From the Burnham Institute, La Jolla, California 92037
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ABSTRACT |
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BAG-1 is a regulator of heat shock protein (Hsp)
70/Hsc70 family proteins that interacts with steroid hormone receptors.
The recently identified BAG-1 long (BAG-1L) protein, an isoform of BAG-1 that arises from translation initiation at a noncanonical CUG
codon, was co-immunoprecipitated with androgen receptors (AR) from
LNCaP prostate cancer cells and other cell lysates, whereas the shorter
originally identified BAG-1 and BAG-1M (RAP 46) proteins were not.
BAG-1L, but not BAG-1 or BAG-1M (RAP46), also markedly enhanced the
ability of AR to transactivate reporter gene plasmids containing an
androgen response element (ARE) in PC3 prostate cancer and other cell
lines. A C-terminal region deletion mutant of BAG-1L failed to
co-immunoprecipitate with AR and functioned as a trans-dominant
inhibitor of BAG-1L, impairing AR-induced transactivation of
ARE-containing reporter plasmids. In addition, BAG-1L significantly
reduced the concentrations of 5-dihydrotestosterone (DHT) required
for AR activity but did not induce ligand-independent transactivation.
BAG-1L also markedly improved the ability of AR to transactivate
reporter genes when cells were cultured with DHT in combination with
the anti-androgen cyproterone acetate. The effects of BAG-1L on AR
could not be explained by detectable alterations in the DHT-induced
translocation of AR from cytosol to nucleus, nor by BAG-1L-induced
increases in the amounts of AR protein. These findings implicate BAG-1L
in the regulation of AR function and may have relevance to mechanisms
of prostate cancer resistance to hormone-ablative and anti-androgen
therapy.
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INTRODUCTION |
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Prostate cancer is the most common malignancy in the United States and the second leading cause of cancer-related death among men (1). The normal prostate gland contains a two-layer epithelium composed of a population of small round stem cells called basal cells, which line the basement membrane, and a population of larger differentiated epithelial cells called secretory cells, which secrete a variety of proteins and other substances into the lumen of the gland (2, 3). Although both basal and secretory cells contain androgen receptors (AR)1 (4, 5), only the luminal secretory epithelial cells are dependent on steroid hormone for their function, growth, and survival (4). In the absence of testosterone or related androgens, which can serve as ligands for AR, the secretory cells undergo rapid programmed cell death (6). Current treatment for metastatic adenocarcinoma of the prostate is predicated on the cell death-inducing effects of anti-androgens and hormone-ablative measures, which reduce endogenous production of androgens. However, nearly all hormone-dependent prostate cancers eventually relapse as fatal hormone-independent disease (7).
Multiple, still largely unidentified mechanisms may account for the complete independence or reduced dependence of prostate cancers on androgens (reviewed in Refs. 8-10). AR gene deletion or sequestration of the AR from the nucleus to the cytoplasm have been described in some hormone-independent tumors, implying that genetic alterations associated with tumor progression can abrogate the necessity for AR in some cases. However, many tumors may rely on other strategies that allow cancer cells to grow in low concentrations of androgens, including AR gene amplification or overexpression (11, 12) and AR mutations that permit transactivation of target genes with little or no requirement for steroid hormones (9, 13). Since most hormone-insensitive prostate cancers still retain a wild-type AR, presumably alterations in the factors that control the levels of AR and its function appear to play a major role in resistance to anti-androgen and hormone-ablative therapies. Thus, a need exists to understand more about the molecular mechanisms that govern the activity of AR.
Steroid hormones mediate their effects by binding to specific intracellular receptors that act as hormone-dependent transcription factors. Upon binding steroid ligands, the AR undergo a conformational change, translocate to the nucleus, and bind to specific DNA sequences located near or in promoter regions of target genes. After binding DNA, the receptor interacts with components of the basal transcription machinery and sometimes sequence-specific transcription factors, resulting in positive or negative effects on gene transcription (14, 15). A number of proteins have been identified that associate with the inactive or hormone-bound hormone-receptor complexes, including several heat shock family proteins and various types of transcription co-activators (reviewed in Refs. 16 and 17). However, many details remain unclear as to the molecular mechanisms by which these proteins modulate the activities of steroid hormone receptors, and even less is known about whether alterations in their expression or function might contribute to the deregulation of steroid hormone responses in cancers.
Recently, an isoform of the human BAG-1 protein (known as RAP46 (see below)) (18, 19) has been reported to bind several steroid hormone receptors in vitro, including AR (19). It is unknown, however, what effect if any, BAG-1 has on the functions of these steroid-dependent transcription factors. Interestingly, BAG-1 and its alternative isoform RAP46 were recently shown to bind tightly to heat shock protein (Hsp) 70/Hsc70 family proteins and modulate their chaperone activity in vitro (20-22). In this regard, BAG-1 appears to function analogously to bacterial GrpE, stimulating the exchange of ADP for ATP on Hsc70 (22). It seems plausible therefore that BAG-1 could alter the bioactivity of AR and other steroid hormone receptors, given that many steroid hormone receptors are constitutively bound to heat shock proteins and that their hormone binding affinity and DNA binding activity can be increased in the presence of Hsp90 and Hsp70, respectively, under some circumstances (23-25).
The human and murine BAG-1 proteins are predicted to be amino acids 230 and 219 base pairs in length, respectively, based on cDNA cloning (18, 19, 26, 27). However, recently longer isoforms of the human and mouse BAG-1 proteins have been identified that can arise by translation initiation from noncanonical CUG codons located upstream and in frame with the originally described BAG-1 open reading frames (27, 28). This longer isoform of BAG-1 contains a basic motif resembling nuclear localization sequences and preferentially targets to nuclei. The human BAG-1 and BAG-1 long (BAG-1L) proteins migrate as ~36-kDa and 57-58-kDa proteins, respectively in SDS-PAGE experiments. In addition, a less abundant isoform of BAG-1 that migrates at ~46-53 kDa has been described and termed either BAG-1M or RAP46. The BAG-1M (RAP46) protein arises from translation initiation at an AUG codon located upstream of the usual start site in the BAG-1 mRNA (27).2 BAG-1M (RAP46) is produced in human, but not mouse, cells.2
Like BAG-1, the BAG-1L and BAG-1M proteins also bind to Hsp70 and Hsc70.2 BAG-1 is ubiquitously expressed, whereas BAG-1L is found preferentially in steroid hormone-dependent tissues such as testis, ovary, breast, and prostate.2 Although little is known about the expression of BAG-1 and BAG-1L in cancers, both proteins were detected by immunoblotting in 9 of 9 prostate cancer cell lines tested.2 In this report, we present evidence that the BAG-1L protein may play an important role in the AR signaling pathway, in that it can form complexes with AR and enhance the androgen-dependent transactivation function of this steroid hormone receptor.
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MATERIALS AND METHODS |
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Plasmids--
The plasmids pcDNA3-hu-BAG-1L and
pcDNA3-hu-BAG-1 were generated as described previously
(26).2 Translation of the longer form (BAG-1L) was forced
by mutation of the noncanonical in frame first CTG codon of the
cDNA to ATG.2 pcDNA3-BAG-1/BAG-1M lacks the
upstream CTG-containing region of the cDNA and encodes both the
originally described ~36-kDa form of BAG-1 and ~the 46-53-kDa
BAG-1M (RAP46) proteins. The plasmid pcDNA3-hu-BAG-1L (C)
(lacking the last 47 amino acids of the human BAG-1 protein) was
generated by polymerase chain reaction using pcDNA3-BAG-1L as a
template and the EcoRI-containing forward primer
5'-GGGAATTCAGTGCGGGCATGGCTC-3' together with the XhoI
containing reverse primer 5'-CCCTCGAGTTATGGCAGGATCAGTGTGTG-3'. After
digestion of the polymerase chain reaction product at the EcoRI and XhoI sites, the resulting
~0.8-kilobase pair fragments were subcloned into
EcoRI/XhoI-digested pcDNA3. pSG5-AR contains the cDNA for the wild-type AR (29). The reporter pLCI plasmid contains the full-length mouse mammary tumor virus long terminal repeat
sequence linked with the chloramphenicol acetyltransferase (CAT) gene
(29, 30). pCMV-p53wt expression vector, MYH101-81 containing the p53
response element and the TATA box from the BAX promoter, and
pUCSV3-CAT containing a SV40 early region promoter have been described
(31, 32).
Cell Culture--
The human prostate cancer cell lines LN-CaP
and PC3, the transformed human embryonal kidney 293, and the monkey
kidney COS7 cell lines were obtained from the American Type Culture
Collection (Rockville, MD). The ALVA31 human prostate cancer cell line
was generously provided by Dr. G. Miller (University of Colorado, Denver, CO). Cells were maintained in a humidified atmosphere with 5%
CO2 in RPMI 1640 or Dulbecco's modified Eagle's medium (293 and COS7) supplemented with 10% FCS, 3 mM glutamine,
100 units/ml penicillin, and 100 mg/ml streptomycin (Life Technologies, Inc.). Two days prior to experiments, cells were transferred into CT-FCS to reduce background levels of steroids.
5-Dihydro-testosterone (DHT) (Sigma) and cyproterone acetate (CPA)
(Sigma) were dissolved in dimethyl sulfoxide and added to the cultures
at a minimum dilutions of 0.0001% (v/v). Control cells received an
equivalent amount of solvent only.
Transfections and Enzyme Assays--
COS7, PC3, and 293T cells
at 60% confluency were transfected by a standard calcium phosphate
precipitate method (33). The medium was replaced with fresh
charcoal-treated fetal calf serum/Dulbecco's modified Eagle's medium
1 h before transfection. The total amount of plasmid DNA used was
normalized to 2.5 µg/well and 8 µg/plate for transfection in
12-well and 6-cm2 plates, respectively, by the addition of
empty plasmid. For reporter gene assays, 0.2 µg of a
-galactosidase expression plasmid pCMV-
gal was co-transfected
with the CAT reporter gene to normalize the transfection efficiency.
Cells were exposed to the precipitate for 5 h at 37 °C. For
COS7 and PC3 cells, a glycerol shock was applied. Cells were exposed to
15% glycerol in HBS buffer (25 mM HEPES pH 7.05, 0.75 mM Na2HPO4, 140 mM
NaCl) for 4 min. The glycerol was removed by washing three times with
PBS and replacement with fresh charcoal-treated fetal calf serum
medium. For 293 cells, the medium was replaced without applying a
shock.
Cell Extracts and Subcellular Fractionation-- For gene expression experiments, cells were washed two times in PBS and lysed in radioimmune precipitation buffer (35) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.28 trypsin inhibitory units/ml aprotinin, 50 µg/ml leupeptin, 1 µM benzamidine, 0.7 µg/ml pepstatin). For protein localization experiments, nuclear and nonnuclear fractions were prepared according to the method of Schreiber et al. (36). Briefly, cells were collected and washed two times with ice-cold PBS. Cell pellets were resuspended in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 2.5 mM dithiothreitol, protease inhibitors) and left on ice for 15 min prior to the addition of Nonidet P-40 to 0.6% (v/v) final concentration. After centrifugation, supernatants (cytoplasmic fractions) were collected, and the nuclear pellets were washed twice in the same buffer. Pellets were finally resuspended in buffer B (20 mM HEPES, pH 7.9, 400 mM NaCl, 25% glycerol, 0.1 mM EDTA, 0.1 mM EGTA, 2.5 mM dithiothreitol, protease inhibitors) and vigorously shaken for 10 min, and the postnuclear supernatants were cleared by centrifugation. Fractions were normalized based on the bicinchoninic acid method (Pierce) prior to SDS-PAGE/immunoblot assay.
Immunoblotting-- Aliquots containing 25 µg of protein were subjected to SDS-PAGE using 10% gels, followed by electrotransfer to Immobilon-P transfer membranes (Millipore Corp., Bedford, MA). Immunodetection was accomplished using 1:1000 (v/v) of anti-BAG-1 monoclonal antibody ascites (26, 37)2 or polyclonal rabbit AR antiserum (Clone AR N20, Santa Cruz Biotechnology, Inc., Santa Barbara, CA), followed by horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech). Detection was performed using an enhanced chemiluminescence detection method (ECL; Amersham Pharmacia Biotech) or the Vector SG substrate (Vector Laboratories, Burlingame, CA).
Co-immunoprecipitations-- LN-CaP cells (2 × 107) were collected at 70% confluency and lysed in HKMEN buffer (10 mM HEPES, pH 7.2, 142 mM KCl, 5 mM MgCl2, 2 mM EGTA, 0.2% Nonidet P-40, protease inhibitors). Cell lysates were passaged several times through a 301/2-gauge needle to disrupt the nuclei. Altnernatively, COS7 cells were transiently transfected with AR and BAG-1 expression plasmids, washed several times in PBS, and treated with 1 mM dimethyl-3,3'-dithiobispropionimadate (Pierce) in PBS for 30 min on ice. After extensive washing in ice-cold PBS, cells were lysed in radioimmune precipitation buffer containing protease inhibitors. Immunoprecipitations were performed in HKMEN either using the IgG1 anti-BAG-1 monoclonal KS6C8 (26)2 or a polyclonal rabbit AR antiserum (clone AR PA1-110 ABR, Inc.) conjugated to protein G-agarose (Zymed, San Francisco, CA). Control immunoprecipitations were performed using IgG1 or rabbit preimmune serum. Immune complexes were analyzed by SDS-PAGE/immunoblot assay using anti-BAG-1 monoclonal antibody with an enhanced chemiluminescence detection method.
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RESULTS |
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BAG-1L Enhances AR-mediated Transactivation of an Androgen Response Element (ARE)-containing Reporter Gene-- The human BAG-1M (RAP46) protein had been shown to bind to AR in vitro (19). We therefore asked whether BAG-1 family proteins can influence the transcriptional activity of this steroid hormone receptor. For these experiments, three different cell lines were transiently co-transfected with plasmids encoding various BAG-1 isoforms and AR, together with a ARE-containing CAT reporter plasmid. The cells were then cultured in the presence or absence of DHT. In the presence of hormone, BAG-1 family proteins increased the transcriptional activity of AR in a concentration-dependent manner, with the plasmid producing the BAG-1L protein displaying far more effect than the plasmid encoding for both BAG-1 and BAG-1M (Fig. 1). The extent of BAG-1L-mediated up-regulation of AR-induced transactivation varied among cell lines, with COS7 and PC3 demonstrating as much as ~ 5-fold increases when transfected with BAG-1L but 293T cells exhibiting only a modest effect. Immunoblot analysis confirmed the production of the BAG-1, BAG-1M, BAG-1L, and AR proteins in the transfected cells and demonstrated production of similar amounts of BAG-1 and BAG-1M compared with BAG-1L (see below for examples). Thus, differences in the relative amounts of BAG-1, BAG-1M, and BAG-1L proteins produced could not account for the greater potency of BAG-1L.
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BAG-1L Decreases the Response of AR to Anti-androgen CPA-- The observation that BAG-1L increased the sensitivity of AR to its ligand DHT (Fig. 2) prompted us to explore the effects of BAG-1L on the suppression of AR transactivity by the anti-androgen cyproterone acetate. For these experiments, AR and ARE-CAT were transfected into COS7 cells with either pcDNA3 control DNA or an equivalent amount of pcDNA3BAG-1L. The cells were treated ~1.5 days later with 1 nM DHT alone or in combination with various concentrations of CPA. Relative CAT activity was then measured 12-14 h later. As shown in Fig. 4, CPA reduced in a concentration-dependent manner the DHT-induced transactivation of the ARE-CAT reporter gene plasmid in both control and BAG-1L-transfected COS7 cells. However, because AR-mediated reporter gene transactivation started at higher levels in BAG-1L transfectants, approximately 2 log higher concentrations of CPA androgens were generally required to reduce reporter gene activity to levels comparable with control-transfected cells (Fig. 4).
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In Vivo Binding of BAG-1L to the AR-- Although BAG-1M (RAP46) has been reported to bind AR in vitro, the interaction of these proteins has not been demonstrated previously in cells. Co-immunoprecipitation assays were therefore performed using lysates prepared from untransfected LN-CaP cells, which constitutively express high levels of the BAG-1, BAG-1M, BAG-1L, and AR proteins (39).2 A polyclonal anti-AR antiserum or a preimmune control serum was employed for immunoprecipitations, and the resulting immune complexes were subjected to SDS-PAGE/immunoblot analysis using the anti-BAG-1 monoclonal antibody KS6C8. As a control, BAG-1 proteins were also immunoprecipitated using the same anti-BAG-1 monoclonal antibody. Alternatively, an IgG1 control antibody was employed to confirm specificity.
As shown in Fig. 5, the BAG-1L protein was readily detected in association with anti-AR immune complexes (lane 5). In contrast, the BAG-1 and BAG-1M proteins did not co-immunoprecipitate with AR but were found in anti-BAG-1 immune complexes, confirming their presence in LN-CaP cells under these conditions. The specificity of these results was confirmed by the absence of BAG-1 family and AR proteins in immune complexes prepared using IgG1 control monoclonal antibody or the preimmune control serum. Although BAG-1L could be detected in AR-containing immune complexes, the reciprocal experiment involving the use of anti-BAG-1 antibody in attempts to co-immunoprecipitate AR proved unsuccessful. Additional experiments suggested that this was due to antibody-induced disruption of BAG-1L interactions with AR (data not shown). Attempts to determine whether BAG-1L can associate with AR in the absence of steroid hormone have been hampered by the rapid turnover of unliganded AR, resulting in lower levels of AR and making quantitative comparisons difficult. However, thus far, we have detected association of BAG-1L with AR only when androgens have been present.
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The C-terminal Hsc70-binding Domain of BAG-1L Is Required for
Interactions with AR--
Previously, we showed that the last 47 amino
acids of the BAG-1 protein are required for binding to the ATPase
domain of Hsc70 (20). We therefore compared a mutant of BAG-1L lacking
this carboxyl-terminal domain, BAG-1L (C), with the wild-type BAG-1L protein. Association with AR was examined after treatment with the
reversible chemical cross-linker dimethyl-3,3'-dithiobispropionimadate in total cell lysates derived from transiently transfected COS7 cells.
As shown in Fig. 6A, anti-AR
immunoprecipitates contained BAG-1L protein, as determined by
immunoblot analysis using anti-BAG-1 antibody. In contrast, the BAG-1L
(
C) protein was not detected in anti-AR immune complexes. The BAG-1
and BAG-1M isoforms of BAG-1 also did not co-immunoprecipitate with AR
(Fig. 6A; lanes 7-9). Successful production of
the BAG-1L, BAG-1L (
C), BAG-1, and BAG-1M proteins was confirmed by
immunoblot analysis of whole cell lysates derived from transfected COS7
cells (Fig. 6A). We conclude therefore that the C-terminal
domain of BAG-1L is required for complex formation with AR.
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Effects of BAG-1L on the Subcellular Localization of the AR-- To investigate the mechanisms underlying the stimulatory effect of BAG-1L on AR transcriptional activity, we ascertained the effects of BAG-1L overexpression on DHT-induced nuclear translocation and stabilization of this steroid hormone receptor. COS7 monkey kidney cells were transiently transfected with plasmids encoding BAG-1/BAG-1M, BAG-1L, and AR at the same concentrations used for reporter gene assays. The cells were then stimulated with 0, 0.1, or 10 nM DHT, and the relative amounts of AR protein in the cytosol and nucleus were determined by immunoblot analysis of nuclear and nonnuclear fractions. As shown in Fig. 7, translocation of AR to the nucleus was induced in a concentration-dependent manner by DHT but was unaffected by overexpression of either BAG-1/BAG-1M or BAG-1L. At these same concentrations of DHT, however, BAG-1L promoted marked increases in AR-dependent transactivation of ARE-CAT (see above). Note also that the total levels of AR were not different when comparing cells that had been transfected with BAG-1L, BAG-1/BAG-1M, or Neo control plasmids (Fig. 7). DHT also did not appear to alter the relative proportions of the BAG-1, BAG-1M, and BAG-1L proteins present within the nuclear and nonnuclear compartments.
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DISCUSSION |
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The data presented here provide the first evidence that a recently identified longer isoform of the human BAG-1 protein (BAG-1L) can modulate the function of a steroid hormone receptor. In particular, we found that the BAG-1L protein can be co-immunoprecipitated with AR and significantly enhances AR-induced transactivation of a reporter gene, whereas the shorter BAG-1 and BAG-1M isoforms of the protein did not. Thus, despite evidence that the human BAG-1M (RAP46) protein can bind to AR and several other steroid hormone receptors in vitro (19), only the long form appears to physiologically interact with AR in cells and to regulate its function.
One factor that could contribute to the preferential binding of BAG-1L to AR in cells is that this longer isoform of BAG-1 contains a nucleoplasmin-like nuclear localization sequence within the NH2-unique domain, which is missing from the shorter BAG-1 and BAG-1M (RAP46) protein.2 In previous studies, we observed that BAG-1L targets preferentially but not exclusively to nucleus when transfected in COS7 and other cell lines, whereas BAG-1 and BAG-1M had a greater tendency to reside in the cytosol.2 This is also true for the LN-CaP cells used in this study for co-immunoprecipitations, which contain all three isoforms of BAG-1, i.e. BAG-1, BAG-1M, and BAG-1L (data not shown). Since in the presence of androgen the AR resides almost exclusively in the nucleus (40), it is conceivable that under these conditions AR interacts with nuclear and not cytoplasmic proteins. Thus, the higher nuclear levels of BAG-1L compared with BAG-1 and BAG-1M may be largely responsible for its physical and functional interactions with AR protein complexes in cells.
Alternatively, another explanation for the observation that BAG-1L but
not BAG-1 or BAG-1M (RAP46) was detected in association with AR in
cells could be that the unique N-terminal region of the BAG-1L protein
is required for binding to AR under physiological conditions. In this
regard, it should be noted that the interaction of the BAG-1M (RAP46)
protein with AR and other steroid hormone receptors has only been
demonstrated in vitro and only then after treatment of
steroid hormone receptor complexes with high salt at elevated
temperature or with urea-containing solutions, conditions that could
cause protein unfolding. In contrast, conformations of BAG-1 that are
competent to bind AR complexes in vivo may only be achieved
when the N-terminal unique region of BAG-1L is present. The N-terminal
unique domain within BAG-1L could also directly bind to AR. However,
clearly the N-terminal domain of BAG-1L is insufficient for AR binding,
since deletion of the C-terminal last 47 amino acids abolished
interactions BAG-1L with AR in cells. The failure of the BAG-1L (C)
protein to form complexes with AR was not due to altered subcellular
localization of this protein compared with wild-type BAG-1L (data not
presented).
The mechanism by which BAG-1L enhances the function of the AR remains to be determined. Clearly, the ability of BAG-1 proteins to bind to and modulate the function of Hsp70/Hsc70 family molecular chaperones by increasing ADP/ATP exchange and facilitating peptide release may provide some clues. As shown here, a carboxyl deletion mutant of BAG-1L lacking the last 47 amino acids, which are required for Hsc70 binding (20), was unable to form complexes with AR. This observation therefore suggests that Hsc70 bridges BAG-1L to AR, as has been proposed for its interactions with many other proteins (21). It is known that at least three members of the Hsp family, namely Hsp90, Hsp70, and Hsp56, are associated with the inactive forms of several steroid hormone receptors in the cytoplasm and may be important for maintaining the stability of these proteins in the absence of ligand and inducing conformations that are competent to bind steroid hormone ligands. Hsp70 has also been detected in the nucleus in association with receptor-DNA complexes where it putatively increases DNA binding affinity (23, 25). Thus, BAG-1L may alter AR interactions with molecular chaperones in ways that modulate the conformation of this steroid hormone receptor and enhance its responses to steroid ligands, e.g. by stabilizing hormone binding, increasing the affinity of AR interactions with DNA target sequences, or facilitating association with coactivator proteins (39). BAG-1L, however, did not appear to detectably increase the proportion of AR that translocates into the nucleus after the addition of DHT or to cause elevations in AR protein levels as might occur if BAG-1L stimulated nuclear translocation or prolonged the half-life of this protein. Also, expression of BAG-1L did not appear to increase the amount of Hsc70 or Hsp70 that would be co-immunoprecipitated with AR (not shown). These mechanisms, therefore, seem not to be involved in the potentiation of AR function by BAG-1L.
Alternatively, BAG-1L could conceivably bind directly to AR and exert
its potentiating effect on AR independently of Hsp70. An
Hsp70-independent mechanism of action is suggested by at least two
observations. First, androgen has been reported to induce dissociation
of not only Hsp90, but also Hsc70 from AR in concert with translocation
of hormone-bound receptor into the nucleus (39). Since BAG-1L forms
complexes with AR in the presence of hormone, this observation implies
that BAG-1L may be able to interact with and modulate AR function
within the nucleus after dissociation of Hsc70. One notable caveat,
however, is that Hsp70 reportedly remains associated with estrogen and
progesterone receptors while bound to their target DNA elements in the
nucleus (16, 23, 25). Thus, unlike Hsp90, the Hsc70 family molecular
chaperones may not always dissociate from nuclear hormone receptors
upon binding ligand. Indeed, we have been able to co-immunoprecipitate at least small amounts of Hsc70/Hsp70 with AR in cells cultured with
androgens.3 Second, a
C-terminal deletion mutant of BAG-1L that fails to bind Hsc70
functioned as a trans-dominant inhibitor of BAG-1L and reduced
AR-mediated transactivation, thus suggesting the possibility of a
Hsc70-independent mechanism. Although the reason why the BAG-1L (C)
mutant protein interferes with AR function requires further
exploration, at least two possibilities can be considered. First, the
BAG-1L (
C) protein may form dysfunctional complexes with endogenous
wild-type BAG-1L, abrogating its effects on AR. However, biophysical
characterization of the BAG-1 protein strongly suggests it is a
monomer, unlike the functionally similar GrpE protein of prokaryotes,
which is known to be a dimer (41). Thus, trans-dominant inhibition of
endogenous BAG-1L may not explain why the BAG-1L (
C) protein
inhibits AR function. Second, if BAG-1L normally bridges AR to other
proteins such as transcriptional co-activators (reviewed in Ref. 28)
and if the N-terminal unique domain of BAG-1L is necessary for this
function, then the BAG-1L (
C) protein could theoretically sequester
a co-factor essential for AR-mediated transcriptional activation. In
this case, the BAG-1L (
C) protein, which does not bind to AR, would
presumably prevent this hypothetical co-factor from binding to
endogenous wild-type BAG-1L·AR complexes.
The role of BAG-1L in the fetal development of male reproductive organs
and in the pathogenesis of prostate cancer remains to be established.
In contrast to most androgen-unresponsive tissues, testes and the
normal prostate gland, as well as 9 of 9 prostate cancer lines thus far
tested, have been shown to express BAG-1L, in addition to the shorter
ubiquitously expressed BAG-1 protein (20). The observation that BAG-1L
significantly reduced the net suppressive effects of an anti-androgen
on AR-mediated transactivation raises the possibility that
overexpression of BAG-1L could provide a selective growth advantage for
some prostate cancers during hormone ablation therapy. Although much
remains to be learned about the specific mechanisms involved, the
observations that (i) BAG-1L markedly enhances
androgen-dependent transactivation by AR, (ii) BAG-1L
reduces the efficacy of anti-androgens with respect to their
suppression of AR-reporter gene transactivation, and (iii) BAG-1L
(C) antagonizes AR-mediated transactivation all suggest that further
studies of BAG-1 and BAG-1L expression and function in normal and
malignant prostate and other androgen-responsive tissues are
warranted.
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ACKNOWLEDGEMENTS |
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We thank Dr. Chawnshang Chang for the AR and ARE-CAT plasmids, Drs. X.-K. Zhang and D. Knee for helpful discussions, and H. Gallant and T. Potter for manuscript preparation.
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FOOTNOTES |
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* This work was supported by NCI, National Institutes of Health, Grant CA-67329, the Swiss Science National Foundation, and Cancer Research Switzerland Grant BIL KFS 19891995.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Scientific Director,
The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-646-3140; Fax: 619-646-3194; E-mail:
jreed{at}burnham-inst.org.
1
The abbreviations used are: AR, androgen
receptor(s); ARE, androgen response element; DHT,
5-dihydrotestosterone; CAT, chloramphenicol acetyltransferase; Hsp,
heat shock protein; PAGE, polyacrylamide gel electrophoresis; FCS,
fetal calf serum; CPA, cyproterone acetate; PBS, phosphate-buffered
saline; CMV, cytomegalovirus.
2 S. Takayama, S. Krajewski, M. Krajewska, S. Kitada, J. Zapata, K. Kochel, D. Knee, D. Scudiero, G. Tudor, G. J. Miller, M. Yamada, T. Miyashita, and J. Reed, submitted for publication.
3 B. A. Froesch, S. Takayama, and J. C. Reed, unpublished data.
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REFERENCES |
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