Chromatin Recycling of Glucocorticoid Receptors: Implications for Multiple Roles of Heat Shock Protein 90

Jimin Liu and Donald B. DeFranco

Departments of Biological Sciences (J.L., D.B.D.), Neuroscience (D.B.D.), and Pharmacology (D.B.D.) University of Pittsburgh Pittsburgh, Pennsylvania 15260


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Unliganded glucocorticoid receptors (GRs) released from chromatin after hormone withdrawal remain associated with the nucleus within a novel subnuclear compartment that serves as a nuclear export staging area. We set out to examine whether unliganded nuclear receptors cycle between distinct subnuclear compartments or require cytoplasmic transit to regain hormone and chromatin-binding capacity. Hormone-withdrawn rat GrH2 hepatoma cells were permeabilized with digitonin to deplete cytoplasmic factors, and then hormone-binding and chromatin-binding properties of the recycled nuclear GRs were measured. We found that recycled nuclear GRs do not require cytosolic factors or ATP to rebind hormone. Nuclear GRs that rebind hormone in permeabilized cells target to high-affinity chromatin-binding sites at 30 C, but not 0 C, in the presence of ATP. Since geldanamycin, a heat shock protein-90 (hsp90)-binding drug, inhibits hormone binding to recycled nuclear GRs, hsp90 may be required to reassemble the receptor into a form capable of productive interactions with hormone. Geldanamycin also inhibits GR release from chromatin during hormone withdrawal, suggesting that hsp90 chaperone function may play multiple roles to facilitate chromatin recycling of GR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The transduction of steroid hormone signals is mediated by cognate receptor proteins, which belong to a large superfamily of nuclear receptors (1, 2, 3). These receptors play an important role in many metabolic, developmental, proliferative, and behavioral responses and exert their effects primarily via their regulation of gene expression (1, 4). Members of the nuclear receptor superfamily have been subdivided into distinct classes based primarily on their sequence recognition elements and dimerization properties. For example, steroid hormone receptors interact primarily as homodimers with inverted repeat DNA elements containing an internal spacing of 3 bp, while retinoid, thyroid hormone, and vitamin D receptors bind primarily as heterodimers to direct repeats of variable spacing (3). In this case, the extent of spacing between direct repeats influences the selection of nuclear receptor heterodimer (5, 6). While these DNA-binding characteristics are useful for the placement of various nuclear receptors within discrete subclasses, in many natural contexts, hormone response elements may vary considerably from these idealized cases.

Steroid receptor proteins are also distinguished from other members of the nuclear receptor family by their maturation and intracellular trafficking properties. Unliganded steroid receptor proteins are generally inert, although steroid receptor function can be uncovered not only by hormone binding, but also upon the activation of nonsteroidal signaling pathways (7, 8, 9, 10). Specific molecular chaperones and other proteins that are associated with unliganded receptors (11, 12) supply a major impediment to steroid receptor function. Proteins associated with fully mature, unliganded steroid receptors include the 90-kDa heat shock protein (hsp 90), a 23-kDa acidic protein, and one of three immunophilins (FKBP52, FKBP51, or CyP-40) (11, 13). Thyroid hormone receptors, and perhaps other members of the nuclear receptor superfamily exclusive of steroid hormone receptors, are not maintained in an inactive state when unliganded by associated proteins. In fact, the nonsteroid nuclear receptors often function as transcriptional repressors when unliganded through their association with transcriptional corepressors (14, 15).

The assembly of steroid receptors into a heteromeric complex is a multistep process that has been dissected in elegant in vitro reconstitution experiments by the Pratt, Smith, and Toft laboratories (16, 17, 18). Intermediate steroid receptor complexes may contain other molecular chaperones including the 70-kDa heat shock protein, hsp70, and utilize chaperone partners to facilitate the ordered assembly of receptor heteromeric complexes (17, 19, 20). For glucocorticoid and progesterone receptors (GRs and PRs), the formation of a fully mature heteromeric complex is required for the receptors to attain hormone binding competence (16, 21).

Although steroid receptor heteromeric complexes can be isolated from whole cell extracts prepared in low-salt buffer, these complexes are quite dynamic. For example, PR heteromeric complexes have been found to turn over in vitro with a t1/2 of approximately 10 min (16). The dynamic nature of steroid receptor heteromeric complexes has been postulated to play an important role in the intracellular trafficking of steroid receptors (12, 22). PR (23, 24), GR (25), and estrogen receptor (ER, Ref. 26) are shuttling proteins that exchange between the nuclear and cytoplasmic compartments. Disruption of the dynamic exchange between steroid receptors and their associated proteins using pharmacological approaches disrupts receptor shuttling and confines receptors to the cytoplasm (26, 27). The fact that unliganded PR and ER are predominantly localized within the nucleus (28, 29) suggests that their nuclear import is not limited by dynamic interactions with hsp90 or other proteins.

In contrast to PR and ER, unliganded GRs are mainly localized within the cytoplasm (30, 31, 32, 33). It has been postulated that the association of hsp90, and perhaps other proteins, may be limiting the import of unliganded GRs (12, 22). Accordingly, hormone binding would be expected to stimulate the release of GR-associated proteins and expose the receptor to the nuclear import machinery. While hormone-bound GRs still maintain the capacity to shuttle between the nuclear and cytoplasmic compartments (25), their nuclear import rate greatly exceeds their rate of nuclear export, leading to the predominant localization of the ligand-bound GRs within nuclei (25). Thus, it appears that the limitation in GR nuclear export may not be mediated by a slow passage across the nuclear pore complex, but more likely is influenced by the retention of the receptor within distinct subnuclear compartments (34, 35).

Once GRs arrive within the nucleus, they rapidly locate and bind to high-affinity target sites within native chromatin and alter the transcriptional activity of linked promoters (1, 36). GR binding to chromatin appears to be strictly hormone dependent (37). The withdrawal of hormone leads to the release of receptors from high-affinity chromatin-binding sites (38, 39). While the kinetics of GR chromatin release appear to correlate with the kinetics of hormone dissociation (34, 40), the nuclear export of unliganded receptors remains relatively slow. Thus, nuclear export of GRs is not restricted solely by their interactions with chromatin.

In this report, we have continued our analysis of the subnuclear trafficking of GRs, focusing exclusively on the properties of unliganded receptors that are released from chromatin. Our results suggest that hsp90 chaperone function may play multiple roles to facilitate chromatin recycling of GR.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Unliganded Nuclear GRs in Permeabilized GrH2 Cells Do Not Require ATP or Cytosolic Factors to Rebind Hormone
The dissociation of hormone from liganded nuclear GRs is quite rapid (40) but not associated with a corresponding rapid rate of nuclear export (25, 34). Previously, we found that hormone withdrawal leads to a rapid dissociation of GR from chromatin (34). These unliganded nuclear receptors accumulate within a novel subnuclear compartment and are competent to rebind chromatin in vitro if exposed to hormone in the presence of ATP and cytosolic factors (34).

In vitro reconstitution studies established that hormone-binding competence of PR and GR is only accomplished after a multistep maturation process that results in the assembly of mature aporeceptor oligomeric complexes (13, 16). Is such a maturation process required for chromatin-released (i.e. recycled) nuclear receptors to regain hormone-binding capacity? We set out to address this question using a digitonin-permeabilized cell system (41, 42) in which the hormone-binding properties of GR could be manipulated by a regimen of hormone withdrawal and reexposure. We initially examined the role of ATP and cytosolic factors in the maturation of recycled nuclear GR. As shown in Fig. 1AGo, neither ATP nor cytosol was required for unliganded nuclear GRs to rebind hormone in digitonin-permeabilized GrH2 cells. In experiments assessing the ATP dependence of hormone rebinding, permeabilized GrH2 cells were pretreated with apyrase to eliminate residual ATP. The effectiveness of the apyrase incubation in reducing ATP levels was confirmed by direct measurements of ATP (not shown).



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  		Figure 1. Unliganded Nuclear GRs in Permeabilized GrH2 Cells Do Not Require Cytosolic Factors or ATP to Rebind Hormone

A, Hormone-withdrawn GrH2 cells were permeabilized with digitonin and treated with apyrase to remove ATP where indicated (-ATP). B, Hormone-withdrawn GrH2 cells were permeabilized with digitonin and pretreated with 0.2 mg/ml WGA for 10 min on ice. Permeabilized cells were then incubated in transport buffer with [3H]Dex ± HeLa cell cytosol, for 20 min at 30 C. In panel A, ATP was either added or omitted, while in panel B, ATP was added and WGA was either added or omitted during incubation. Nuclei were then collected and radioactivity (in cpm) measured by scintillation counting. Background radioactivity (in cpm) obtained from control samples containing unlabeled Dex has been subtracted from values shown. Data shown are the average of at least two separate experiments performed with duplicate samples.

 
Although digitonin-permeabilized GrH2 cells are not competent to support nuclear import in the absence of cytosolic factors (41, 42), import of residual cytosolic factors might contribute to hormone binding of nuclear GRs. To more convincingly eliminate this possibility, we included the general nuclear transport inhibitor, wheat germ agglutinin (WGA), in our in vitro hormone-binding experiments to further impede the nuclear import capacity of permeabilized cells. We had previously shown that WGA treatment of permeabilized GrH2 cells effectively blocked hormone-dependent import of GR (42). As shown in Fig. 1BGo, WGA treatment of digitonin-permeabilized GrH2 cells had no effect on the in vitro hormone-binding activity of unliganded nuclear GR, either in the presence or absence of cytosol. Thus, recycled, unliganded nuclear GRs in permeabilized GrH2 cells do not require cytosolic factors to rebind hormone.

Chromatin Rebinding, but Not Hormone Rebinding, of Nuclear GRs Is Temperature Dependent in Vitro
Once steroid receptors have assembled into mature heteromeric complexes, hormone binding can proceed even at lower temperatures. As shown in Fig. 2AGo, the binding of dexamethasone (Dex) to unliganded nuclear GRs in permeabilized GrH2 cells is as efficient at 0 C as it is at 30 C. Longer incubation times at 0 C did not significantly enhance hormone binding (not shown). Thus, receptor maturation, which should not proceed at 0 C, most likely occurred during the 30-min period of in vivo hormone withdrawal of GrH2 cells.



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  		Figure 2. Chromatin Rebinding, but Not Hormone Rebinding, of Nuclear GRs Is Temperature Dependent in Vitro

Hormone-withdrawn GrH2 cells were permeabilized with digitonin and incubated in transport buffer containing ATP and [3H]Dex for 40 min at 0 C (lanes 1–3) or 30 C (lanes 4–6). In panel A, cells were collected for scintillation counting to measure [3H]Dex binding. Background radioactivity (in cpm) obtained from control samples containing unlabeled Dex has been subtracted from values shown. In panel B, cells were either unextracted (lanes 1 and 4) or extracted with hypotonic (lanes 2 and 5) or CK (lanes 3 and 6) buffer, and subjected to Western blot analysis to visualize GR remaining within nuclei. The nuclear protein, NuMa, provides an internal control for the relative amount of nuclear protein recovery between samples.

 
To ascertain whether these hormone-bound GRs in permeabilized GrH2 cells were associated with chromatin, a differential extraction paradigm was used that distinguishes the chromatin-binding state of the receptor (34). Briefly, receptors that are bound with high affinity to chromatin are resistant to a low-salt, hypotonic extraction, but sensitive to high-salt cytoskeletal (CK) extraction (34). As shown in Fig. 2BGo, hormone-bound GRs in permeabilized GrH2 cells were resistant to hypotonic extraction (compare lanes 4 and 5), and thus associated with chromatin, if subjected to a 40-min incubation at 30 C. The fact that receptors in these cells were sensitive to CK buffer extraction (Fig. 2BGo, lane 6) demonstrated they were chromatin bound and not associated with the nuclear matrix (34). In contrast, incubation of hormone-bound GRs in permeabilized cells at 0 C for 40 min (Fig. 2BGo) or longer (not shown) did not lead to the high-affinity binding of receptors to chromatin (compare lanes 1 and 2). Thus, recycled, ligand-bound nuclear GRs do not spontaneously associate with chromatin, but require an additional temperature-dependent step for high- affinity chromatin binding.

In previous studies, we found it difficult to assess the ATP dependence of GR chromatin binding in permeabilized cells due to the increased extent of receptor targeting to the matrix under ATP depletion conditions (43) (J. Yang and D. B. DeFranco, unpublished data). Nonetheless, we have found that appropriate chromatin rebinding of recycled, nuclear GRs in permeabilized GrH2 cells has a strict requirement for ATP hydrolysis. Thus, neither GTP, ADP, nor the nonhydrolyzable ATP analog, AMP-PNP, could substitute for ATP to generate nuclear GRs that were resistant to hypotonic buffer extraction, but sensitive to high-salt buffer extraction (not shown).

Geldanamycin, an hsp90-Binding Benzoquinoid Ansamycin, Inhibits Hormone Rebinding of Unliganded Nuclear GRs in Permeabilized GrH2 Cells
Various molecular chaperones are involved in the maturation of GRs and PRs in vitro (13). hsp90 participates in a number of discrete steps in this pathway and is found not only associated with mature, hormone-binding competent receptors, but also in intermediate inactive aporeceptor complexes (13, 19). Establishing a role for hsp90 in steroid receptor activation has been greatly facilitated by the use of the hsp90-binding benzoquinoid ansamycin, geldanamycin (GA). GA occupies the nucleotide-binding site on hsp90 and prevents the switch to its ATP-bound conformation (44, 45), which is required for production of steroid-binding competent aporeceptor complexes (46). We therefore used GA to ascertain whether hsp90 is involved in the generation of hormone-binding competent nuclear GRs after their release from chromatin.

As shown in Fig. 3AGo, when GA is added to permeabilized GrH2 cells, hormone binding to nuclear GRs is reduced by 50% (lane 2). If GA is added to GrH2 cells during the withdrawal period, a more potent inhibitory effect on in vitro binding of hormone to GR (i.e. a 90% decrease) was observed (Fig. 3AGo, lane 3). Although prolonged GA treatment has been associated with accelerated down-regulation of GR protein (47), the brief (i.e. 20 min) exposure that we employed did not lead to any dramatic change in steady state GR levels (Fig. 3Go, B and C). Thus, the effects of GA on in vitro hormone binding to GR reflect a decrease in hormone-binding activity of the receptor. Including GA in the in vitro binding assay as well as during the withdrawal in vivo was no more effective than inclusion of GA during the withdrawal period alone (Fig. 3AGo, lanes 3 and 4). If cells are maintained in the continuous presence of hormone (i.e. 90 min), GR hormone-binding activity is still reduced upon a 15- or 30-min incubation with GA (not shown). Thus, as observed in reconstituted cell-free systems (12), the association of hormone with GR is dynamic and sensitive to disruptions in hsp90 function.



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  		Figure 3. GA Inhibits Hormone Rebinding to Recycled Nuclear GR

Hormone-treated GrH2 cells were withdrawn from hormone in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 1 µg/ml GA and then permeabilized with digitonin. Permeabilized cells were incubated with (lanes 2 and 4) or without (lanes 1 and 3) GA in transport buffer containing ATP and [3H]Dex for 20 min at 30 C. Nuclei were collected and subjected to scintillation counting to measure [3H]Dex binding (A) or Western blot analysis (B) to reveal GR remaining within nuclei. In panel A, background radioactivity (in cpm) obtained from control samples containing unlabeled Dex has been subtracted from values shown. Data shown are the average of at least two separate experiments performed with duplicate samples. In panel C, relative GR levels are normalized to NuMa protein levels detected on the costained Western blot (B).

 
GA Inhibits the Release of Unliganded GR from Chromatin
The effectiveness of GA, particularly when given during in vivo hormone withdrawal, in preventing hormone binding to recycled nuclear GRs in permeabilized GrH2 cells suggests that hsp90 may be required for these receptors to reacquire hormone-binding competence. As shown in Fig. 4Go, whole-cell hormone-binding assays confirmed that glucocorticoid hormone was dissociated from GRs upon hormone withdrawal of GrH2 cells in the presence of GA. Thus, nuclear GRs in GA-treated, hormone-withdrawn GrH2 cells were predominantly unliganded, yet incapable of effectively interacting with hormone. This result also demonstrates that hsp90 is unlikely to be required for hormone dissociation from nuclear receptors. To identify the subnuclear compartment where these unliganded nuclear receptors reside, we used differential extractions both in situ for indirect immunofluorescence assays (Fig. 5AGo) and with GrH2 cells in suspension for Western blot analysis (Fig. 5Go, B and C). As shown in Fig. 5Go, the inclusion of GA during the in vivo withdrawal of hormone from GrH2 cells dramatically inhibited GR release from chromatin. As shown previously, and confirmed in Fig. 5AGo (panel b) and Figs. 5BGo and 5CGo (lane 5), GRs in hormone-withdrawn GrH2 cells were sensitive to a hypotonic buffer extraction. The sensitivity of GR to hypotonic buffer extraction was dramatically reduced when GA was included during hormone withdrawal (Fig. 5AGo, panel d; Figs. 5BGo and 5CGo, lane 8). Unliganded nuclear receptors in GA-treated GrH2 cells were sensitive to extraction by high-salt CK buffer (Fig. 5AGo, panel e; Figs. 5BGo and 5CGo, lane 9) and are therefore likely to remain associated with chromatin after hormone dissociation. Thus, while hormone can be dissociated from chromatin-bound GRs, their release from high- affinity interactions with chromatin may require the chaperoning function of hsp90.



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  		Figure 4. GA Does Not Affect the Dissociation of Hormone from Nuclear GR in Hormone-Withdrawn GrH2 Cells

GrH2 cells were treated with 10-7 M [3H]corticosterone for 1 h. Cells were then either kept in hormone-containing medium (-withdrawal) or withdrawn from hormone, in the absence or presence of 1 µg/ml GA, for 30 min. Each sample was then collected in 200 µl lysis buffer, with 100 µl used for scintillation counting to measure [3H]corticosterone binding and 10 µl used for Western blot analysis (not shown). The measurements shown have been normalized to relative GR content determined from Western blots. Data shown are the average of at least two separate experiments performed with duplicate samples.

 


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  		Figure 5. Addition of GA during Hormone Withdrawal Inhibits GR Release from Chromatin

GrH2 cells were treated with 10-7 M corticosterone for 1 h, and then either untreated (panels B and C, lanes 1–3) or withdrawn from hormone (panel A and panels B and C, lanes 4–9) for 30 min. GA (1 µg/ml) was either omitted (A, panels a and b; B and C, lanes 4–6) or included (A, panels c, d, and e; B and C, lanes 7–9) during the hormone withdrawal. Cells were then extracted with hypotonic (Hypo) or CK buffer before processing for immunofluorescence staining (A) or Western blot analysis (B). In panel C, average measurements of relative GR levels (±SD) from two to three separate Western blots are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Steroid receptor proteins can be reutilized and regain their competence to respond to hormone when recycled from the nuclear to cytoplasmic compartment (32, 48). Munck and co-workers (48) have also hypothesized the existence of a nuclear bypass pathway that permits the reutilization of nuclear GRs without an obligatory passage through the cytoplasm. Although direct experimental evidence for recycling of nuclear GRs has recently been provided in experiments utilizing digitonin-permeabilized cells (34), fundamental questions regarding the mechanism of GR recycling within the nucleus remain unanswered. It is well established that hormone-binding competent cytoplasmic GRs exist as heteromeric complexes that include molecular chaperones, such as hsp90, immunophilins, and various chaperone partners (11, 13). How do receptors that release hormone, yet remain associated with the nucleus, regain the capacity to bind hormone? What are the requirements for recycling of nuclear GRs?

Through the use of a permeabilized cell system, where receptor exchange between the nuclear and cytoplasmic compartments can be minimized, we have provided definitive evidence for nuclear recycling of GR. The inhibition of nuclear GR hormone binding by GA suggests that hsp90 may function in the nucleus to assemble recycled receptors into hormone-binding competent heteromeric complexes (see model in Fig. 6Go). Future studies directed toward the characterization of such putative nuclear aporeceptor complexes will reveal whether maturation of nuclear GR, or other steroid receptors, is related to the cytoplasmic receptor maturation pathway established in cell-free systems.



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  		Figure 6. Recycling of Nuclear GR

A proposed model for the recycling of nuclear GRs is shown with hps90 implicated in chromatin release and reassembly of aporeceptor heteromeric complexes. ATP may be required for hsp90 chaperone function at the level of GR chromatin release and for maintaining appropriate dynamic association of GR with the nuclear matrix.

 
As observed in various in vitro systems (16, 17, 18), the binding of hormone to unliganded nuclear GRs in permeabilized cells is temperature independent and does not require ATP. Thus once matured, nuclear receptors are fully primed to respond to a hormonal signal and do not require active cellular processes to do so. In contrast, the subsequent high-affinity interaction of liganded receptors with chromatin appears to be an active process. The ATP dependence of in vitro GR chromatin binding was difficult to assess as in vitro incubations of permeabilized cells at ambient temperatures in the absence of ATP leads to increased nuclear matrix binding of receptors. The in vitro binding of GR to reconstituted nucleosomes does not require ATP (49, 50), suggesting that ATP may be used in intact nuclei not necessarily to facilitate appropriate receptor targeting to chromatin binding sites, but to prevent inappropriate targeting of receptors to alternative subnuclear compartments (i.e. the nuclear matrix). ATP hydrolysis appears to be essential in our permeabilized cell system to restrict GR interactions with the matrix. Furthermore, GTP can not substitute for ATP, suggesting that Ran (51, 52, 53) and perhaps other small GTP-binding proteins are not involved in directing GR to appropriate chromatin-binding sites.

Recent work from our laboratory has established a role for hsp70 and one of its chaperone partners, DnaJ, in the correction of protein misfolding, or aggregation within the nucleus (54, 55). Such chaperone systems may be required for the efficient subnuclear trafficking of steroid receptors, as relatively high protein concentrations and limited free diffusion space within the nucleus may favor inappropriate protein-protein interactions that could lead to aggregation.

Our studies have also uncovered another potential function of molecular chaperones in subnuclear trafficking of GR. Hormone withdrawal leads to rapid chromatin release of both bulk GRs (Ref. 34 and this report) and receptors associated with high-affinity target sites (39). However, despite the fact that chromatin-associated GRs release their bound hormone in the presence of GA, their subsequent release from chromatin is dramatically inhibited. It should be noted that nuclear receptors in GA-treated, hormone-withdrawn cells that resist low-salt extraction are high-salt extractable, eliminating the possibility of increased nuclear matrix association of the receptors under these conditions. We therefore postulate that the chaperoning activity of hsp90 may be needed to facilitate GR release from high-affinity chromatin-binding sites upon hormone dissociation (see model, Fig. 6Go). Since our studies examined the chromatin-binding properties of bulk receptors, it is unknown whether this hsp90 dependence applies to GR associated with chromatin of specific target genes. However, the techniques used to examine GR association with target gene chromatin (39) can be applied in our permeabilized cell system to address this issue.

Additional studies of GA effects illustrate the dynamic nature of unliganded and liganded GR interactions with chromatin. For example, GRs are resistant to hypotonic extraction, and therefore chromatin associated, if GA is added to cells after a 30-min hormone withdrawal (not shown). This observation suggests that unliganded nuclear receptors may also have some limited capacity to interact with chromatin. We hypothesize that as long as hsp90 function is not compromised, the release of unliganded receptors from high-affinity chromatin-binding sites is more favored than their association with these sites. This could explain why unliganded nuclear receptors, while not detected on chromatin, can be driven to high-affinity chromatin binding sites by GA treatment after hormone withdrawal. Since there is a minimal loss of chromatin-bound receptors when cells are incubated with GA in the continuous presence of hormone (not shown), some fraction of hormone-bound receptors may release from high-affinity chromatin-binding sites. Once hormone dissociates from these chromatin-released liganded receptors, their hormone and chromatin rebinding would be inhibited by GA.

Despite the fact that hsp90 has always been considered to be an abundant (i.e. 1–2%) cytoplasmic protein, a small but significant fraction (~3%) of hsp90 is found within nuclei (56). Thus, there appears to be sufficient nuclear pools of hsp90 to perform biologically relevant functions. In vitro studies have revealed a role for hsp90 in DNA binding of helix-loop-helix transcription factors (57, 58). Interestingly, hsp90 has been found to facilitate the release of GR (59) and ER (60) from DNA in vitro. In these studies, the ATP dependence of hsp90 effects on receptor release from DNA was not strictly addressed. GA inhibits ATP binding to hsp90 through its occupancy of the ATP-binding site of hsp90 (61). Given the GA effects on GR chromatin binding that we observed in our permeabilized cell system, it will be relevant to consider the ATP requirement for hsp90 effects on steroid receptor release from chromatin and DNA.

What property of steroid receptors could account for the putative hsp90 requirements for chromatin release? Upon hormone binding, steroid receptor ligand-binding domains (LBDs) undergo a conformational change (62) that is characterized by the movement of a particular exposed {alpha}-helix (i.e. helix 12) toward the hormone-binding pocket (63, 64). As a result, hydrophobic segments of the LBD, as well as the bound hormone itself, are no longer solvent exposed. Furthermore, previously inaccessible LBD surfaces become exposed and available for interactions with appropriate coactivators and other components of the transcriptional machinery. It is unclear how the elaborately networked LBD structure responds to the release of bound hormone. Does the LBD simply "relax" to reaquire the conformation it possessed when initially unliganded? In such a scenario, the exposure of hydrophobic segments of the unliganded receptor’s hormone-binding pocket might increase the propensity for receptor aggregation unless molecular chaperones such as hsp90 are present to prevent such inappropriate interactions. Unliganded nuclear receptors in GA-treated, hormone-withdrawn cells remain salt extractable and thus are unlikely to be significantly aggregated.

The results presented in this report establish that steroid receptor encounters with molecular chaperones are not restricted solely to the cytoplasm. As receptors traffic through distinct subnuclear compartments, their individual domains may adopt various folded or unfolded states. Thus, nuclear molecular chaperone complexes, which may bear some resemblance to cytoplasmic complexes or possess unique compositions, may be called upon to maintain nuclear receptors in biologically active conformations.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture
The GrH2 rat hepatoma cell line, which expresses elevated levels of GR (62), was maintained at 37 C in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 5% FBS (Irvine Scientific, Santa Ana, CA).

Antibodies and Chemicals
The BuGR2 monoclonal antibody (63) was used to detect GR. Ab-1 is a mouse monoclonal antibody directed against the NuMA nuclear matrix protein (Oncogene Science, Cambridge, MA). GA was provided by the Drug Synthesis & Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute.

In Vivo Hormone Treatment and Hormone Withdrawal
For all hormone treatments, GrH2 cells were treated with 10-7 M corticosterone (Sigma Chemical Co., St. Louis, MO) for 1 h. For hormone withdrawal, hormone-treated cells were briefly rinsed three times with phenol red-free DMEM (Life Technologies) plus 5% charcoal-stripped FBS and then incubated with this medium at 37 C for 30 min, unless otherwise noted. GA (1 µg/ml) was included in the withdrawal medium where indicated.

Cell Permeabilization
GrH2 cells were collected using PBS containing 2 mM EDTA. Cells were rinsed with ice-cold transport buffer (20 mM HEPES, pH 7.3, 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM dithiothreitol (DTT), and 1 µg/ml each of protease inhibitors, aprotinin, leupeptin, and pepstatin A), and then permeabilized using ice-cold transport buffer containing 40 µg/ml digitonin (Sigma Chemical Co.) for 5 min on ice. Cell pellets were then rinsed with transport buffer before indicated incubations with hormone.

In Vitro and in Vivo Hormone Binding
Permeabilized cells were incubated at 0 C or 30 C for 20 min in 50–100 µl transport buffer containing 10-7 M [3H]Dex (41.0 Ci/mmol, Amersham Life Science, Arlington Heights, IL), 10 µg/ml BSA, 5 mM creatine phosphate, 20 U/ml creatine phosphokinase, in the presence or absence of 2 mM ATP. To deplete ATP, permeabilized cells were incubated with 3–5 U of apyrase (Sigma) in 100 µl of transport buffer for 3 min on ice. In some experiments 25% (vol/vol) HeLa cell cytosol (42) or 0.2 mg/ml WGA (Sigma) was also included. All in vitro hormone binding experiments included a control in which a 1000-fold molar excess of unlabeled Dex was included to monitor nonspecific binding of [3H]Dex, which typically represented 10–20% or lower of total binding.

Nuclei were recovered from hormone-treated permeabilized cells as described previously (34). Intact nuclei isolated after centrifugation at 400 x g were resuspended in 200 µl ethanol and added to 5 ml ScintiSafe Solution (Fisher Scientific, Pittsburgh, PA) for liquid scintillation counting. Specific binding of [3H]Dex was determined after the subtraction of nonspecific binding.

For in vivo measurements of hormone binding, GrH2 cells, grown in DMEM containing charcoal-stripped serum, were incubated with 20 nM [3H]corticosterone (77.0 Ci/mmol, Amersham Life Science) for 1 h. Nonspecific binding was assessed by the inclusion of 1000-fold excess of unlabeled corticosterone (Sigma). Where indicated, cells were withdrawn from hormone for various lengths of time by incubation in withdrawal medium. Cells were then extensively washed with culture medium and lysed by sonication in high-salt lysis buffer [10 mM HEPES, pH 7.0, 450 mM NaCl, 5 mM EDTA, 0.05% SDS, 1% Triton X-100, 2 mM DTT, and protease inhibitors, (42)]. One half of the lysate was added to ScintiSafe Solution to measure [3H]corticosterone binding, while the other half was used to determine relative GR levels by Western blot analysis (42).

Biochemical Extractions
For low-salt, hypotonic extraction, nuclei isolated from digitonin-permeabilized GrH2 cells were treated with 150 µl hypotonic (Hypo) buffer [10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.1% Triton X-100, and protease inhibitors (43)] for 3 min on ice. For high-salt extraction, nuclei were treated for 3 min on ice with 150 µl of CK buffer [10 mM piperazine-N,N'-bis[2-ethanesulfonic acid], pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, and protease inhibitors, (43)]. The Hypo or CK buffer-extracted nuclei, as well as an aliquot of unextracted nuclei, were dissolved in 1x SDS sample buffer (132 mM Tris-HCl, pH 6.8, 20% glycerol, 10% SDS, 10.4% ß-mercaptoethanol, 0.02% pyronin Y), boiled for 5 min, and then subjected to SDS-PAGE. Occasionally, whole cells were directly lysed on culture plates using high-salt lysis buffer.

Western Blot Analysis
GRs in intact or extracted nuclei were detected by Western blot analysis using the BuGR2 antibody as previously described (42). To provide an internal control for gel loading and transfer efficiency, NuMA (nuclear matrix protein) was also detected on the same blots using the Ab-1 antibody. GR or NuMA on Western blots was visualized using the enhanced chemiluminescence (ECL) (Amersham International, Little Chalfont, Buckinghamshire, U.K.) detection system.

Indirect Immunofluorescence
GrH2 cells were fixed with -20 C methanol for 5 min at room temperature. Fixed cells were incubated with the BuGR2 anti-GR antibody. An fluorescein isothiocyanate-coupled antimouse IgG antibody (Boehringer Mannheim Biochemicals, Indianapolis, IN) was used as secondary antibody to detect GR. Stained cells were observed by fluorescence microscopy through an Optiphot-2 microscope (Nikon Inc., Garden City, NY) and photographed with T-Max 400 film (Eastman-Kodak Co., Rochester, NY).


   ACKNOWLEDGMENTS
 
Geldanamycin was provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute. We thank Drs. David Toft and Péter Csmerley for helpful discussions.


   FOOTNOTES
 
Address requests for reprints to: Donald B. DeFranco, Department of Biological Sciences, University of Pittsburgh, A234 Langley Hall, Fifth and Ruskin Streets, Pittsburgh, Pennysylvania 15260. E-mail: dod1{at}vms.cis.pitt.edu

This work was supported by NIH Grant CA-43037 (to D.B.D.).

Received for publication October 1, 1998. Revision received December 2, 1998. Accepted for publication December 16, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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