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(Investigative Ophthalmology and Visual Science. 2006;47:700-708.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.05-0697
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Heat Shock Protein 90 Is an Essential Molecular Chaperone for Nuclear Transport of Glucocorticoid Receptor ß

Xinyu Zhang,1 Abbot F. Clark,1,2 and Thomas Yorio1

1From the Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas ; and 2Alcon Research, Ltd., Fort Worth, Texas.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. Glucocorticoid sensitivity in glaucoma has been attributed to differences in the expression of the two glucocorticoid receptors, GR{alpha} and GRß. GR{alpha} undergoes steroid-dependent nuclear translocation by associating with a heat shock protein (Hsp)90 multiprotein heterocomplex. The nuclear transport of the non–ligand-binding GRß is still unknown. In this study, the roles of Hsp90 in the nuclear transport of GRß were investigated.

METHODS. Immunocytochemistry and Western blot analysis were performed to detect the subcellular expression of GRß and Hsp90 in normal and glaucomatous trabecular meshwork (TM) cells, as well as in TM cells overexpressing GRß. The role of Hsp90 in GRß transport and stability were determined with the Hsp90 inhibitor, 17-AAG and the proteasome inhibitor lactacystin. Coimmunoprecipitation was performed to study GRß-Hsp90 complexes.

RESULTS. In normal and glaucomatous TM cells, the nuclear concentration of Hsp90 correlates with the nuclear expression of GRß. Transfection with a GRß expression construct produces an overexpression and accumulation of GRß in the nucleus with a corresponding increase in nuclear Hsp90 amount. 17-AAG, a specific Hsp90 inhibitor, completely blocks the nuclear accumulation of GRß and consequently leads to the degradation of GRß in proteasomes. Coimmunoprecipitation experiments verify that GRß complexes with Hsp90 and the microtubule motor protein dynein.

CONCLUSIONS. These data provide strong evidence that Hsp90 is an essential molecular chaperone for the nuclear transport of GRß. This transport appears to occur along micotubular tracks. Because nuclear GRß is important in regulating the glucocorticoid responsiveness, changes in GRß nuclear transport could influence subsequent responses that are often seen clinically, such as glucocorticoid resistance in some inflammatory and autoimmune diseases or enhanced glucocorticoid sensitivity in glaucoma.

Glucocorticoids have long been associated with the development of ocular hypertension and glaucoma.1 Individuals vary in their ocular sensitivity to glucocorticoids, and patients with glaucoma are more prone to development of glucocorticoid-induced ocular hypertension than is the normal population.2 3 4 In addition, normal individuals that are steroid responsive are at greater risk of development of glaucoma.5 The elevated IOP associated with glaucoma and induced by glucocorticoids is due to increased aqueous humor outflow resistance at the trabecular meshwork (TM).6 Although glucocorticoids have been shown to induce a variety of changes in the TM that may account for increased aqueous outflow resistance and elevated IOP, the mechanism(s) responsible for differences in steroid sensitivity are unknown. In a previous study, we demonstrated differences in the levels and distribution of an alternatively spliced isoform of the glucocorticoid receptor, GRß, between normal and glaucomatous TM (GTM) cells. GRß acts as a dominant negative regulator of glucocorticoid activity, which may explain the differences in steroid sensitivity among normal individuals and patients with glaucoma.7

The glucocorticoid receptor (GR) belongs to the superfamily of steroid-thyroid-retinoid acid receptor proteins.8 9 In humans, alternative splicing of the glucocorticoid receptor gene generates two receptor isoforms termed glucocorticoid receptor-{alpha}(GR{alpha}) and glucocorticoid receptor-ß(GRß), which differ only at their carboxyl terminus.10 11 GR{alpha} functions as a ligand-dependent transcription factor that regulates diverse effects of glucocorticoids controlling human development and physiology.12 13 The actions of exogenous glucocorticoids used in the treatment of a wide variety of diseases, including asthma and rheumatoid arthritis, are achieved by binding to the GR{alpha} receptor. In contrast, the GRß receptor does not bind glucocorticoids and lacks transcriptional activity.10 14 GRß has been reported to suppress GR{alpha} activity14 15 16 17 and has been implicated in several glucocorticoid resistance diseases including asthma, arthritis, and inflammatory bowel disease.18 Moreover, GRß also appears to play a critical role in regulating glucocorticoid responsiveness in the trabecular meshwork (TM) cells by affecting the transcriptional activity of the GR{alpha} response.7

Conditional nuclear transport of GR{alpha} is the process used for glucocorticoids to regulate targeted gene activity. GR{alpha} resides predominantly in the cytoplasm as a multiprotein heterocomplex that contains heat shock protein (Hsp)90, Hsp70, and an immunophilin, such as FKBP51, FKBP52 or Cyp-40.19 20 21 22 The activation of GR{alpha} appears to involve the chaperone, Hsp90.23 24 Hsp90 is a chaperone for maintaining an appropriate ligand-binding conformation for steroid receptors, but it also participates in the nuclear-cytoplasmic shuttling of steroid receptors.25 26 It has been proposed that GR{alpha} moves through the cytoplasm to the nucleus in this heterocomplex form, with Hsp90 and the immunophilin acting as a protein transport unit of the transportosome.27 28 The GR{alpha}-Hsp90 complex is in a dynamic state of assembly and disassembly20 ; however, association with Hsp90 is necessary for receptor movement.26 29 30 The dynamic heterocomplex of GR{alpha}-Hsp90-FKBP52 associates with tubulin and the microtubule motor protein dynein,31 32 33 which shuttles the complex along microtubular tracks toward the nucleus with ultimately releasing GR{alpha} into the nucleus.33 34

The localization and translocation of GRß is less clear. GRß has been found in the cytoplasm and the nucleus7 11 16 35 and is associated with Hsp90.16 36 How GRß is transported into the nucleus is completely unknown. GRß is homologous with GR{alpha} through all of its sequence except the last 27 amino acids.11 The possible association of GRß with Hsp9016 36 could account for GRß stability and subcellular distribution.

Presently, we have evaluated the role of Hsp90 in regulating the transport of GRß from the cytoplasm to the nucleus in TM cells. Our results demonstrate that Hsp90 is the molecular chaperone for the nuclear import of GRß.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Materials and Antibodies
17-AAG (17-allylamino, 17-demethoxygeldanamycin, a geldanamycin derivative) was the kind gift of Thomas Mueller, Kosan Biosciences, Inc. (Hayward, CA). Lactacystin was purchased from Calbiochem (San Diego, CA); dexamethasone (DEX) from Sigma-Aldrich (St. Louis, MO); (4',6'-diamidino-2-phenylindole (DAPI) from Molecular Probes (Eugene, OR); polyclonal anti-GRß antibody from Affinity Bioreagents (Golden, CO); monoclonal antibodies to Hsp90, histone1, and ß-tubulin from Santa Cruz Biotechnology (Santa Cruz, CA); and Alexa Fluor 594 and 633 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG from Molecular Probes.

Cell Lines and Cell Culture
Human trabecular meshwork cell lines were generated as previously described.37 38 39 Primary normal TM (NTM) cell line and a stable transformed TM cell line (NTM-5)39 were derived from donors (ages 79 and 18 years, respectively) with no reported history of glaucoma. The primary (GTM) cell line was derived from a 79-year-old donor with a documented history of primary-open angle glaucoma. TM cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% fetal bovine serum (FBS); penicillin, streptomycin, and glutamate; and 44 mM NaHCO3 in 5% CO2 at 37°C.

Plasmid and Transfection
A pCMX-hGR{alpha} expression vector was converted into pCMX-hGRß by the following steps: (1) generation of a C-terminal PCR fragment that overlaps an EcoRI restriction site on the common GR{alpha}/GRß sequence on exon 9 (forward primer) and has the unique GRß carboxyl terminal sequence containing a 3' BamHI restriction site; (2) restriction digestion (EcoRI/BamHI) of GRß PCR product and purification of fragment; (3) restriction digestion (EcoRI/BamHI) of pCMX-hGR{alpha} plasmid and purification of digested plasmid; (4) ligation of restriction digested pCMX-hGR{alpha} with GRß PCR fragment; and (5) DNA sequencing to confirmation of the GRß sequence in the plasmid. This GRß plasmid was transfected into Escherichia coli DH5{alpha}, to amplify the GRß plasmid DNA. NTM-5 cells were transiently transfected with pCMX-hGRß overnight using calcium phosphate reagent according to the manufacturer’s procedures (BD Biosciences, Mountain View, CA). After transfection, cells were incubated in 10% FBS-DMEM for another 24 hours to allow overexpression of GRß protein.

Immunofluorescence
TM cells grown on glass coverslips were fixed in 4% paraformaldehyde for 30 minutes, permeabilized in 0.2% Triton X-100 for 15 minutes, and incubated in 0.2 M glycine for 30 minutes After blocking for 20 minutes with 5% bovine serum albumin+5% normal goat serum, these cells were incubated overnight at 4°C with anti-GRß+anti-Hsp90 antibodies. Subsequently, the cells were treated for 1 hour with Alexa Fluor 633 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG for confocal microscopy. For conventional microscopy, the cells were incubated with anti-GRß antibody and Alexa Fluor 594 goat anti-rabbit IgG. To visualize nuclei, the cells were incubated for 10 minutes with DAPI. Conventional immunofluorescence images were viewed with a fluorescence microscope (Diaphot; Nikon), and image analysis was performed with image analysis software (IPLab; Scanalytics, Billerica, MA). Confocal immunofluorescence microscopy was performed with a confocal scanning laser microscope system (model LSM-410; Carl Zeiss Meditec, Dublin, CA).

Immunoblot Analysis and Coimmunoprecipitation
For transfected cells, cytoplasmic, and nuclear fractions were isolated as described previously.33 After 17-AAG and lactacystin treatments, whole-cell lysate was prepared by solubilization of cell pellets in RIPA buffer supplemented with 1% Nonidet P40 (NP-40) and 2% SDS. SDS-PAGE was performed on 4% to 15% gradient gels. Coimmunoprecipitation was performed according to methods described previously.16 Initially, immunoprecipitation was performed with the anti-GRß antibody followed by a Western blot with the anti-Hsp90 antibody. The sequence was then reversed by using the anti-Hsp90 antibody to immunoprecipitate and the anti-GRß for Western blot analysis. For detecting the GRß-dynein complex, immunoprecipitation was performed with anti-GRß antibody followed by a Western blot analysis with an anti-dynein antibody.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Subcellular Distribution of Hsp90 and GRß
The expression and distribution of GRß between NTM cell lines and GTM cell lines are significantly different, with NTM cell lines having a much higher amount of GRß, especially in the nucleus, compared with GTM cell lines.7 The higher differential distribution of nuclear GRß among normal versus GTM cell lines may be the result of a varied efficiency of receptor nuclear transport, which could affect the stability and the subcellular distribution of GRß. However, little information is available concerning the normal nucleocytoplasmic trafficking of GRß. Because Hsp90 is an important molecular chaperone for steroid receptors and regulates GR{alpha} translocation from the cytoplasm to the nucleus, we investigated the role of Hsp90 in the nuclear transport of GRß. GRß immunostaining was prominent in the nucleus of NTM (primary NTM) cells (Fig. 1) . Of interest, Hsp90 also was prominent in the nucleus and both GRß and Hsp90 colocalized in the nucleus in these NTM cells (Fig. 1) . In contrast, GTM (primary GTM) cells appeared to have much lower GRß nuclear staining, and similarly, nuclear Hsp90 staining also appeared lower, with very little colocalization of GRß with Hsp90 in these nuclear regions. This suggested that the nuclear distribution of Hsp90 correlates with the nuclear amount of GRß in these different TM cell lines.


Figure 1
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  		FIGURE 1. The subcellular distribution of Hsp90 correlated with the expression of GR. Primary cultured NTM and GTM cells were fixed, permeabilized, and stained with polyclonal rabbit anti-GRß antibody and monoclonal mouse anti-Hsp90 antibody. Confocal immunofluorescence microscopy was used to detect the distribution of GRß (red), Hsp90 (green), and colabeling of GRß with Hsp90 (yellow) for NTM (top) and GTM (bottom) cells. Insets: higher magnification. An increase in the distribution of Hsp90 was coincident with a higher amount of GRß in the nucleus in NTM cells, whereas in GTM cells, there was relatively low nuclear GRß and Hsp90 immunostaining. The experiment was performed three times. Scale bars, 50 µm.

 
Effect of Overexpression of GRß on the Accumulation of Hsp90 in the Nucleus
To investigate further the relationship between Hsp90 with GRß, we induced overexpression of GRß by transfecting NTM-5 (transformed NTM) cells with the GRß expression construct pCMX-hGRß and compared the nuclear localization of Hsp90 among transfected and untransfected cells. Approximately 40% of the NTM5 cells were effectively transfected with the GRß expression vector and overexpressed and accumulated GRß in the nucleus (Fig. 2A) . Hsp90 also concentrated and colocalized with GRß in the nucleus of the GRß-transfected cells. In contrast, Hsp90 was found mainly in the cytoplasm of the untransfected cells. Therefore, Hsp90 appeared to colocalize with GRß, and Hsp90 localization was changed by GRß overexpression. This coincidence of nuclear accumulation of GRß and Hsp90 by overexpressing GRß suggested that Hsp90 was associated with the nuclear import of GRß.


Figure 2
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  		FIGURE 2. Overexpression of GRß caused the accumulation of Hsp90 in the nucleus. (A) Transient transfection of NTM-5 cells was performed with a GRß expression construct pCMX-hGRß. Primary polyclonal rabbit anti-GRß antibody and monoclonal mouse anti-Hsp90 were used to immunostain the cells. Confocal immunofluorescence microscopy was used to detect the distribution of GRß (red), Hsp90 (green), and costaining of GRß and Hsp90 (yellow). Top: sections observed under low magnification; bottom: same sections under high magnification. Scale bars, 50 µm. (B) NTM-5 cells were transiently transfected with an empty vector pCMX or a GRß expression vector, pCMX-hGRß. Cell nuclear fraction lysates (60 µg protein) were subjected to Western immunoblot using anti-GRß (top) and anti-Hsp90 (middle) antibodies. The lower image was obtained using the anti-histone1 antibody and served as an internal control. Lane 1: control cells no transfection (con); lane 2: empty vector pCMX transfection; and lane 3: pCMX-hGRß transfection. The corresponding bar graphs represent the mean ± SE of three experiments. *P < 0.05 for the difference between the nuclear amount of GRß or Hsp90 in TM cells transfected with pCMX-hGRß versus the empty vector pCMX; t-test, n = 3. (C) NTM-5 cells, after 24 hours of posttransfection incubation at 37°C, were treated with either vehicle control or 100 nM dexamethasone (DEX) for 24 hours. Cell nuclear fraction lysates (60 µg protein) were subjected to Western immunoblot analysis with anti-GRß (top), or anti-Hsp90 (bottom) antibodies. Lane 1: control cells with no transfection and no DEX; lane 2: control cells with no transfection+DEX; lane 3: empty vector pCMX transfection+con); lane 4: pCMX transfection+DEX; lane 5: pCMX-hGRß transfection+con; and lane 6: pCMX-hGRß transfection+DEX. The corresponding bar graphs represent mean ± SE of three experiments. *P < 0.05 for the difference between the nuclear amount of GRß in TM cells transfected with pCMX-hGRß (lanes 5, 6) versus the empty vector pCMX (lanes 3, 4). *P < 0.05 for the difference between the nuclear amount of Hsp90 in TM cells treated with DEX versus vehicle: lane 2 versus lane 1, lane 4 versus lane 3, or in TM cells transfected with pCMX-hGRß (lane 5) versus with pCMX (lane 3). **P < 0.05 for the difference between the nuclear amount of Hsp90 in TM cells transfected with pCMX-hGRß and treated with DEX (lane 6) versus TM cells transfected with pCMX-hGRß without DEX treatment (lane 5); t-test, n = 5.

 
To support these immunocytochemistry observations, we performed analysis of the distribution of GRß and Hsp90 using Western immunoblot analysis of nuclear fraction lysates extracted from NTM-5 cells with or without GRß transfection. Increased expression of GRß was detected after transfection with the GRß expression vector pCMX-hGRß (Fig. 2B , top, lane 3), compared with untransfected cells (lane 1) or empty vector pCMX transfection (lane 2). Similar to the immunocytochemistry data, the amount of Hsp90 protein increased in the nuclear fraction in NTM-5 cells transfected with pCMX-hGRß (Fig. 2B , middle, lane 3), compared with untransfected cells (lane 1) or cells transfected with empty vector (lane 2). Both confocal microscopy and Western immunoblot analysis consistently demonstrated that the amount of Hsp90 was increased in the nucleus of cells overexpressing GRß.

Treatment with 100 nM dexamethasone (DEX), a synthetic glucocorticoid, did not change the amount of GRß in the nuclear fractions (Fig. 2C , top, lanes 2, 4). However, DEX treatment appeared to increase slightly the nuclear amount of Hsp90 in NTM-5 cells (Fig. 2C , middle, lanes 2, 4) compared to with vehicle control (lanes 1, 3). Hsp90 is a known chaperone protein for nucleocytoplasmic trafficking of GR{alpha},34 and this increase of nuclear Hsp90 after DEX treatment may be associated with DEX-induced translocation of GR{alpha} from the cytoplasm to the nucleus. Transfection with pCMX-hGRß increased the expression of GRß in the nuclear fraction (Fig. 2C , top, lanes 5, 6). When GRß transfection was combined with DEX treatment, an even further increase of nuclear Hsp90 (Fig. 2C , middle, lane 6) was observed compared with other NTM-5 cells without GRß transfection (Fig. 2C , lanes 1, 2, 3, 4). These data suggested that both DEX treatment and GRß transfection could increase the nuclear accumulation of Hsp90, but probably through different pathways. DEX treatment activated the GR{alpha} complex, which includes the chaperone Hsp90, with subsequent translocation to the nucleus. In contrast, increased expression of GRß caused translocation of both GRß and Hsp90 to the nucleus, independent of a glucocorticoid signal.

Effect of the Hsp90 Inhibitor 17-AAG on the Nuclear Transport of GRß
17-AAG is an Hsp90-specific inhibitor, that binds to Hsp90 and inhibits its chaperone activity. 17-AAG treatment leads to the degradation of Hsp90 and client proteins that are associated with Hsp90.40 To test directly whether Hsp90 is necessary for nuclear transport of GRß, we blocked the activity of Hsp90 by treating NTM-5 cells with 17-AAG (1 µM) during and after transfection with pCMX-hGRß. Similar to previous results, NTM-5 cells transfected with pCMX-hGRß overexpressed GRß and accumulated Hsp90 in the nucleus (Fig. 3C) . 17-AAG treatment completely blocked the nuclear localization of Hsp90 and consequently blocked the nuclear accumulation of GRß, even in the GRß-transfected NTM-5 cells (Fig. 3D) . 17-AAG treatment also decreased Hsp90 nuclear expression in cells transfected with the control pCMX empty vector and blocked nuclear import of the endogenous of GRß (Figs. 3A 3B) . These data confirmed that Hsp90 was an essential molecular chaperone for nuclear transport of GRß in TM cells.


Figure 3
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  		FIGURE 3. The Hsp90 inhibitor, 17-AAG, blocked the nuclear transport of GRß. NTM-5 cells were transiently transfected with empty vector pCMX or pCMX-hGRß and stained with primary rabbit anti-GRß antibody and mouse anti-Hsp90 antibody. Confocal microscopy was used to detect GRß (red), Hsp90 (green), and DAPI nuclear staining (blue). The rightmost column represents the overlay staining of red, green, and blue. Cells were transiently transfected with (A) empty vector pCMX as a control experiment; (B) empty vector pCMX and treated with 1 µM 17-AAG during and after transfection; (C) GRß expression vector pCMX-hGRß; and (D) GRß expression vector pCMX-hGRß and treated with 1 µM 17-AAG during and after transfection. Magnification bars, 50 µm.

 
Effect of the Proteasome Inhibitor Lactacystin on the Degradation of GRß after 17-AAG Treatment
17-AAG treatment prevented the nuclear localization and accumulation of GRß as well as cytoplasmic accumulation of GRß in GRß-transfected NTM-5 cells. This could have resulted from the instability and degradation of GRß in the cytoplasm after 17-AAG-mediated inhibition of nuclear translocation. Other studies have shown that 17-AAG treatment leads to proteasome-mediated client protein degradation.41 42 43 44 45 To investigate whether proteasome degradation accounts for the lack of GRß in the cytoplasm, we treated NTM-5 cells with 2 µM lactacystin, a specific proteasome inhibitor, along with 1 µM 17-AAG during and after transfection with the GRß expression vector pCMX-hGRß. Transfection of NTM-5 cells with the GRß expression vector caused the accumulation of both GRß and Hsp90 in the nucleus (Fig. 4A) . 17-AAG treatment blocked both Hsp90 and GRß transport into the nucleus, with little accumulation of GRß in the cytoplasm (Fig. 4B) . However, GRß accumulated in the cytoplasm when GRß transfected cells were treated with both 17-AAG and the proteasome inhibitor lactacystin (Fig. 4C) . Lactacystin itself did not affect the nuclear transport of Hsp90 and GRß in the GRß-transfected NTM-5 cells (Fig. 4D) .


Figure 4
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  		FIGURE 4. The proteasome inhibitor, lactacystin, inhibited the degradation of GRß after 17-AAG treatment. NTM-5 cells were transfected with pCMX-hGRß and treated with 1 µM 17-AAG and/or 2 µM lactacystin during (overnight) and after transfection (24 hours). Immunofluorescence was performed as described in Figure 3 . Cells were transfected with (A) pCMX-hGRß alone; (B) pCMX-hGRß and treated with 17-AAG during and after transfection; (C) pCMX-hGRß and treated with 17-AAG and lactacystin during and after transfection; and (D) pCMX-hGRß and treated with lactacystin during and after transfection. Scale bars 50 µm. (E) Cells were transfected with empty vector pCMX or pCMX-hGRß and treated with 17-AAG and/or lactacystin during and after transfection. Whole-cell lysates under different conditions were subjected to Western immunoblot analysis with anti-GRß antibody (top). ß-Tubulin was blotted as an internal control (bottom). Lane 1: pCMX transfection control; lane 2: pCMX tranfection control+17-AAG; lane 3: pCMX-hGRß transfection; lane 4: pCMX-hGRß transfection+17-AAG; lane 5: pCMX-hGRß transfection+17-AAG+lactacystin; lane 6: pCMX-hGRß transfection+lactacystin. The corresponding bar graphs represent the mean ± SE of three experiments. *P < 0.05 for the difference between the amount of GRß in TM cells treated with 17-AAG versus ethanol: lane 2 versus lane 1; lane 4 versus lane 3. **P < 0.05 for the differences between the amount of GRß in TM cells treated with 17-AAG+Lactacystin (lane 5) versus those treated with 17-AAG alone (lane 4); t-test, n = 3.

 
To confirm this immunocytochemistry observation, GRß Western immunoblot analysis was performed using whole-cell lysates extracted from NTM-5 cells after 17-AAG and/or lactacystin treatment. Proteins protected from the proteasome degradation appear to be insoluble in buffers containing only nonionic detergents but could be solubilized in SDS-containing buffer.45 46 47 Thus, we used RIPA buffer supplemented with 2% SDS to extract whole-cell lysates. Increased expression of GRß was detected in NTM-5 cells after transfection with pCMX-hGRß (Fig. 4E , lane 3), compared with cells transfected with the control null pCMX vector (lane 1). 17-AAG treatment depleted GRß levels in pCMX-hGRß-transfected cells (Fig. 4E , lane 4). Lactacystin itself did not affect the amount of GRß in these pCMX-hGRß transfected NTM-5 cells (Fig. 4E , lane 6). However, the combination treatment of 17-AAG and lactacystin retained GRß in the cells (Fig. 4E , lane 5), although a higher molecular weight band was observed. The increased molecular weight of GRß after 17-AAG and lactacystin treatment could be due to polyubiquitination of GRß protein, as has been described for other proteins extracted from cells treated with geldanamycin and proteasome inhibitors.45 48 49 17-AAG treatment also depleted endogenous GRß in control pCMX-transfected cells (Fig. 4E lane 2). Both the confocal microscopy and Western immunoblot analysis data provide strong evidence that the Hsp90 inhibitor, 17-AAG, can block the nuclear transport of GRß, and this leads to the degradation of GRß in the cytoplasm through a proteasome-mediated pathway.

Complexing of Hsp90 with GRß
To further support our contention that Hsp90 complexes with GRß and shuttles this complex into the nucleus, we performed coimmunoprecipitation experiments. Cytoplasmic and nuclear fractions were isolated from pCMX-hGRß-transfected NTM-5 cells. Initially, immunoprecipitation was performed with anti-GRß antibody followed by Western immunoblot analysis with anti-GRß or anti-Hsp90 antibodies. Indeed, Hsp90 coprecipitated with GRß in the cytoplasmic fraction of NTM-5 cells transfected with GRß (Fig. 5A , top, lane 3). Probing the GRß-precipitated protein Western blot analysis with anti-GRß antibody clearly demonstrated that GRß was overexpressed in both the cytoplasmic and nuclear fractions of GRß transfected NTM-5 cells (Fig. 5A , bottom, lanes 3, 4) compared with pCMX-transfected control cells (Fig. 5A , bottom, lane 1, 2). When the immunoprecipitation sequence was reversed (i.e., immunoprecipitation with anti-Hsp90 antibody followed by anti-GRß Western immunoblots), we were able to detect GRß coprecipitated with Hsp90 in the cytoplasmic fraction in GRß-transfected NTM-5 cells (Fig. 5B , top, lane 3). These coimmunoprecipitation data consistently showed that GRß can associate with Hsp90 in NTM-5 cells that overexpress GRß.


Figure 5
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  		FIGURE 5. Hsp90 complexed with GRß. NTM-5 cells were transiently transfected with the GRß expression vector pCMX-hGRß. After 24 hours of posttransfection growth, cell cytosol and nuclear fraction lysates (100 µg protein) were prepared and subjected to coimmunoprecipitation and Western immunoblot analysis. (A) Anti-GRß was used to immunoprecipitate GRß from cell lysates and to detect GRß by Western immunoblot analysis (bottom). The Hsp90 that was coimmunoprecipitated with GRß antibody was detected by Western immunoblot analysis using an antibody against Hsp90 (top). The corresponding bar graphs represent the mean ± SE of three experiments. *P < 0.05 for the difference between precipitated Hsp90 in TM cells transfected with pCMX-hGRß versus the empty vector pCMX; t-test, n = 3. (B) Anti-Hsp90 was used to immunoprecipitate Hsp90 from cell lysates and to detect Hsp90 by Western immunoblot analysis (bottom). The GRß that was coimmunoprecipitated with the Hsp90 antibody was detected by Western immunoblot analysis using an antibody against GRß (top). *The corresponding bar graphs represent the mean ± SE of three experiments. *P < 0.05 for the difference between precipitated GRß in TM cells transfected with pCMX-hGRß versus the empty vector pCMX. t-test, n = 3.

 
Western immunoblot analysis with anti-Hsp90 after immunoprecipitation with anti-Hsp90 antibody also was performed (Fig. 5B , bottom). The amount of GRß precipitated with Hsp90 (Fig. 5B , top, lane 3) was much less than total precipitated GRß (Fig. 5A , bottom, lane 3). Similarly, the amount of Hsp90 precipitated with GRß (Fig. 5A , top, lane 3) was much less than the total precipitated Hsp90 (Fig. 5B bottom, lane 3). These data indicated that only a small amount of GRß appears to be associated with Hsp90 in the cytoplasm. In addition, there was apparently a shift of Hsp90 from the cytoplasm to the nucleus after transfection with pCMX-hGRß and overexpression of GRß (Fig. 5B bottom, lane 3,4) compared with the pCMX empty vector control (Fig. 5B bottom, lanes 1, 2).

Pattern of the Cytoplasmic Distribution of GRß
Primary cultured TM cells have a relatively broad, flat, elongated morphology,50 and the cytoplasm and the nucleus are large and quite distinct, providing a good model to study the subcellular location of target proteins. Immunocytochemical analysis of GRß localization showed a fibrous distribution pattern of GRß in the cytoplasm of primary cultured NTM (NTM; Fig. 6A ). It appeared that GRß aligned with cytoskeletal elements. Furthermore, Immunoprecipitation using anti-GRß antibody followed by Western immunoblot analysis detected the microtubule motor protein dynein with the anti-dynein antibody (Fig. 6B) . Indeed, dynein coprecipitated with GRß in the cytoplasmic fraction of NTM-5 cells, and this association was increased when NTM-5 cells overexpressed GRß by transfection with pCMX-hGRß (Fig. 6B) . GR{alpha} has been found to be colocalized with microtubules51 associated with its chaperone Hsp90. The translocation of GR{alpha} has been proposed to occur along microtubule tracks, because binding of GR{alpha}-Hsp90 complex to immunophilins also recruits the retrograde motor protein dynein.33 It is possible that a similar microtubular arrangement may be involved in Hsp90 chaperoning the nuclear transport of GRß.


Figure 6
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  		FIGURE 6. The cytoplasmic distribution of GRß showed a fibrous pattern, and GRß complexed with the microtubule motor protein dynein. (A) Primary cultured NTM cells were cultured in 10% FBS-DMEM. Cells were subjected to fixation and permeabilization and were incubated with polyclonal rabbit anti-GRß antibody followed by goat anti-rabbit IgG Alexa Fluor 594. DAPI was used to define the nuclear region. The image was viewed under a conventional immunofluorescence microscope. Scale bar, 20 µm. (B) NTM-5 cells were transfected with the empty vector pCMX or the GRß expression vector pCMX-hGRß. Cell cytosol and nuclear fraction lysates (100 µg protein) were prepared and subjected to coimmunoprecipitation with the anti-GRß antibody and Western immunoblot analysis with the anti-dynein antibody. The corresponding bar graphs represent the mean ± SE of results in three experiments. *P < 0.05 for the difference between precipitated dynein in TM cells transfected with pCMX-hGRß versus the empty vector pCMX; t-test, n = 3.

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
Although much is known about the glucocorticoid response regulated by GR{alpha}, less is understood about the actions of GRß, including its role in regulating glucocorticoid resistance in some diseases.18 Most studies have focused on the comparison of the expression of GRß between normal subjects and those with disease. However, GRß inhibition of GR{alpha} gene transcription regulation may occur in the nucleus through the formation of transcriptionally impaired GR{alpha}–GRß heterocomplexes.16 In addition to altered expression levels of GRß, the regulation of the transport of GRß to its site of action could also be a mechanism for regulating glucocorticoid responsiveness.

Unlike other steroid receptors, little is known about the transport of GRß from the cytoplasm to the nucleus. The present report demonstrates that Hsp90 serves as a chaperone for GRß in TM cell lines. We observed good correlation between nuclear levels of GRß and Hsp90 in TM cell lines. Those cells containing high levels of nuclear GRß also contained high nuclear Hsp90 levels. Conversely, those cells expressing lower GRß had lower levels of Hsp90 in their nuclei. Transfection with a GRß expression construct caused an overexpression and accumulation of GRß protein in the nucleus that also led to a concomitant increase in Hsp90 in the nucleus. Treatment of the transfected cells with 17-AAG, a specific inhibitor of Hsp90 that binds to and inhibits the chaperone activity of Hsp90,40 52 53 54 completely blocked the accumulation of GRß in the nucleus of the transfected cells. In addition, coimmunoprecipitation experiments demonstrated that GRß was physically associated with Hsp90. Thus, our results demonstrate that Hsp90 is an essential chaperone molecule for the transport of non–ligand-bound GRß from the cytoplasm to the nucleus.

Hsp90 is an ATP-dependent molecular chaperone that has inherent adenosine triphosphatase (ATPase) activity.23 55 56 It posses an ATP–adenosine diphosphate (ADP) binding pocket at its N-terminal domain, and ATP binding and hydrolysis are necessary for chaperone function.23 55 56 Blocking ATP binding to this pocket with a class of benzoquinoid ansamycin drugs, such as 17-AAG, prevents the completion of client protein refolding and leads to degradation by proteasomes.40 44 45 46 47 57 58 59 This mechanism is consistent with our current observation that after 17-AAG treatment, the de novo synthesized GRß could not be transported into the nucleus. Instead, it apparently was degraded through a proteasome-mediated pathway in the cytoplasm because lactacystin, a highly specific proteasome inhibitor,42 60 was able to reduce the degradation of GRß and enhanced its accumulation in the cytoplasm. The precise mechanism of 17-AAG blocking the nuclear transport of GRß is not known. It is possible that preventing the ATP binding by occupancy of the ATP binding pocket by 17-AAG inhibits the activity of Hsp90 to refold and maturate the GRß protein as it does for a variety of other signaling molecules.61 Alternatively, the energy from hydrolysis of ATP via its ATPase activity is necessary, to furnish the GRß to travel from the cytoplasm through the nuclear pore to the nucleus, or perhaps both mechanisms are operative.

Our coimmunoprecipitation experiments detected the GRß-Hsp90 heterocomplex only in the cytoplasmic fractions, even after transfection and de novo synthesis of GRß. This heterocomplex was not detected in the nucleus, although there was an accumulation of both Hsp90 and GRß in the nuclear fractions, which suggests that Hsp90 associates with GRß in the cytoplasm and disassociates from GRß on entering the nucleus. In addition, we observed that the amount of GRß that was associated with Hsp90 was much less than the total cytoplasmic GRß pool expressed in the cells. This finding indicates that not all GRß in the cytoplasm was complexed with Hsp90. Most of the GRß was free from Hsp90, indicating that the interaction of GRß with Hsp90 is a dynamic assembly–disassembly process. It is possible that similar to GR{alpha},26 29 30 the dynamic association of GRß with Hsp90 is mainly necessary for the association of GRß to a cytoskeleton motor system for translocation to the nucleus.

Hsp90 generally interacts with a host of cofactors and cochaperones, including Hop; the immunophilins such as FKBP51, FKBP52, cytophilin 40, and p23, and Hsp70 and its cofactors.21 62 63 64 The Hsp90 multimolecular complex is involved in recruiting the retrograde motor protein, dynein, for the translocation of GR{alpha} along microtubular tracks from the cytoplasm to the nucleus.33 34 It is not known which of these cochaperones are involved in the transport of GRß from the cytoplasm to the nucleus. We observed a fibrous cytoplasmic staining pattern of GRß in primary cultured TM cells, and it seems that the GRß receptor is closely associated with a cytoskeleton structure, such as microtubules. Furthermore, coimmunoprecipitation detected that GRß complexed with the retrograde microtubule motor protein dynein. This suggests that the microtubule motor system is involved in Hsp90-chaperoned nuclear transport of GRß. The molecular details of this process, such as which cochaperones and immunophilins participate in GRß nuclear transport remains to be elucidated.

A working model implicating Hsp90 as a chaperone for the nuclear transport of GRß is proposed (Fig. 7) . Initially, Hsp90 or Hsp90 multimolecular complex dynamically associates with GRß in the cytoplasm and chaperones GRß from the cytoplasm through the nuclear pore to the nucleus along a microtubule track. Once it enters the nucleus, Hsp90 disassociates from GRß and releases GRß in the nucleus for it to inhibit GR{alpha} transcriptional activity. Hsp90 inhibitors blocking ATP nucleotide binding to the Hsp90 prevent the nuclear import of GRß and lead to the proteasome-mediated degradation of GRß in the cytoplasm.


Figure 7
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  		FIGURE 7. A model for Hsp90 chaperoned nuclear transport of GRß. Hsp90 weakly binds to GRß but this association may recruit other components, such as cytoskeleton structures, for the transport of GRß through the cytoplasm to the nucleus. Once in the nucleus, Hsp90 dissociates from GRß. 17-AAG, a Hsp90 specific inhibitor, which binds to Hsp90, prevents the chaperon activity of Hsp90 needed for the nuclear transport of GRß, as well as promoting the degradation of GRß by proteasome, which can be blocked by the proteasome inhibitor, lactacystin.

 
In summary, Hsp90 was identified as an essential molecular chaperone for transport of GRß from the cytoplasm to the nucleus. Once in the nucleus, GRß can act to regulate negatively the glucocorticoid activity mediated through the GR{alpha} receptor. This finding also suggests that regulation of the GRß level in the nucleus could influence subsequent responses that are often seen clinically, such as glucocorticoid resistance in some inflammatory and autoimmune diseases18 or enhanced glucocorticoid sensitivity in glaucoma.7


   Acknowledgements
 
The authors thank Allan Shepard (Alcon Research, Ltd.), for help in generating the GRß expression construct pCMX-hGRß; Shaoyou Chu and I-fen Chen Chang for technical support in confocal microscopy; and Ganesh Prasanna, Raghu Krishnamoorthy, and Santosh Narayan for useful discussions.


   Footnotes
 
Supported by grants from National Eye Institute Grants EY11979 and EY016242, Advanced Technology Program, Texas Higher Education Coordinating Board, and Alcon Research, Ltd.

Submitted for publication June 3, 2005; revised October 5, 2005; accepted December 19, 2005.

Disclosure: X. Zhang, Alcon Research, Ltd. (F); A.F. Clark, Alcon Research, Ltd. (E); T. Yorio, Alcon Research, Ltd. (F), Kosan Biosciences, Inc. (F)

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Thomas Yorio, Department of Pharmacology and Neuroscience, UNT Health Science Center, Fort Worth, TX 76107; yoriot{at}hsc.unt.edu.


   References
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 Methods
 Results
 Discussion
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