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Originally published In Press as doi:10.1074/jbc.M401317200 on April 29, 2004

J. Biol. Chem., Vol. 279, Issue 29, 29944-29951, July 16, 2004
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An Hsp27-related, Dominant-negative-acting Intracellular Estradiol-binding Protein*

Hong Chen{ddagger}, Martin Hewison§, Bing Hu¶, Manju Sharma||, Zijie Sun||, and John S. Adams{ddagger}**

From the {ddagger}Division of Endocrinology, Diabetes, and Metabolism and the Department of Pathology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California 90048, the §Division of Medical Sciences, the University of Birmingham, Queen Elizabeth Medical Center, Birmingham, B15 2TH, United Kingdom, and the ||Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305

Received for publication, February 6, 2004 , and in revised form, April 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
New World primates (NWPs) exhibit a compensated form of resistance to gonadal steroid hormones. We demonstrated recently that estrogen resistance in NWP cells was associated with the overexpression of two proteins, a nonreceptor-related, dominant-negative-acting estrogen response element (ERE)-binding protein (ERE-BP) and an intracellular estradiol-binding protein (IEBP). Based on the N-terminal sequences of tryptic fragments of IEBP isolated from a 17{beta}-estradiol (E2) affinity column we cloned a full-length cDNA for IEBP from the estrogen-resistant NWP cell line, B95-8. Subsequent sequence analysis revealed 87% sequence identity between the deduced peptide for IEBP and human Hsp27. When hormone-responsive, wild-type Old World primate (OWP) cells were transiently transfected with IEBP cDNA, E2-directed ERE reporter luciferase activity was reduced by 50% compared with vector only-transfected OWP cells (p < 0.0018). When IEBP and ERE-BP were cotransfected, ERE promoter-reporter activity was reduced by a further 60% (p < 0.0001). Electrophoresis mobility shift analyses showed that IEBP neither bound to ERE nor competed with the estrogen receptor (ER) for binding to ERE. However, there was evidence of protein-protein interaction of IEBP and ER{alpha}; IEBP was coimmunoprecipitated with anti-ER{alpha} antibody in wild-type cells stably transfected with IEBP. A specific interaction between ER{alpha} and IEBP was confirmed in glutathione S-transferase pull-down and yeast two-hybrid assays. Data indicate that the Hsp27-related IEBP interacts with the ligand binding domain of the ER{alpha}. In summary, by inhibiting the ER{alpha}-E2 interaction, IEBP acts to squelch ER{alpha}-directed ERE-regulated transactivation and promote estrogen resistance in NWP cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Compared with Old World primates (OWPs),1 including man, New World primates (NWPs) represent an evolutionarily distinct infraorder of primates confined to the South and Central American subcontinents. A central point of distinction between NWPs and OWPs resides in the fact that NWPs display relative resistance to adrenal, gonadal, and vitamin D sterol/steroid hormones (1–11). The precise mechanism for this remains unclear but does not involve aberrant expression of nuclear receptors for specific steroid hormones (12, 13), the principal cause of hormone resistance in humans (14). Instead, hormone resistance in NWP cells appears to be the result of epigenetic factors, which result either in low affinity receptorsteroid binding kinetics or attenuation of receptor-DNA interaction. For example, studies of glucocorticoid resistance in NWP cells have shown increased expression of the heat shock protein (Hsp)90-associated FK506-binding immunophilin FKBP51, which inhibits ligand binding to glucocorticoid receptors (GR) (15). By contrast, vitamin D resistance in NWPs appears to be caused by aberrant expression of a dominant-negative-acting vitamin D response element-binding protein (VDRE-BP), the protein being homologous to human heterogeneous nuclear ribonucleoprotein A (hnRNPA) (16). Estrogen resistance in NWP is associated with the overexpression of two proteins, a nonreceptor-related estrogen response element (ERE)-binding protein (ERE-BP) (17, 18) and an intracellular estradiol-binding protein (IEBP) (19, 20). ERE-BP is a member of hnRNPC-like family and acts to squelch estrogen receptor (ER)-ERE transactivation by competing with the ER for binding to ERE (16, 17). IEBP is a member of the Hsp27 family and is overexpressed in female and male estrogen-resistant NWP cells and tissue (19).

In recent studies we have sought to clarify the molecular function of these intracellular- and response element-binding proteins. Although ERE-BP and its vitamin D counterpart, VDRE-BP, appear to fulfill dominant-negative, cis-acting functions, the actions of IEBP are less clear. With regard to IEBP we considered two countervailing hypotheses to explain the function of this protein. One postulate held that IEBP competes with the ER for binding of its ligand 17{beta}-estradiol (E2) thus squelching E2-ER-directed signaling. The opposing hypothesis held that IEBP aids in delivery of E2 to the ER and promotes estrogen action in the cell. The latter view holds true with the intracellular vitamin D-binding protein (IDBP) and the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), where IDBP overexpression increases 1,25(OH)2D3-VDR-directed transactivation (21). To determine whether IEBP acts in a fashion analogous to IDBP we have cloned the candidate IEBP cDNA from estrogen-resistant NWP cells. In functional studies we demonstrate that, unlike its vitamin D counterpart, IEBP acts to squelch E2-ER-mediated transcription. Furthermore, we demonstrate that IEBP can cooperate in a dominant-negative mode with the ERE-BP by acting as an intracellular repository for E2 and/or by association with the ligand binding domain of the ER, thereby blocking its transactivating potential. In either case, the net effect is to interrupt ER-ER homodimerization, thereby legislating hormone resistance. These data suggest that the regulation of E2-ER signaling by IEBP is an important prereceptor determinant of estrogen action.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Postnuclear Extracts—All cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD). The estrogen-resistant NWP cell line B95-8, derived from the hormone-resistant common marmoset (Callithrix jacchus), was maintained in RPMI 1640 medium. The estrogen-responsive OWP breast cell line 6299, derived from a rhesus monkey (Macaca mulatta), was maintained in Dulbecco's modified Eagle's medium (Irvine Scientific Irvine, CA). Cell culture conditions and postnuclear cell extraction were performed as described previously (22). In some experiments, confluent cultures were preincubated up to 48 h in medium containing 10 nM E2 prior to harvest and preparation of extracts.

Molecular Cloning of the IEBP—Based on the amino acid sequence of the N-terminal tryptic peptide (RVPFSL) of IEBP, we designed an IEBP-specific sense oligonucleotide primer and its reversion antisense primer for 5'- and 3'-RACE. Poly(A)+ RNA (2.5 µg) from B95-8 cells was used as the template to generate the 5'- and 3'-ends of the IEBP cDNA with the Marathon cDNA amplification kit (Clontech Laboratories Inc., Palo Alto, CA). Second-strand cDNA synthesis and adapter ligation were performed as instructed in the enclosed manual. The adapterligated cDNA was then used as template for annealing adapter- and IEBP-specific primers for the RACE reaction: 5'-CGCAGGAGCGAGAAGGGGACGCG-3' and 5'-CGCGTCCCCTTCTCGCTCCTGCG-3' for the 5'- and 3'-RACE of IEBP, respectively. A cDNA for the IEBP was generated by end-to-end amplification using specific 5'- and 3'-primers. The amplified products were then subcloned into the pcDNA3.1/V5/His/TOPO expression vector and sequenced by the Cedars-Sinai Medical Center Sequencing Core Facility using dye terminator cycle sequence reactions on ABI automated sequencers.

Transient Transfections—5 x 105 estrogen-resistant NWP B95-8 or estrogen-responsive OWP breast 6299 cells were seeded into 6-well plates in phenol red-free medium containing 10% charcoal-stripped fetal calf serum and allowed to proliferate to 80–90% confluence. Transfections were performed in triplicate with the following combinations of DNA preparations to a maximum final concentration of 20 µg DNA/ml in LipoTAXI solution (Stratagene, La Jolla, CA): (i) 5.5 µg of ERE-luciferase reporter plasmid; (ii) 0.5 µg of ER{alpha} expression plasmid (pRShER); (iii) 5.0 µg of IEBP or ERE-BP plasmid (in cDNA3.1/V5/His/TOPO vector); (iv) 5.0 µg of {beta}-galactosidase expression construct as internal control; and (v) pGEM-3z vector DNA as carrier (Promega, Madison, MI). An equal volume of 20% fetal calf serum-supplemented, antibiotic-free medium was added to each well 5 h after transfection followed by the addition of 10 nM E2. After an additional 48 h at 37°C, the cells were lysed, and luciferase and {beta}-galactosidase activities were measured.

Generation of Cell Lines Overexpressing the IEBP—E2-responsive OWP 6299 breast cells were incubated with 5.0 µg of pcDNA3.1/V5/His/TOPO IEBP plasmid in LipoTAXI solution for 5 h followed by the addition of an equal volume of 20% fetal calf serum-supplemented medium. After incubation overnight, cells were split (1:10 ratio) and incubated with fresh medium containing 500 µg/ml Geneticin-selective antibiotic G418 sulfate (Invitrogen). This medium was replaced every 3–4 days until stable colonies formed. Single colonies were picked, transferred into a new dish, and incubated with medium containing the selection antibiotic G418 until confluence was attained.

Ligand Binding Analysis—Specific [3H]E2 binding was measured in postnuclear extracts of vector alone and the three IEBP stably transfected cell lines. Briefly, postnuclear extracts isolated as described above were reconstituted in NaCl-containing ETD buffer (pH 8.0) to achieve a final salt concentration of 0.5 M NaCl and incubated overnight at 4 °C with 4 nM [3H]E2 in the presence or absence of 0.1–100 nM unlabeled competitive ligand. Protein-bound [3H]E2 was separated from unbound sterol by incubation with dextran-coated charcoal. Experiments were conducted in triplicate.

Western Blot Analysis—Denatured cell extracts were subjected to electrophoresis using 4–12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes as described previously (16). The membranes were blocked with 5% nonfat dry milk for 1 h and then incubated with monoclonal anti-human Hsp27 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 2 h and with horseradish peroxidase-conjugated secondary antibody for another 1 h prior to detection of antibody-reactive proteins with chemiluminescence reagent (ECL, Amersham Biosciences).

Co-immunoprecipitation—Cells were washed with phosphate-buffered saline twice and lysed with radioimmune precipitation assay buffer (1x phosphate-buffered saline containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1 mM phenylmethylsulfonyl fluoride, 30 µl/ml of aprotinin, and 10 mM sodium orthovanadate (Sigma) by incubation on ice for 10 min. The resulting lysates were then disrupted by repeated aspiration through a 23-gauge needle, and cell supernatants were obtained by centrifugation (14,000 x g for 10 min). Aliquots of supernatant (containing 50 µg of protein each) were then incubated with anti-ER{alpha} or anti-Hsp27 antibody overnight at 4 °C. 20 µl of protein A/G-agarose (Santa Cruz) was added and incubated at 4 °C for another 1 h. The protein mixtures were then washed by repeated centrifugation in radioimmune precipitation assay buffer (four times) and phosphate-buffered saline (once). The resulting pellet was resuspended in 2x SDS sample buffer. After boiling, samples were analyzed by 4–20% SDS-PAGE, and separated proteins were transferred to nitrocellulose membranes. Western blot analyses were then carried out using anti-ER{alpha} and anti-Hsp27 antibodies and visualized by ECL.

Yeast Two-hybrid Screening—The full-length ER{alpha} cDNA was amplified using the oligonucleotides 5'-GGGGAATTCCATATGACCATGACCCTCCACACCAAAGCATCAGGG-3' and 5'-GCCAGGGGGATCCTCAGACTGTGGCAGGGAAACCCTC-3'. The ER{alpha} cDNA was cloned into the NdeI and BamHI site of GAL4 DNA binding domain vector (GAL4 DNA-BD/ER). The full-length human Hsp27 cDNA was amplified using oligonucleotides 5'-GCCGAATTCGCCCAGCGCCCCGCACTTTT-3' and 5'-CCCCTCGAGGGTGGTTGCTTTGAACTTTATTTGAG-3'. The IEBP cDNA was cloned into EcoRI and XhoI site of GAL4 DNA activation domain vector. GAL4 DNA-BD/ER was cotransformed with the GAL4 DNA-AD/Hsp27 plasmid using a Yeast Transformation System 2 kit (Clontech, Palo Alto, CA) according to the manufacturer's instructions; some of the plates were treated with water (control), 10–100 nM E2, or 10–100 nM tamoxifen.

GST Pull-down Assay—A GST fusion protein with the ligand-binding domain of ER{alpha} (residues 246–595) was expressed in Escherichia coli strain DH5{alpha} and purified by glutathione-Sepharose beads according to the manufacturer's instructions (Amersham Biosciences). Postnuclear extracts were applied to the GST beads and incubated for 1 h. The loaded GST-extract mixture was then washed repetitively (five times) with phosphate-buffered saline containing 5 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride and resuspended in 2 x SDS sample buffer and boiled for 5 min. Denatured protein were resolved on 4–20% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, probed with appropriate antibody (Santa Cruz Biotechnology), and visualized by ECL.

Statistics—Where indicated, experimental means were compared statistically using an unpaired Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning and Sequence Analysis of IEBP cDNA—In previous studies we identified and characterized tryptic fragments of an E2 affinity column-purified protein that corresponded to the putative IEBP in NWP B95-8 cells (19). To clone the cDNA corresponding to this protein we used degenerate oligonucleotides corresponding to the amino acid sequence of tryptic peptides (see "Experimental Procedures") to carry out RACE generation of candidate cDNAs. Analysis of the resulting deduced amino acid sequence for IEBP (Fig. 1) revealed 87% sequence identity to the human Hsp27 and 40 and 44% sequence identity to small Hsp-related human {alpha}-crystallin A and B, respectively (Fig. 2; 23–25). The greatest degree of sequence identity among the NWP IEBP, human Hsp27, and the {alpha}-crystallins (Fig. 2) was observed in two places (shaded areas in Fig. 1), at residue positions 26–34 near the N terminus and in the central portions, {alpha}-crystallin cores (residue positions 101–138) of the molecule. The greatest sequence identity (94%) between IEBP and Hsp27 was found in the middle of the two molecules. This region of IEBP and Hsp27 bears (i) a portion of a sequence homologous to the ATP binding/ATPase domain characteristic of all heat shock proteins (26), residue positions 65–116, underlined, Fig. 1); (ii) a region of sequence homology (residue positions 147–181, underlined, Fig. 1) that retains 18–35% sequence identity with the proximal reaches of the substrate binding domains of human Hsp90 and Hsp70 families (26); and (iii) a 16-amino acid-long region from residue positions 107 to 122 (superior dotted line, Fig. 1), which exhibits 38% sequence identity to the ligand binding domain of the human ER{alpha} (27, 28). It should be noted, however, that there was no LXXLL motif in this region of either IEBP or Hsp27 prototypical of a steroid hormone receptor (29). Furthermore, no tryptic peptides bearing a high degree of sequence identity with any of the known ER gene products or their naturally occurring, alternatively spliced derivatives (30, 31) were identified from E2 affinity chromatography. These data suggest that the E2 affinity support was interactive with the ER{alpha}-like ligand binding domain of the IEBP.



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  		FIG. 1.
The NWP IEBP shares homology with human Hsp27 and the {alpha}-cystallins A and B. The full-length, deduced amino acid sequence for IEBP is compared with human Hsp27 and the human {alpha}-crystallins A (hCrysA) and B (hCrysB). The shaded areas depict regions of sequence identity among all four molecules. The underlined regions denote areas of sequence homology with the ATP and substrate binding domains of human Hsp90 and Hsp70 families.

 



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  		FIG. 2.
Degree of sequence identity among members of the sHsp family. The high degree of sequence identity between IEBP and human Hsp27 suggests that IEBP is the NWP homolog of Hsp27.

 
Functional Characterization—As stated above, purification of IEBP using E2 affinity chromatography confirmed its capacity for E2 binding (19) but did not elucidate the functional relevance of this protein in NWP cells. Therefore, experiments were carried out to clarify whether the transient overexpression of IEBP in NWP cells antagonized, facilitated, or did nothing to estrogen-induced transactivation (Fig. 3). As expected, estrogen-responsive OWP cells demonstrated a significant increase in ERE-directed reporter activity when exposed to an ER-saturating concentration of E2. When estrogen-responsive OWP cells were transiently cotransfected with IEBP cDNA as well as an ERE-promoter-reporter construct, ERE-directed luciferase activity was reduced 50% compared with vector alone-transfected OWP cells. Despite the down-regulation of reporter activity under the influence IEBP expression, transfected cells retained some responsiveness to E2. Transient overexpression of the known, NWP dominant-negative-acting ERE-BP (18) in estrogen-responsive OWP cells also suppressed ERE-mediated transcription. When IEBP and ERE-BP were cotransfected there was an additive decrease in ERE-luciferase reporter activity to only 11% of basal activity.



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  		FIG. 3.
Transient expression of IEBP and ERE-BP squelch ERE-directed transactivation. Expression constructs containing cDNA for the NWP ERE-BP and/or NWP IEBP were transiently cotransfected with an estrogen-responsive luciferase reporter plasmid into the ER{alpha}-positive human MCF-7 breast cancer cell line, in the absence or presence of 10 nM E2. Data are the mean of triplicate determinations of luciferase activity. Significant differences among sample means are noted.

 
Similar results were also obtained after stable transfection of IEBP into OWP cells (Fig. 4A); increased expression of IEBP in each of the three subclones was confirmed by Western blot analyses using an anti-human Hsp27 antibody (data not shown). Promoter-reporter data showed that ERE-luciferase activity was decreased 2–3-fold in the presence of constitutively overexpressed IEBP compared with wild-type, vector alone-transfected cells (left panel, Fig. 4A). As with the transient transfectants (Fig. 3), this effect was only partially abrogated by pretreatment with E2 (right panel, Fig. 4A). Because they represented a stable resource for IEBP, the IEBP stable transfectant variants were useful models for assessment of specific E2 binding by IEBP. Data presented in Fig. 4B revealed variable but significantly increased E2 binding in postnuclear extracts of all three IEBP stable transfected OWP clones compared with control cells, with maximal displacement being greatest in the clone 1 cells, which exhibited the most profound squelching of basal and E2-stimulated ERE-luciferase activity. In each case the concentration of E2-required to achieve half-maximal displacement of [3H]E2 from IEBP was between 0.1 and 1.0 nM (Fig. 4B).



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  		FIG. 4.
Stable overexpression of IEBP suppresses ERE-directed transactivation and is associated with increased cytoplasmic binding of E2. A, effect of IEBP on ERE promoter-reporter luciferase activity in wild-type 6299 breast cancer cells from an OWP host stably transfected with vector alone (open bars) and three different subclones of cells stably transfected with the full-length IEBP cDNA in the presence or absence of 10 nM E2 (closed bars). Data are the mean of triplicate determinations of luciferase activity. * indicates statistical difference from vector alone transfectants, p < 0.001. B, displacement of [3H]E2 in postnuclear extracts of IEBP stably transfectant cell lines. Data are the mean of triplicate determinations of percent maximal [3H]E2 displaced by increasing doses of E2 (0.1–100 nM) in vector only controls and the three IEBP stable transfectant cell lines.

 
Evidence for an Intracellular ER{alpha}-IEBP (Hsp27) Interaction—Analysis of both transient and stable transfectants indicated that IEBP acted to squelch ERE-directed transcription. Data presented in Fig. 4B suggest that this may be caused by competition of ER{alpha} and IEBP for ligand. However, it is also possible that IEBP interrupted ER-ERE-regulated transcription by a dominant-negative interaction with the ER{alpha}, the ERE, or both. To determine whether IEBP functions as a direct competitor with ER{alpha} for ERE binding, we carried out electrophoresis mobility shift analyses (EMSAs) using an idealized ERE as probe with (i) recombinant human ER{alpha}; (ii) ERE-BP purified by ERE-DNA affinity chromatography from postnuclear extracts of estrogen-resistant NWP cells (18); (iii) IEBP-enriched postnuclear extracts from the OWP-IEBP stable transfectant clone 1; and/or (iv) recombinant human Hsp27 as competitive binding proteins (Figs. 5 and 6A). Unlike the ER{alpha}, which demonstrated specific, anti-ER{alpha}"supershiftable" ERE binding in EMSA, the IEBP-containing clone 1 extract exhibited no ERE binding (7th lane, Fig. 5A). Data in Fig. 5B demonstrate that the addition of 100 nM E2 to the IEBP-containing EMSA reaction mixtures did nothing to alter the lack of an IEBP-ERE interaction; the positive control for these experiments was another NWP protein, the ERE-BP, which is known to interact in a specific manner with the consensus ERE (18).



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  		FIG. 5.
Effects of IEBP on ERE-directed transcription are not caused by direct interaction with the ERE or disruption of ER·ERE complex formation. Shown are EMSAs using double-strand consensus ERE as probe and IEBP, recombinant human ER{alpha}, and/or ERE affinity-purified ERE-BP as protein. IEBP (A, 7th lane; B, 3rd and 4th lanes) neither bound to ERE nor competed with the ER for binding to ERE (A, 6th lane; C, 2nd and 3rd lanes) in the presence or absence of 100 nM E2. The ER·ERE complex was supershifted by adding anti-Hsp27 antibody with or without 100 nM E2 (C, 4th and 5th lanes).

 



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  		FIG. 6.
Direct association between the human ER{alpha} and Hsp27-like proteins. A, EMSA using double-strand consensus ERE as probe and recombinant human ER{alpha}, human Hsp27, as well as anti-human Hsp27 antibody as complexing protein in the presence or absence of 100 nM E2. B, immunoprecipitation of the protein constituents of postnuclear extracts of vector alone and IEBP-transfected OWP 6299 breast cancer cells with anti-human Hsp27 (left panel) and anti-human ER{alpha} (right panel) followed by detection with anti-human ER{alpha} antibody anti-human Hsp27 antibody, respectively.

 
Furthermore, the combination of IEBP-containing clone 1 extract with the human ER{alpha} did nothing to compete away ER{alpha}-ERE probe binding (6th lane, Fig. 5A); this failure of IEBP to affect ER{alpha}-ERE probe binding was unaltered by the presence of added E2 (Fig. 5C). To determine whether IEBP might participate in the ER{alpha}·ERE probe complex, anti-Hsp27 antibody was added to the reaction mixture for EMSA (4th and 5th lanes, Fig. 5C). The ER{alpha}·ERE complex was supershifted, indicating that anti-Hsp27-reactive protein (i.e. IEBP) was likely part of the ER{alpha}·ERE probe complex. However, unlike the addition of anti-ER{alpha} antibody to ER{alpha}·ERE complex, which resulted in a supershift of the entire ER{alpha}·ERE complex (fifth lane, Fig. 5A), there was residual ER{alpha}·ERE complex that was not supershifted upon addition of anti-Hsp27 antibody. Preliminary data (not shown) indicate that failure to achieve a "complete supershift" with anti-Hsp27 antibody results from an Hsp27-mediated reduction in the affinity of the ER{alpha} for ERE probe. Finally, the addition of E2 to the reaction mixture did little to alter the mobility of anti-Hsp27 antibody-IEBP·ER{alpha}·ERE complex, although there was some indication that the addition of E2 did diminish the intensity of that complex. The IEBP and ER{alpha} mixing experiments presented in Fig. 5C involved the combination of proteins of NWP and human origin (IEBP and ER{alpha}, respectively). To ensure that EMSA results were not the consequence of a species difference between the proteins of interest, a similar experiment was performed with recombinant human Hsp27, a human homolog of IEBP, and the human ER{alpha} (Fig. 6A). Like IEBP, human Hsp27 showed no ERE binding capability, and, when added to ERE probe in combination with ER{alpha}, there was no alteration in ER{alpha}·ERE binding, whether in the absence or presence of hormone E2. As was the case with IEBP (Fig. 5C), addition of anti-Hsp27 antibody to the ER{alpha}·ERE·Hsp27-containing EMSA reaction mixture resulted in a supershift in the complex to the top of the gel, indicating that human Hsp27, like its NWP primate counterpart, IEBP, was a part of the ER{alpha}·ERE complex. As was the case with the ER{alpha}·ERE·Hsp27-containing EMSA reaction mixture, addition of E2 to the appeared to do little to alter the mobility or the intensity of the ER{alpha}·ERE complex.

To confirm a direct protein-protein interaction between ER{alpha} and IEBP, coimmunoprecipitation of the complex from the postnuclear extract of vector-transfected wild-type and IEBP-overexpressing clone 1 cells was attempted with either anti-human ER{alpha} or anti-human Hsp27 antibody for immunoprecipitation and anti-Hsp27 and anti-ER{alpha} antibody for immunoblot detection, respectively (Fig. 6B). Wild-type cell extracts demonstrated the presence of both the ER{alpha} and Hsp27 and their interaction with one another. Extracts of clone 1 IEBP-overexpressing cells exhibited an enhancement of this interaction. These data confirmed an association between the two proteins even in estrogen-responsive wild-type cells, which are recognized to be relatively depleted of IEBP-related proteins (see Fig. 4B).

Ligand-dependent Interaction of ER{alpha} with Hsp27—When added acutely to EMSA reaction mixtures containing the ER{alpha}, the IEBP, or both, the presence of E2 did little to alter the ER{alpha}-ERE interaction (Figs. 5 and 6A). However, overnight exposure of ER{alpha}-expressing wild-type cells to E2 strongly enhanced IEBP expression (Fig. 7A), highlighting the possible importance of E2 itself as an endogenous regulator of IEBP expression; because it was already maximally expressed in IEBP transfectants, E2, at concentrations as high as 100 nM, had no observable stimulatory effect on IEBP expression in cells already constitutively overexpressing the protein (data not shown). Although data presented in Fig. 7A confirmed transregulation of IEBP expression by E2, they did not address the potential for prolonged E2 exposure to promote or inhibit IEBP-ER{alpha} binding. Hence, the yeast two-hybrid system was employed to determine whether the ability of E2 to promote IEBP expression resulted in a ligand-driven interaction between the ER{alpha} and IEBP "in vivo." The full-length human ER{alpha} cDNA was cloned as a fusion protein with the DNA binding domain of GAL4. This, together with a full-length human Hsp27-GAL4 activation domain fusion, was used in yeast cotransformations. To identify proteins that interacted with ER{alpha} in a ligand-specific fashion, yeast colonies were selected on SD Leu/Trp/His/Ade-medium supplemented with or without 10 or 100 nM E2 or the ER antagonist tamoxifen (Fig. 7B). The full-length Hsp27 and ER{alpha} insert grew only in the presence of E2. No growth was observed in the presence of tamoxifen or in selective media without E2. To confirm a direct interaction between Hsp27 and ER{alpha}, GST pull-down assays were performed. Protein extracts of clone 1 IEBP-overexpressing cells were incubated with a GST-ER{alpha} ligand binding domain fusion protein. Control assays employed a human GR fusion protein or GST protein alone. In each case, SDS-PAGE separation of the GST reaction mixtures was assessed using anti-Hsp27 antibody. Data confirmed that Hsp27 was immunoprecipitated only by GST-ER{alpha}, not by GST-GR or by GST alone (Fig. 7C).



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  		FIG. 7.
E2-regulated expression of IEBP and its interaction with ER{alpha}. A, increased expression of Hsp27 in wild-type breast cells. Shown is Western blot analysis of Hsp27 in wild-type breast cells in the presence or absence of increasing doses of E2 (0.1–100 nM). B, yeast two-hybrid analysis of ligand E2-dependent interaction of Hsp27 with ER. AH109 yeast cells were cotransfected with a GAL4 DNA binding domain-ER{alpha} fusion protein plasmid in the presence or absence of a corresponding Hsp27 fusion protein plasmid, and colonies were growth selected using Leu/Trp/His/Ade-medium containing 10 nM E2, 10 nM ER antagonist tamoxifen (tamox), or vehicle only (–). C, GST pull-down analysis of ER-interacting proteins. Protein extracts of cells overexpressing IEBP were incubated with either a GST-ER or GST-GR fusion protein or GST protein alone. GST-bound proteins were separated on SDS-PAGE and probed with an anti-Hsp27 antibody.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Steroid receptors belong to a superfamily of ligand-inducible transcription factors that regulate expression of a wide variety of target genes (32). Within this system, hormone specificity is provided not only by ligand-selective domains within cognate steroid hormone receptor proteins and coregulatory proteins (33) but also by DNA sequence variations within cis-acting hormone response elements in target gene promoters (34). In common with other transcription factors, recent studies have shown that tissue and disease specificity of steroid hormone receptors may derive from epigenetic factors that act to "fine tune" hormone signaling. For example, local concentrations of steroid hormones are now known to be highly dependent on prereceptor regulation by steroid-metabolizing enzymes that act in an autocrine or intracrine fashion to dictate local levels of receptor ligands (35, 36). Steroid hormone responses are also highly dependent on the expression and function of repressor and activator accessory proteins that act to facilitate postreceptor signal transduction (28, 29, 37, 38).

In recent studies, using steroid hormone-resistant NWP cells as a model, we have characterized two further mechanisms involved in the modulation of steroid hormone receptor action; these NWP cells demonstrate tissue resistance to vitamin D and estrogen in the face of normal expression of receptors for 1,25(OH)2D3 and E2, respectively (8, 10). Hormone resistance is caused by overexpression of two distinct classes of epigenetic factors. In the first class of epigenetic factors are dominant-negative-acting inhibitors of 1,25(OH)2D3- and E2-directed transactivation. These proteins are in the hnRNP family of cis-acting proteins (16, 18). They squelch steroid hormone-directed transactivation by competing in trans with activated receptor proteins for the cognate hormone response elements of the receptor in the promoter of hormone-regulated genes. Expression and function of these proteins do not appear to be restricted to NWP, as we have described recently the first case of vitamin D-resistant rickets in a VDR-normal human subject who presented with constitutive overexpression of an hnRNPA1-like VDRE-BP (39). In the second class of epigenetic factors is the heat shock family of chaperone proteins (Hsp). These proteins serve as relatively high affinity and high capacity carriers of sterol/steroid hormones in the intracellular environment, governing the availability and delivery and hormones to specific cellular destinations and the ligand-interacting proteins that reside there; these Hsp-like intracellular binding proteins are to be distinguished from those proteins that transport the same steroid hormones throughout the circulation (20–22). In the case of the IDBPs, these proteins act to move 25-hydroxylated vitamin D metabolites to the VDR and mitochondrial vitamin D 1- and 24-hydroxylases, promoting 25(OH)D3 1- and 24-hydroxylation (40). Although four distinct IDBPs with a high degree of sequence identity to members of the Hsp70 family have been characterized recently (41), some of which are known to be capable of interacting with steroid hormones other than the 25-hydroxylated vitamin D metabolites (22), the existence of a distinct intracellular chaperone protein(s) for estrogen in estrogen-resistant NWP cells has only recently been described (19). As such, the aim of the current study was to clone, sequence, express, and functionally characterize IEBP to determine whether it exhibited the same protransactivating potential exhibited by its vitamin D counterpart.

Based on the amino acid sequence of tryptic fragments of a protein purified by E2 affinity chromatography from postnuclear extracts of estrogen-resistant NWP cells, we were able to design degenerate primers that enabled amplification of an IEBP cDNA with similarity to the "small Hsps" including human Hsp27 and crystallins A and B (Fig. 1). The extraordinarily high degree of sequence identity, 87% (Fig. 2), between the NWP IEBP and the human Hsp27 indicates that IEBP is likely the interspecies homolog of human Hsp27. The family of small Hsps (sHsps; 15–42 kDa), which includes Hsp27, is encountered in both pro- and eukaryotes (42–45). In the human genome, Hsp27 is encoded by four different genes on chromosomes 3, 7, 9, and X (46); such redundancy indicates that the encoded protein(s) is critical for survival of the host. Like their counterparts in the 70-kDa, 90-kDa and 60-kDa range (42, 47, 48), the sHsps (i) are up-regulated by cell "stress," including heat stress, which is transduced by heat shock factors interacting with specific heat shock enhancer elements in the promoter of Hsp genes and (ii) can function as "molecular chaperones" protecting the structural and functional integrity of the intracellular proteins to which they are bound. Unlike Hsps in the 70-, 90-, and 60-kDa families, sHSPs appear to (i) be less homologous in their amino acid sequence; (ii) play a central role in preventing apoptosis (49); (iii) be crucial for the organization of the cytoskeleton and microfilamental structures therein (50, 51); and (iv) be needed for self-oligomerization, providing for the refractory nature of the human lens (52, 53).

Although the N- and C-terminal amino acid sequences may vary considerably among sHsp family members and between species, the general domain structure of the sHsp molecules remains highly conserved through evolution (54). A central {alpha}-crystallin domain of ~90 residues is bounded by the variable N-terminal and C-terminal extensions. The C-terminal extension, a polar structure, is now considered to be the prime mediator of the chaperone function of the molecule, whereas the conserved {alpha}-crystallin and variable N-terminal domains are thought to be essential for multimerization of the sHsps. In addition to being highly homologous with human Hsp27, compared with the {alpha}-crystallins (Figs. 1 and 2) the IEBP isolated from estrogen-resistant NWP cells here was determined to possess the typical conserved central {alpha}-crystallin core domain flanked by variable N- and C-terminal extensions. Considering that IEBP was isolated by its ability to adhere to an E2 affinity support, the IEBP sequence was scanned for the presence of an estrogen binding site. As shown in Fig. 1, a sequence with 38% identity to a 21-amino acid stretch of the ligand binding domain of the ER{alpha} was detected in the highly conserved {alpha}-crystallin core domain of the molecule. Although formal mapping studies have not yet been completed, it is presumed that it is this part of the IEBP, and its human homolog Hsp27, that is responsible for the enhancement of E2 binding in cells constitutively overexpressing IEBP (see Fig. 4B). Moreover, because this putative E2 binding subdomain resides in the conserved {alpha}-crystallin domain of the sHsps, it is possible that estrogen binding may be a function of other members of the sHsp27 family. Although there resides some sequence identity to the ATP-binding/ATPase domain prototypical of the larger Hsps (i.e. Hsp70, Hsp90, and Hsp60) in the conserved {alpha}-crystallin domain, the role, if any, ATP has in governing the function of IEBP and other Hsp70-like proteins remains to be determined.

In addition to bearing the above mentioned enhancer cis-elements that can interact with heat shock factors, the Hsp27 promoter also contains an ERE half-site in direct proximity to an Sp1 site and the TATA box (55, 56). Although this ERE half-site can be shown to interact with the ER{alpha}, E2-directed transactivation of the Hsp27 gene, as reported by a number of laboratories (57), does not require the ERE half-site (57). Our studies confirm that the anti-human Hsp27-reactive IEBP is an estrogen-responsive gene product, being markedly up-regulated after overnight exposure to ER-saturating concentrations of E2 (Fig. 6A). So, IEBP, and presumably its human homolog Hsp27, is an estrogen up-regulated gene product that can, in turn, bind the same hormone. These results indicate that E2 can up-regulate expression of an E2-interacting protein that, in turn, can squelch E2-ER{alpha}-ERE-directed transactivation (see Figs. 3 and 4). This suggests that transcriptional down-regulation of IEBP or Hsp27 gene expression might be achieved by the product of that gene through its ability to squelch, but not completely subdue (see 3rd and 4th bars in Fig. 3), estrogen-driven expression. In other words, it is possible that Hsp27 or IEBP could be autoregulated; i.e. when E2-ER{alpha}-directed Hsp27 expression goes up, it produces a protein(s) that dampens subsequent E2-ER enhancer action at the level of the Hsp27 promoter. Such a negative feedback system would serve to regulate E2-promoted transactivation reostatically.

If this is the case, then how does IEBP function to squelch ER{alpha}-directed transactivation? Here we considered three of the likely possibilities. (i) IEBP (Hsp27) was bound by the ERE, preventing access of the E2-liganded ER{alpha} homdimer to the ERE; results depicted in Fig. 5A and Fig. 6A failed to demonstrate a direct interaction between the ERE and IEBP and human Hsp27, respectively. (ii) IEBP somehow promoted the interaction of dominant-negative-acting ERE-BP with the ERE. In this case EMSA failed to demonstrate any evidence that the IEBP altered the ERE-BP-ERE binding interaction. And (iii) ER{alpha}-ERE interaction was subverted (i.e. rendered nonfunctional) by a direct interaction of the IEBP itself with the ER{alpha}-ERE. The last appeared to the case. EMSA demonstrated that IEBP and human Hsp27 were included in an ER{alpha}·ERE complex (Figs. 5 and 6A, respectively) and that the degree of interaction between the two proteins could be increased in the presence of more of the Hsp27-like protein (Fig. 6B). Furthermore, squelching of ER{alpha}·ERE-directed transactivation of IEBP was additive to that observed under the influence of another NWP E2 resistance-causing protein, the ERE-BP (Fig. 3). Our current working hypothesis holds that interaction of the IEBP (or human Hsp27) with the ER{alpha} acts to diminish the capacity and/or affinity of the ER{alpha} for ligand E2, perhaps by limiting access of E2 to the ligand binding domain of the receptor while leaving the IEBP E2 binding available for ligand binding (Fig. 8). Competitive ligand binding studies between IEBP/Hsp27 and ER are currently under way to determine whether the former acts to "steal" ligand away from the ER. This would explain the inability of added E2 to reverse the decreased transactivation caused by the relative overexpression of IEBP (see Figs. 3 and 4A) and raises the possibility that Hsp27 and related molecules can function as corepressors. As such, it is possible that sHsps provide a basic infrastructure for intracellular hormone binding that is distinct from their more established function as cytoplasmic chaperones to classical steroid hormone receptors such as the GR and ER (58, 59).



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  		FIG. 8.
Normal and IEBP-mediated squelching of ER-ERE-directed transactivation. A, normal transcriptional events with E2-bound ER homodimer interacting with ERE to increase transcription. B, "squelched" transcriptional events under the influence of IEBP; the interposition of the E2-binding IEBP between ER and ligand leads to disruption of ER dimerization, the ER-ERE interaction, and transactivation.

 
Human breast cancers that harbor the ER{alpha} are susceptible to estrogen-directed growth advantage (60). This has led to the broad usage of selective estrogen receptor modulators or SERMs, which occupy but do not effectively activate the ER, as adjuvant chemotherapeutic agents in this disease (61). The mechanism(s) by which occupancy of the ER{alpha} by E2 affects this change in tumor cell growth and proliferation remain an area of intense investigation (62). One of the genes that is activated in human ER{alpha}.-expressing breast cancer cells by E2 exposure is Hsp27 (42), the human homolog of the NWP IEBP reported here; similarly, estrogen-driven Hsp27 expression can be squelched by exposure of cells to SERMs (57). This has led to the investigation of Hsp27, like ER{alpha}, as a human breast cancer tumor marker with Hsp27 tumor expression currently suggested to be a "downstream" indicator of estrogen-ER{alpha} interaction in tumor cells (55). In some, but not all studies (63, 64), Hsp27 expression has been shown to correlate with ER{alpha} expression. Therefore, it is of note that in the coimmunoprecipitation, yeast two-hybrid and GST pull-down assays carried out as part of the current studies (see Figs. 6B and 7) there was evidence for a direct protein-protein interaction between the Hsp27-like IEBP and ER{alpha}, which was promoted by the presence of E2 and hindered by exposure to the clinically useful SERM tamoxifen. These data suggest that coexpression of the ER{alpha} and IEBP by breast cancer cells may be functionally as well as temporally linked to one another. The consequences of the additive, dominant-negative-acting squelching of E2-ER{alpha}-ERE-directed transcription of IEBP (Hsp27) and the hnRNP-related ERE-BP on breast cancer cell behavior in vitro are currently under investigation.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant RO1DK55843 (to H. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY518309 [GenBank] . Back

** To whom correspondence should be addressed: Division of Endocrinology, Diabetes, and Metabolism, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Rm. B-131, Los Angeles, CA 90048. Tel.: 310-423-8970; Fax: 310-423-0440; E-mail: adamsj{at}cshs.org.

1 The abbreviations used are: OWP, Old World primate; E2, 17{beta}-estradiol; EMSA, electromobility shift assay; ER, estrogen receptor; ER{alpha}, estrogen receptor {alpha}; ERE, estrogen response element; ERE-BP, estrogen response element-binding protein; GR, glucocorticoid receptor; GST, glutathione S-transferase; hnRNP heterogeneous nuclear ribonucleoprotein; IDBP, intracellular vitamin D-binding protein; IEBP, intracellular estradiol-binding protein; NWP, New World primate; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; RACE, rapid amplification of cDNA ends; SERM, selective estrogen receptor modulator; sHsp, small heat shock protein; VDR, vitamin D receptor; VDRE-BP, vitamin D response element-binding protein. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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