movement machinery, and it may be that protein solutes move through the cytoplasm by both mechanisms.

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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Dexamethasone-Dependent Movement and Transcriptional Activation by GFP-GR
The E82.A3 cell line is a subclone of L929 selected for glucocorticoid resistance, it does not contain GR mRNA or GR protein, and it responds to glucocorticoid only when transfected with a GR expression plasmid (39). In Fig. 1Go, 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. 1AGo), and this distribution is unaffected by dexamethasone (Fig. 1BGo). The GFP-GR chimera, however, is retained predominantly in the cytoplasm in the absence of hormone (Fig. 1CGo), and it moves to the nucleus when cells are treated with dexamethasone (Fig. 1DGo). Figure 1EGo 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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Figure 1. Dexamethasone-Dependent GFP-GR Translocation from Cytoplasm to Nucleus and Transcriptional Activation in L Cells

The E82.A3 subline of L cells was transfected with GFP or GFP-GR expression plasmid as described in Materials and Methods. Cells expressing GFP (A and B) or GFP-GR (C and D) were incubated for 20 min with 0.1% ethanol (A and C) or 1 µM dexamethasone (B and D), and the fluorescence was photographed from the living cells. Panel E shows the CAT activity in E82.A3 cells transfected with GFP expression plasmid (bars on left), with GFP-GR (hatched bars in middle), or with the wild-type GR (WTGR) expression plasmid (SV2Wrec) (solid bars on right) after 20 h incubation in the absence (-) or presence (+) of 1 µM dexamethasone. CAT activity is normalized to ß-galactosidase activity in the same samples, and the bars are the average of two experiments with the range of values shown by the vertical line.

 
Geldanamycin Inhibits Translocation of the GFP-GR
Although the E82.A3 cells are an optimal system for testing the biological activity of the GFP-GR in the absence of any endogenous GR, these L cells are not optimal for visualizing cytoskeleton. 3T3 cells are better for visualizing cytoskeleton because they have a flatter shape with a higher ratio of cytoplasmic to nuclear volume. The effect of geldanamycin on GFP-GR translocation from the cytoplasm to the nucleus in 3T3 cells is shown in Fig. 2Go. 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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Figure 2. Inhibition of Dexamethasone-Mediated Movement of GFP-GR from Cytoplasm to Nucleus by Geldanamycin in 3T3 Cells

3T3 cells expressing GFP-GR were placed on ice and maintained at 0 C for 1 h with vehicle (0.1% ethanol) or 1 µM dexamethsaone (Dex), with 10 µM geldanamycin (GA) or vehicle (0.1% dimethylsulfoxide) being added at 30 min. At the end of the 1-h incubation at 0 C, cells were shifted to 37 C for 20 min to allow the steroid-bound receptors to translocate to the nucleus. Fluorescence was then photographed from the living cells. Conditions are: A, control exposed only to vehicle; B, dexamethasone only; C, geldanamycin only; D–F, dexamethasone and geldanamycin. Panel G presents a bar graph of the nuclear translocation score for each condition determined according to a scale from 0 to 4 shown on the right, where C represents cytoplasmic and N represents nuclear fluorescence. The values represent the mean ± SEM from three experiments in which >100 cells were scored per experiment. Translocation scores for A–F are: A, 0; B, 4; C, 0; D, 0; E, 1; F, 2.

 
Under hormone-free conditions, GFP-GR expressed in 3T3 cells is predominantly cytoplasmic (Fig. 2AGo), and dexamethasone causes it to move to the nucleus (Fig. 2BGo). Treatment with geldanamycin alone does not affect the cytoplasmic localization of GFP-GR in hormone-free cells (Fig. 2CGo), but geldanamycin does inhibit nuclear transfer of the GFP-GR in dexamethasone-treated cells (c.f. Fig. 2Go, D–F, with Fig. 2BGo). By 20 min at 37 C, essentially all cells treated with dexamethasone alone show a concentrated nuclear fluorescence like that shown in Fig. 2BGo, but cells treated with both dexamethasone and geldanamycin show the range of fluorescence patterns illustrated in Fig. 2Go, 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. 2Go. The bar graphs in Fig. 2GGo 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 3Go 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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Figure 3. Disruption of Cytoskeletal Networks in 3T3 Cells

Cells were incubated for 1 h in the presence of the indicated cytoskeletal disrupting agent. The cells were then fixed, and tubulin (microtubules), actin (microfilaments), and vimentin (intermediate filaments) were visualized by indirect immunofluorescence as described in Materials and Methods. Conditions are: A, no treatment; B, treatment with 0.6 µg/ml colcemid (left), 1 µg/ml cytochalasin D (middle), or 1% IDPN (right); C, treatment with all three agents together; D, after treatment with all three agents together, cells were incubated for 1 h in normal (inhibitor-free) growth medium.

 
Figure 4Go 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. 4Go, bottom panels).



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Figure 4. Loss of Geldanamycin Inhibition of Dexamethasone-Mediated GFP-GR Movement from Cytoplasm to Nucleus When All Three Cytoskeletal Networks of 3T3 Cells Are Disrupted

3T3 cells expressing GFP-GR were incubated for 1 h at 37 C with vehicle (top row) or with colcemid, cytochalasin D, and IDPN (bottom row). Cells were then incubated 1 h at 0 C with dexamethasone, with geldanamycin being present for the last 30 min. The incubation temperature was then increased to 37 C for 20 min to permit GFP-GR transfer from cytoplasm to nucleus. The bar graphs present the nuclear translocation scores from dexamethasone-treated cells in the absence (solid bar) or presence (hatched bar) of geldanamycin determined as described in the legend to Fig. 2Go.

 
Disruption of Any Individual Cytoskeletal Network Makes GFP-GR Translocation Geldanamycin Insensitive
Inasmuch as 1-h treatment with individual cytoskeletal disrupting agents produced relatively selective disruption (by indirect immunofluorescence) of individual cytoskeletal networks in 3T3 cells, we asked whether treatment with any single agent or combination of agents would convert GFP-GR translocation to geldanamycin insensitivity. In the experiments of Fig. 5Go, 3TGo3 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. 5Go, 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. 3DGo) has been returned to a geldanamycin-inhibited mode.



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Figure 5. Any Single Cytoskeletal Disrupting Agent Renders GFP-GR Movement in 3T3 Cells Geldanamycin Insensitive, and Geldanamycin Inhibition of Movement Returns upon Withdrawal of the Disrupting Agent

3T3 cells expressing GFP-GR were treated with colcemid (A), cytochalasin D (B), IDPN (C), or all three agents (D) (open bars). For the cells shown in the black bars, cytoskeletal disrupting agents were washed away after 1 h, and cells were incubated for 3 h in medium without disrupting agents to permit restoration of cytoskeletal networks. All cells were then incubated with dexamethasone in the absence or presence of geldanamycin as described in the legend to Fig. 4Go. Nuclear translocation scores represent the mean ± SEM from three experiments.

 
We also observed that treatment of 3T3 cells with geldanamycin inhibited dexamethasone-mediated translocation of the endogenous GR visualized by indirect immunofluorescence. As with the chimera, microtubular disruption with colcemid restored steroid-induced GR translocation in geldanamycin-treated cells (data not shown).

Effects of Geldanamycin and Cytoskeletal Disruption on the Rate of GFP-GR Translocation
As shown in Fig. 6AGo, 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. 6AGo), 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. 6BGo, 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. 6CGo).



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Figure 6. Effect of Disruption of Cytoskeleton and Effect of Geldanamycin on the Rate of Dexamethasone-Mediated Movement of GFP-GR from Cytoplasm to Nucleus

A, 3T3 cells expressing GFP-GR were incubated for 1 h without ({square}) or with ({blacksquare}) 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 ({square}) or presence ({blacksquare}) of 10 µM geldanamycin, and dexamethasone-mediated movement of GFP-GR at 37 C was assayed at the indicated times. C, 3T3 cells expressing GFP-GR were incubated with all three cytoskeletal disrupting agents (colcemid, cytochalasin D, and IDPN). Dexamethasone-dependent movement of GFP-GR was then assayed under three conditions: without geldanamycin treatment ({circ}) or treatment with 10 µM (•) or 500 µM ({square}) geldanamycin. D, Cells expressing GFP-GR were treated with 1 µM colchicine (black bars) or 1 µM {gamma}-lumicolchicine (hatched bars), and dexamethasone-dependent movement was then assayed in the presence or absence of 10 µM geldanamycin (GA). Values are the average of two experiments with the range of values shown by the vertical line.

 
It can be argued that the cytoskeletal disrupting agents in some way inactivate geldanamycin, e.g. by metabolism or by exclusion from the cell. It could also be argued that disruption of any of the three cytoskeletal networks causes a common change in some cellular component such that geldanamycin is inactivated. However, in Fig. 6CGo, 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 ({square}). 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. 6DGo. In this case, 3T3 cells were treated either with colchicine (black bars) or with {gamma}-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 {gamma}-lumicolchicine do not (data not shown). As shown in Fig. 6DGo, geldanamycin did not inhibit GFP-GR translocation in colchicine-treated cells, but translocation is inhibited in {gamma}-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. 7Go, 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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Figure 7. Geldanamycin Does Not Inhibit GH-Dependent Movement of GFP-STAT5B from Cytoplasm to Nucleus of 3T3 Cells

3T3 cells expressing GHR and GFP-STAT5B were treated for 1 h at 37 C with colcemid (+COLC) or vehicle (CONTROL). Cells were then placed on ice and incubated with 500 ng/ml of GH for 1.5 h (+GH) with 10 µM geldanamycin being present for the last 30 min (+GA). The temperature was then shifted to 37 C for 30 min, and the nuclear translocation score was quantified as described in the legend to Fig. 2Go. The three black bars on the left are from control cells, and the hatched bars on the right are from cells treated with colcemid.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GFP-GR chimera we have used here to study cytoplasmic-nuclear translocation in living cells moves in a dexamethasone-dependent manner and is transcriptionally active (Fig. 1Go). Translocation of GFP-GR is inhibited in cells with intact cytoskeleton by the inhibitor of hsp90 chaperone function, geldanamycin (Fig. 2Go). However, when 3T3 cells are treated with cytoskeletal disrupting agents, translocation of GFP-GR (Figs. 4Go and 5Go) 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. 3Go) and geldanamycin sensitivity (Fig. 5Go). 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. 5Go). 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 {gamma}-lumicolchicine, the biologically inactive isomer of colchicine, to affect geldanamycin sensitivity of GFP-GR movement (Fig. 6DGo). 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. 5Go), 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. 6AGo), 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. 6BGo, 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. 7Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Rhodamine-conjugated donkey antimouse IgG and rhodamine phalloidin were from Molecular Probes (Eugene, OR). TUB2.1 monoclonal anti-ß-tubulin IgG, the V9 monoclonal antivimentin IgG, colcemid, cytochalasin D, colchicine, {gamma}-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. 7Go.

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.


    ACKNOWLEDGMENTS
 
The authors thank Kenneth Rinehart, Bernd Groner, Gunnar Norstedt, Li-yuan Yu-Lee, and The Eli Lilly Company for providing reagents used in this work. The authors gratefully acknowledge the technical assistance of Cheryl DiCapua and Mary Morales.


    FOOTNOTES
 
Address requests for reprints to: William B. Pratt, Department of Pharmacology, The University of Michigan Medical School, Medical Science Research Building III, Ann Arbor, Michigan 48109-0632.

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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