Overexpression of Unliganded Steroid Receptors Activates Endogenous Heat Shock Factor

Nianqing Xiao and Donald B. DeFranco

Department of Biological Sciences University of Pittsburgh Pittsburgh, Pennsylvania 15260


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The synthesis of a number of heat shock proteins is induced in response to various environmental stresses. The resultant induction of heat shock protein gene transcription is brought about by the activation of specific transcription factors termed heat shock factors (HSFs) that exist in a latent form in nonstressed cells. Multiple mechanisms are likely to contribute to negative regulation of HSF activity. One model, which remains controversial, proposes the existence of a negative feedback loop by which one of the products of HSF activation, the 70-kDa heat shock protein (hsp70), acts as one of its negative regulators. Accordingly, HSF activation would proceed upon sequestration of hsp70 by substrates (i.e. unfolded proteins) that may accumulate to relatively high levels in stressed cells. To examine whether putative native substrates of hsp70 (e.g. steroid receptors) could impact the regulation of HSF activity, we have examined whether steroid receptors could activate endogenous HSF. We have found that overexpression of androgen (AR), glucocorticoid (GR), mineralocorticoid, and progesterone receptors in transiently transfected COS-1 cells induced HSF activity. With the exception of AR, which was competent to activate HSF when either liganded or unliganded, all other steroid receptors tested only activated HSF when unliganded. This activity was mapped to the ligand-binding domain of rat GR, making it unlikely that HSF activation results from the induction of a novel gene product by unliganded receptors. As overexpression of hsp70 can eliminate HSF activation by AR, GR, and progesterone receptors, we favor the view that HSF activation can result from the sequestration, by steroid receptor ligand-binding domains, of a negative regulator of HSF, such as hsp70 or an hsp70-associated protein.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Unliganded steroid hormone receptors are poised to respond to hormonal signals and alter the expression of unique target genes, often in a tissue- or cell type-specific manner (1, 2, 3). Typically, the binding of cognate hormone is required to unleash the transcriptional regulatory activity of the receptor, although in some cases, receptors can modulate transcription in response to the activation of other signal transduction pathways (4, 5). Activation of the estrogen receptor by epidermal growth factor is associated with direct phosphorylation of the receptor by mitogen-activated protein kinase (6, 7). Whether steroid receptor phosphorylation accounts for ligand-independent activation in other systems (8) has not been established.

The carboxyl-terminal ligand-binding domain (LBD) of steroid receptors is responsible for maintaining unliganded receptors in an inactive form (9). The dominant inactivation function associated with unoccupied steroid receptor LBDs is imparted upon linked receptor DNA-binding and trans-activation domains (10), but can also impair the activity of heterologous functional domains (10, 11). Upon hormone binding, the LBD undergoes a conformational change (12) that not only leads to the relief of its repressive effects on DNA-binding and amino-terminal trans-activation domains, but also exposes a dimerization interface (13) and a trans-activation domain (14, 15, 16, 17) encoded within the LBD.

The LBD of steroid receptors also serves to assemble a heteromeric complex comprised of various members of the heat shock and immunophilin families of proteins (18, 19). Principal protein components associated with glucocorticoid receptors (GRs) and progesterone receptors (PRs) include the 90-kDa heat shock protein (hsp90), a 52-kDa immunophilin (FKBP52), and a 23-kDa protein (p23) (18, 19). The association of these proteins with GR and PR is required for the receptors to attain a high affinity ligand-binding capacity (20, 21).

The assembly of GR and PR heteromeric complexes is a multistep process that proceeds in a defined order in vitro (21, 22). GR and PR associate with a 70-kDa heat shock protein (hsp70) in one of the earliest steps in the formation of receptor-heteromeric complexes (21, 23). This binding appears to be only transient, as intermediate complexes are then generated composed of a the 48-kDa Hip protein and p60 (24). Hormone binding competence is finally acquired as mature forms of GR and PR are generated possessing stably associated hsp90, FKBP52, and p23 (21, 24).

Although PR and GR heteromeric complexes of various composition have been visualized using in vitro assembly reactions (21, 25), confirmation that such an ordered pathway operates in vivo has proven difficult to obtain (26, 27). Coimmunoprecipitation experiments have identified hsp90 (28, 29) and FKBP52 (30, 31) as components of unliganded steroid receptor heteromeric complexes, but the association of other proteins with unliganded steroid receptors in vivo remains controversial (32). For example, there are conflicting reports regarding the association of hsp70 with steroid receptors such as GR and PR (28, 33, 34, 35, 36). As hsp70 does not appear to be a component of hormone binding-competent steroid receptor heteromeric complexes reconstituted in vitro (21, 24), it remains to be established whether hsp70 association with steroid receptors in vivo is functionally significant.

Heat shock proteins are involved in steroid receptor signal transduction in nonstressed cells (9), indicating that constitutive levels of these stress-activated proteins are sufficient to impact receptor function. In response to various environmental stresses, the levels of inducible forms of heat shock proteins increase dramatically (37, 38). Does the elevation in hsp90 and hsp70 levels in stressed cells alter steroid hormone signaling? An increased association of hsp90 and hsp70 with PR was found to result from environmental stresses that induced expression of heat shock proteins (39). Under these conditions, steroid receptor-mediated trans-activation was potentiated (39). Heat shock potentiation of GR trans-activation has also been observed and is representative of effects that result from other types of environmental stress (40). Although the mechanistic basis for these effects remains obscure, these results illustrate that steroid hormone signaling is sensitive to conditions that activate particular cellular stress responses.

The transcriptional activation of heat shock protein genes such as hsp90 and hsp70 is regulated by a family of DNA-binding proteins termed heat shock factors (HSFs) (41, 42). HSF-1 is the principal member of this family that is thought to regulate hsp70 gene transcription (43, 44) via its binding, as a trimer, to a unique sequence within the hsp70 promoter, termed heat shock response elements (HSEs) (41, 42). HSF-1 is present in nonstressed cells and is, therefore, maintained in a dormant state until activated by the appropriate environmental stress (41, 42). A specific regulatory domain has been identified within HSF-1 that is responsible for negative regulation of is activity in nonstressed cells (45, 46, 47). The activity of the human HSF-1 regulatory domain is influenced by direct phosphorylation mediated via the raf/ERK pathway (48). In addition to this level of control, there is considerable evidence for the involvement of hsp70 in negative regulation of HSF activity (44, 49). In this case, a negative feedback loop may be established that limits the extent and duration of HSF activity and perhaps partially contributes to the maintenance of inactive HSF (50). Although results from in vivo studies in yeast (51) and mammalian cells (52) lend support to this model, results that contradict this model have also been obtained (53).

The association of hsp70 with distinct transcriptional regulators (i.e. GR and HSF) implicates an involvement for this stress-activated protein in signal transduction that may extend beyond its well established chaperone functions. As hsp70 pools in vivo are likely to be dynamic, dramatic alterations in the bioavailability of hsp70 that are likely to accompany the mobilization of a cellular stress response may have an impact on GR- or HSF-mediated transcriptional responses. We, therefore, examined whether manipulation of steroid receptor levels affects HSF activity in cultured cells. Our observed activation of HSF activity by unliganded steroid receptors provides evidence for an unique level of cross-talk between steroid hormone signaling and cellular responses to environmental stresses.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Induction of HSF Activity by Unliganded GR
Activated HSFs bind to heat shock elements (HSEs) as trimers and stimulate the transcription of linked promoters (42). HSF-induced transcription can be easily monitored in cells transfected with HSE-containing reporter plasmids. As shown in Fig. 1Go, a 1-h heat shock (i.e. 43 C) of COS-1 cells activated endogenous HSF activity, leading to the robust induction of chloramphenicol acetyltransferase (CAT) activity from a reporter that possess a minimal human hsp70 gene promoter with an intact HSE (54). A CAT reporter lacking the HSE sequences was unaffected by an analogous heat shock of transfected COS-1 cells (data not shown). Interestingly, cotransfection with a rat GR expression vector (i.e. 6RGR) also led to induced transcription from the HSE-CAT reporter plasmid (Fig. 1Go) in the absence of heat shock. This activation of HSF activity was dependent upon the presence of the HSE, as no change in basal transcription from a CAT reporter lacking the HSE was noted upon cotransfection with the GR expression vector (Fig. 1Go). GR maintained its property as a hormone-dependent trans-activator under our transfection conditions, as revealed by the hormone-dependent activation from a cotransfected glucocorticoid response element (GRE)-linked CAT reporter (Fig. 1Go).



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Figure 1. Overexpression of Unliganded GR Activates HSF

COS-1 cells were transfected with 2 µg CAT reporter plasmids that either lacked upstream regulatory elements (CAT) or possessed an upstream GRE (GRE-CAT) or HSE (HSE-CAT). Where indicated (GR), 4 µg of the rat GR expression plasmid p6RGR were cotransfected. In all cases, 0.2 µg of a cytomegalovirus-ßgal reporter plasmid was also cotransfected to provide a standard to normalize transfection efficiency. Total DNA was constant in all transfections. Transfected cells were grown in the presence or absence of 1 µM Dex or RU486 as indicated for 36 h and then harvested, lysed, and assayed for CAT activity. Some transfected cells (HS) were heat shocked at 43 C for 1 h and then recovered for 3 h before harvesting. Results are an average of at least three independent experiments.

 
As unliganded GR did not appear to function as a trans-activator in transfected COS-1 cells (Fig. 1Go), it was unlikely that GR induction of HSE-CAT activity resulted from receptor-dependent synthesis of a gene product that activated HSF activity. In fact, in the presence of dexamethasone, GR-dependent induction of HSE-CAT promoter activity was suppressed (Fig. 1Go). As will be shown below (Fig. 8BGo), GR levels were not dramatically reduced upon dexamethasone treatment of transfected COS-1 cells, indicating that the hormone-dependent relief of GR-dependent activation of HSF was not due to decreased synthesis of the receptor. The glucocorticoid antagonist RU486 is also effective in relieving GR-dependent HSE-CAT promoter activation (Fig. 1Go), although the suppression was somewhat less than that observed with dexamethasone.



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Figure 8. GR Expression in Dexamethasone (Dex)-treated and hsp70 Cotransfected Cells

COS-1 cells were cotransfected with p6RGR DNA, HSE-CAT, and, where indicated, an hsp70 expression plasmid (pßact-Hsp70). Cells (either untreated or treated with 1 µM Dex) were harvested after 36 h, and equivalent amounts of total protein in cell-free extracts were separated by SDS-PAGE. GR was visualized by Western blot analysis.

 
To determine the relative effectiveness of GR effects on GRE- vs. HSE-linked promoters, varying amounts of p6RGR DNA were independently cotransfected into COS-1 cells with either a GRE-linked or a HSE-linked CAT reporter. Although the induction of GRE-linked promoter activity by ligand-bound GR reached its maximum at relatively low amounts of 6RGR plasmid (i.e. 1–2 µg/transfection; Fig. 2Go), robust induction of HSE-linked promoter activity required at least 5-fold higher amounts of transfected p6RGR DNA (i.e. 8 µg/transfection; Fig. 2Go). This nonlinear dose-response curve of GR-dependent induction of HSE-linked promoter activity resembles that obtained when induction from an HSE-linked reporter was analyzed after microinjection of varying amounts of denatured proteins into Xenopus laevis oocytes (55). As higher amounts of p6RGR DNA reduced transfection efficiency and had adverse affects on cell survival (data not shown), we did not examine induction of HSE-CAT activity at higher amounts of transfected p6RGR DNA. As it is difficult to definitively measure GR levels in transient transfection experiments, particularly at the level of individual cells, we cannot relate the increased amount of transfected p6RGR DNA with absolute GR levels. Nonetheless, it appears likely that effective activation of HSF activity required GR levels exceeding those required for effective induction from a GRE-linked promoter.



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Figure 2. Dose-Response Analysis of Transfected p6RGR DNA Effects on HSE- and GRE-Linked Promoters

COS-1 cells were transfected with different amounts of p6RGR DNA, as indicated, 2 µg HSE-CAT or GRE-CAT, and 0.2 µg cytomegalovirus-ßgal. Cells were grown in the presence or absence of Dex for 36 h and then harvested, lysed, and assayed for CAT activity.

 
The GR LBD Is Required for Activation of HSF Activity
To reveal whether a unique domain within the rat GR is responsible for activation of HSF activity, receptor deletion mutants were cotransfected into COS-1 cells with the HSE-CAT reporter. As shown in Fig. 3AGo, deletion of the carboxyl-terminal LBD (i.e. GR mutant VAN525) dramatically reduced the ability of GR to activate HSF, whereas deletion of the amino-terminal 407 amino acids (i.e. VA407C) actually led to an increase in HSF activation. For both full-length wild type GR (VARO) and VA407C, dexamethasone treatment abolished HSF activation (Fig. 3AGo). The inability of VAN525 to activate HSF activity was not due to differences in its expression, as Western blot analysis showed that VARO, VA407C, and VAN525 were expressed equivalently in transfected COS-1 cells (Fig. 3BGo). To confirm that the GR LBD was primarily responsible for HSF activation, this domain of the receptor was expressed separately as a ß-galactosidase (ßgal) chimera. As shown in Fig. 3CGo, the GR LBD-ßgal chimera (i.e. VAL-HBD) was as effective as the full-length GR-ßgal chimera (i.e. VARL) in activating HSF activity in transfected COS-1 cells. The ßgal protein (i.e. VAL) did not activate HSF (Fig. 3CGo), establishing that the LBD portion of VAL-HBD was responsible for this activity. The LBD-ßgal chimera lacks a DNA-binding domain and, thus, is unable to activate target gene transcription through DNA binding. Thus, these results support our contention that activation of HSF does not result from the induction of a gene product by unliganded GR.



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Figure 3. Mapping of the GR Domain Responsible for Activation of HSF

A, COS-1 cells were cotransfected with 4 µg of plasmids expressing full-length GR (VARO), amino-terminal-truncated (VA407C) or carboxyl-terminal-truncated GR (VAN525), 2 µg HSE-CAT, and 0.2 µg cytomegalovirus-ßgal. Cells were harvested after 36 h and then lysed and assayed for CAT activity. Dexamethasone (Dex; 1 µM) was added, where indicated, during the 36-h incubation. Results are an average of at least three independent experiments. B, Cell-free extracts were prepared from COS-1 cells transfected as described in A, and total protein was separated by SDS-PAGE. Full-length GR as well as 407C and N525 deletion derivatives were visualized by Western blot analysis. C, COS-1 cells were cotransfected with HSE-CAT and expression plasmids encoding the ßgal gene alone (VAL), a full-length GR-ßgal chimera (VARL), or a GR LBD-ßgal chimera (VAL-HBD). Cells were harvested after 36 h, lysed, and assayed for CAT activity. Results are an average of at least three independent experiments.

 
Activation of HSF Activity by Other Steroid Receptors
The LBDs of steroid receptors, although sharing some degree of homology, possesses multiple amino acid determinants that permit discrimination between closely related steroidal ligands (56). It was, therefore, of interest to reveal whether activation of HSF activity was unique to unliganded GR or shared by other steroid receptors. As shown in Fig. 4Go, in addition to GR, unliganded PR, mineralocorticoid receptor (MR), and androgen receptor (AR) activated HSF activity in transfected COS-1 cells. HSF activity was not activated by ligand-bound GR, PR, or MR, but curiously, AR maintained its ability to activate HSF activity when ligand bound (Fig. 4Go). To confirm this result, a DNA titration was performed for both AR and GR expression plasmids, and HSF activation was monitored in both the presence and absence of hormone. In these transfections, a HRE-luciferase (luc) reporter was also cotransfected into cells with the HSE-CAT reporter so that hormone-dependent transcriptional induction mediated by receptor binding to its cognate HRE could be monitored in the same population of transfected cells as HSE activation by HSF. As shown in Fig. 5Go, A–D, activation of HSF by both AR and GR required higher amounts of transfected DNA than hormone-dependent receptor induction from the HRE-linked reporter. This experiment provided an independent confirmation of our previous experiment with GR (Fig. 2Go) that was performed with separate transfected cell populations. Likewise, no induction of HSF activity was noted for ligand-bound GR regardless of the amount of GR expression plasmid transfected (Fig. 5CGo). In contrast, the hormone-binding state of AR had little effect on its ability to activate HSF activity at all DNA concentrations examined (Fig. 5DGo). Thus, although AR, GR, MR, and PR have the capacity to activate HSF activity when unliganded, only AR maintained this activity when ligand bound.



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Figure 4. Activation of HSF by Other Steroid Receptors

COS-1 cells were cotransfected with GR, PR, MR, and AR expression plasmids along with either an HSE-CAT or a HRE-CAT reporter gene. GR-, PR-, MR-, and AR-transfected cells were grown either untreated or treated (+H) with 1 µM dexamethasone (Dex), R5020, aldosterone, or DHT, respectively. Cells were harvested after 36 h, lysed, and assayed for CAT activity. Results are an average of at least three independent experiments.

 


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Figure 5. Dose-Response Analysis of Transfected p6RGR and p6RAR DNA Effects on HSE- and HRE-Linked Promoters

COS-1 cells were cotransfected with varying amounts of GR (A and C) or AR (B and D) expression plasmids along with HSE-CAT and HRE-luciferase reporters. Cells were grown in the presence or absence of 1 µM Dex (A and C) or DHT (B and D) for 36 h. A and B show results from hormone-treated cells. Cells were then harvested and assayed for both CAT and luciferase activities. A, B, and D show the results of a typical experiment; C shows the average results from three independent experiments.

 
Activation of HSF Activity by Steroid Receptors Is Overcome upon Coexpression of hsp70
HSFs are present in nonstressed cells, but are maintained in an inactive state (41, 42). A specific regulatory domain has been characterized within HSF-1 that is responsible for negative regulation of its activity (45, 46, 47). It has been postulated that hsp70, one of the products of HSF activation, participates in a negative feedback loop to inactivate HSFs and thereby limits the extent of heat shock protein induction (50). Members of the hsp70 family may also play a role in nonstressed cells to inactivate HSFs through direct interactions with their regulatory domain (50). According to this view, HSF activation might be initiated once hsp70 is released and becomes recruited to other targets (i.e. misfolded or aggregated proteins) that are generated after various environmental stresses. Interactions between steroid receptors and hsp70 have been detected in vivo, but not in all studies (33, 34, 36). Could overexpression of unliganded steroid receptors initiate activation of HSFs through a sequestration of hsp70? As shown by the indirect immunofluorescence micrographs in Fig. 6Go, expression of GR in transfected COS-1 cells led to an increased accumulation of endogenous hsp70, which was predicted from the activation of HSF activity as measured using a model cotransfected HSE-linked reporter (see Fig. 1Go). Interestingly, the subcellular localization of induced hsp70 in these cases coincided with the compartmentalization of GR. Thus, in transfected cells in which GR was cytoplasmic, hsp70 accumulated predominantly within the cytoplasm (Fig. 6Go), whereas in cells with unliganded nuclear GR, hsp70 correspondingly accumulated within the nucleus. Thus, it seems likely that in transiently transfected COS-1 cells, GR may stably associate with hsp70 and sequester it into either the cytoplasmic or the nuclear compartment.



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Figure 6. Visualization of GR and hsp70 Subcellular Localization in Transfected COS-1 Cells by Indirect Immunofluorescence

COS-1 cells were transfected with p6RGR DNA, fixed with methanol, and processed for indirect immunofluorescence to visualize transiently expressed GR and endogenous hsp70. Cells were grown in the absence or presence of Dex for 36 h.

 
If sequestration of hsp70 by unliganded steroid receptors leads to the induction of HSF activity, we might expect to overcome this induction upon elevation of hsp70 levels. As shown in Fig. 7Go, cotransfection of an hsp70 expression vector along with either GR, PR, or AR expression vectors led to a dramatic reduction in the extent of HSF activation from the HSE-linked reporter in COS-1 cells that were not treated with hormone. In addition, the induction of HSF activity by ligand-bound AR was overcome upon cotransfection with an hsp70 expression plasmid. As Western blot analysis demonstrated that GR expression was not affected by either hormone treatment or hsp70 DNA cotransfection of COS-1 cells (Fig. 8Go), the alleviation of unliganded GR-dependent HSF activation was not due to a reduction in receptor levels.



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Figure 7. Suppression of Steroid Receptor-Induced HSF Activation by Coexpression of hsp70

COS-1 cells were cotransfected with GR, PR, or AR expression plasmids; HSE-CAT; cytomegalovirus-ßgal; and an hsp70 expression plasmid (pßact-Hsp70). Cells were harvested after 36 h, lysed, and assayed for CAT activity. Where indicated, cells were treated with 1 µM dihydrotestosterone (DHT). Results are an average of at least three independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The HSF family of transcription factors regulates heat shock protein gene expression and is activated in response to a variety of environmental stresses (41, 42). The accumulation of unfolded proteins within stressed cells may provide one of the intracellular signals that mediate HSF activation (55). As chaperone proteins, such as members of the hsp70 family, are recruited to unfolded protein substrates in stressed cells (37), HSFs may be released from their association within an inhibitory complex, which could include hsp70 (44, 49). This putative mechanism is most likely not solely responsible for HSF activation, as alterations in HSF phosphorylation (48) or association with other regulatory factors (42) may also contribute to the regulation of HSF activity.

In this report we demonstrate that overexpression of steroid receptors in transiently transfected cells can lead to activation of HSF activity. The extent of receptor overexpression required to elicit this effect is difficult to assess on a single cell basis. However, DNA titration experiments suggest that the extent of receptor overexpression to generate measurable HSF activation probably exceeds that required to generate maximum hormonal induction from HRE-linked promoters by approximately 5-fold. This overexpression does not dramatically dampen the ability of functional receptors to maximally activate transcription, indicating that receptors and HSF are not competing for limiting cofactors or coactivators. Importantly, at the same relative level of receptor overexpression, HSF is not activated if receptors are ligand bound, with one notable exception (i.e. AR). Thus, the spare receptors that may be responsible for bringing about HSF activation are maintained in a conformation competent to bind hormone and are not grossly misfolded. Curiously, activation of HSF that results from overexpression of unliganded steroid receptors or the accumulation of unfolded proteins (55) appears to be mediated by a common mechanism (see below). Under our transfection conditions, the unliganded steroid receptors were ineffective as trans-activators from HRE-driven reporters, making it unlikely that HSF activation was brought about by the action of a receptor-induced gene product. This contention was supported by the observation that overexpression of the unliganded GR LBD as a ßgal chimera was sufficient to effectively activate HSF.

What mechanism is responsible for activation of HSF by unliganded steroid receptors? The LBD, which mediates HSF induction by GR, is known to associate with many proteins, particularly in the unliganded state (19). Thus, analogous to the mechanism thought to be responsible for HSF activation by unfolded proteins (50), this domain may sequester a negative regulator of HSF. Hsp70 has been postulated to be at least one such negative regulator of HSF activity, as activation of HSF brought about by the accumulation of unfolded proteins is abrogated upon overexpression of hsp70 (57). Our experiments also support a role for hsp70 in negative regulation of HSF activity, as activation by unliganded steroid receptors was likewise abrogated by overexpression of hsp70.

The implication from our findings is that HSF activation may result from the sequestration of members of the hsp70 family by unliganded steroid receptors. Although members of the hsp70 family of chaperone proteins have been found to be associated with steroid receptors in some in vivo studies (33, 35, 36), the specificity of such receptor/hsp70 complexes has been questioned (34). In vitro, the association between hsp70 and steroid receptors appears to be only transient and involved principally in the earliest stages of receptor maturation (21, 23). We would hypothesize that upon overexpression of unliganded receptors in transfected cells, such normally transient steroid receptor/hsp70 complexes may be stabilized to a certain extent. If this results in a significant depletion of available hsp70 pools, negative regulation of HSF may be overcome.

Regardless of the mechanism responsible for steroid receptor-mediated activation of HSF, we feel that caution must be applied toward the interpretation of any results regarding receptor function that are generated in cells that overexpress receptors. Such cells may be mobilizing a stress response, particularly if unliganded receptors are allowed to accumulate in excess of physiologically relevant levels. We would not expect that this level of receptor overexpression (i.e. ~4–5 fold) sequesters all of the cellular hsp70 pools. However, given the multitude of protein folding and trafficking pathways that use the chaperone activity of hsp70 (37), any significant departure from the putative equilibrium compartmentalization of these chaperones may exert profound effects on hsp70-regulated events.

A unique aspect of our manipulation of HSF activity was the fact that steroid receptor-dependent activation of HSF was reversible and completely abolished in all but one case by the binding of ligand. We, therefore, speculate that ligand binding might facilitate the release of GR-bound hsp70, which would then become available to participate in negative regulation of HSF activity. AR was unique in its ability, when overexpressed, to activate HSF activity in both the absence and presence of hormone. Both hormone-independent and -dependent activations of HSF activity by AR were reduced upon cotransfection with hsp70 DNA, suggesting that both unliganded and ligand-bound AR function analogously to sequester available hsp70. The physiological significance of this unique behavior of AR is unknown, but may become apparent once the role of molecular chaperones in the maturation of AR is established.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The rat GR, AR, and MR complementary DNA (cDNA) expression plasmids (provided by K. R. Yamamoto, University of California-San Francisco; R. Miesfeld, University of Arizona, Tucson, AZ; and D. Pearce, University of California-San Francisco, respectively) were in the same plasmid backbone (i.e. p6R) that possesses the adenovirus major late promoter to drive receptor expression (58). The chicken PRA cDNA (provided by B. O’Malley, Baylor College of Medicine, Houston, TX) (59) was cloned into the p6R vector. Plasmids encoding full-length and truncated GR, including VARO, VAN525, and VA407C, and the ßgal gene fused to full-length (VARL) and the HBD (VAL-HBD) of GR (provided by K. R. Yamamoto and D. Picard, University of Geneva, Geneva, Switzerland) have been described previously (60, 61). HSE-CAT (R. Kingston, Harvard University, Boston, MA) encodes a CAT reporter gene driven by a segment of the human HSP70 gene promoter (i.e. -34 to -1) that contains a potent HSE (54). G140CAT (provided by R. Kingston) is from the same parental plasmid as HSE-CAT, except it contains nucleotides -40 to -1 of the HSP70 promoter with a Gal4-binding site in place of the HSE (61A ). MMTV-CAT contains a CAT gene linked to MMTV LTR (62), whereas the pLC-Luc (provided by J. Dudley, University of Texas, Austin, TX) plasmid has the luciferase gene attached to mouse mammary tumor virus long terminal repeat (63). Unless otherwise noted, the mouse mammary tumor virus-CAT and pLC-Luc plasmids are referred to in the text as GRE (or HRE)-CAT and HRE-Luc, respectively. The hsp70 expression plasmid pßacHSP70 (provided by R. Morimoto, Northwestern University, Evanston, IL) was generated by cloning the human hsp70 cDNA fragment downstream of the human ß-actin promoter (64).

Cell Culture, Transfection, and Heat Shock Treatment
COS-1 monkey kidney fibroblasts were grown at 37 C in DMEM (Life Technologies, Bethesda, MD) supplemented with 10% FBS (Irving Scientific, Santa Ana, CA). Transient transfections were performed using the calcium phosphate precipitation method (65). For all transfections, a ßgal expression plasmid was cotransfected with CAT and Luc expression plasmids, so that transfection efficiency could be normalized from the comparison of ßgal activity (65). Each transfection was performed at least three or four times, and averages ± SD are indicated in all figures. Cells were incubated with the DNA-calcium phosphate precipitate for 6 h and then treated with 15% glycerol for 30 sec before feeding with DMEM supplemented with 10% charcoal-stripped FBS (Sigma Chemical Co., St. Louis, MO). Transfected cells were typically harvested after an additional 36- to 48-h incubation at 37 C. Dexamethasone, R5020, or dihydrotestosterone (Sigma) was added to a final concentration of 1 µM. For heat shock, cells were incubated at 43 C for 1 h, 36 h after transfection, and then recovered at 37 C for 3 h before harvesting.

CAT, Luciferase, and ßgal Assays
Thirty-six to 48 h after transfection, cells were harvested and resuspended in 0.25 M Tris-HCl, pH 7.8. Cells were then sonicated for 5 sec and centrifuged at 14,000 rpm for 5 min in a microfuge. The supernatant was used for enzyme activity assays. CAT assays were performed as described previously (66), and the resultant CAT activity was quantified using a FUJIX BS 200 PhosphoImager Analyzer (Fuji Photo Film, Tokyo, Japan). Luciferase and ßgal assays were performed as described previously (65).

Western Blotting Analysis
Western blot analysis was performed as described previously (36). Briefly, equivalent amounts of total protein in cell-free extracts were separated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA). Wild type and deletion mutant derivatives of GR were then detected using the BuGR2 anti-GR monoclonal antibody (67) and visualized using an enhanced chemiluminescence system (Amersham International, Aylesbury, UK).

Indirect Immunofluorescence
Cells grown on glass coverslips were fixed with cold methanol 36 h after transfection and processed for indirect immunofluorescence as described previously (68). GR and hsp70 were detected with a rabbit polyclonal anti-GR antibody (Affinity Bioreagents, Neshanic, NJ) and the SPA810 anti-hsp70 monoclonal antibody (Stressgen, Victoria, Canada), respectively. SPA810 specifically recognizes hsp70 and does not recognize hsc70. A tetramethyl rhodamine isothiocyanate-coupled antirabbit IgG (Boehringer Mannheim, Indianapolis, IN) and fluorescein isothiocyanate-coupled antimouse IgG antibody (Boehringer Mannheim) were used to visualize GR and hsp70, respectively.


    ACKNOWLEDGMENTS
 
Drs. J. Dudley, R. Kingston, R. Miesfeld, R. Morimoto, B. W. O’Malley, D. Pearce, D. Picard, and K. R. Yamamoto are thanked for their kind gifts of DNA. R. Morimoto is also thanked for his critical review of this manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Donald B. DeFranco, Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260.

This work was supported by USPHS Grant CA-43037 from the NIH.

Received for publication March 28, 1997. Revision received May 8, 1997. Accepted for publication May 10, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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