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. 2007 Dec 20;22(3):559–569. doi: 10.1210/me.2007-0297

Control of Estradiol-Directed Gene Transactivation by an Intracellular Estrogen-Binding Protein and an Estrogen Response Element-Binding Protein

Hong Chen 1, Martin Hewison 1, John S Adams 1
PMCID: PMC2262167  PMID: 18096692

Abstract

New World primates exhibit a form of resistance to estrogens that is associated with overexpression of an estrogen response element (ERE)-binding protein (ERE-BP) and an intracellular estradiol (E2)-binding protein (IEBP). Both proteins suppress E2-mediated transcription when overexpressed in estrogen receptor-α (ERα)-positive cells. Although ERE-BP acts as a competitor for ERE occupancy by liganded ERα, the function of IEBP and its human homolog, heat-shock protein 27 (hsp27), is less clear. In data presented here, we have used E2-responsive human MCF-7 breast cancer cells to show that IEBP/hsp27 can regulate estrogen signaling as a cytosolic decoy for E2 and as a protein chaperone for ERα. Furthermore, co-immunoprecipitation, colocalization, yeast two-hybrid, and glutathione S-transferase pull-down analyses indicate that IEBP/hsp27 also interacts with ERE-BP to form a dynamic complex that appears to cycle between the cytoplasm and nucleus during normal estrogen signaling. Overexpression of either IEBP/hsp27 or ERE-BP in MCF-7 cells resulted in abnormal subcellular distribution of the IEBP/hsp27 and ERE-BP, with concomitant dysregulation of ERE occupancy as determined by chromatin immunoprecipitation. We hypothesize that IEBP/hsp27 and ERE-BP not only cause hormone resistance in New World primates but are also crucial to normal estrogen signaling in human cells. This appears to involve a physical association between the two proteins to form a complex that is able to interact with both E2 and ERα in cytosolic and nuclear compartments.

THE EFFECTS OF estrogen in vivo are largely mediated by an intracellular estrogen receptor-α (ERα), which belongs to the steroid hormone receptor superfamily of ligand-inducible transcription factors (1). ERα modulates gene expression by binding to specific estrogen response elements (ERE) in the promoter regions of target genes. The selectivity and efficacy of ERE-mediated gene regulation is therefore dependent on a variety of factors. One important determinant is the level of expression of ERα and its alternative form ERβ, with the latter demonstrating a pattern of ligand binding and gene transactivation that is quite distinct from ERα (2,3,4,5,6). The two forms of ER also exhibit specific patterns of ligand binding. This has been exploited clinically by the development of selective ER modulators such as tamoxifen and raloxifene, which bind ER in a similar fashion to its naturally occurring ligand, the active estrogen 17β-estradiol (E2), and exhibit estrogen action in some tissues and antiestrogen action in others (7). In common with other steroid hormone receptors, estrogen signaling is also influenced by extra-receptor proteins that act to fine-tune ER-mediated transactivation. These include receptor-associated coregulatory proteins such as coactivators and corepressors that enhance or repress transcription via the recruitment of other elements of the chromatin remodeling and transcriptional machinery (6,8,9,10,11).

In recent studies, using steroid hormone-resistant New World primate (NWP) cells, we have characterized two additional mechanisms that contribute to the modulation of steroid hormone receptor action (12). The first of these involves cis-acting comodulator proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that compete with steroid hormone receptors for binding to their cognate response elements, thus attenuating hormone-stimulated transactivation (13,14,15,16,17,18). At least 30 different hnRNPs have been identified in mammalian cells, where their principal function appears to be as single-strand intranuclear RNA-binding proteins involved in the stabilization and handling of pre-mRNA (19). However, hnRNPs have also been shown to bind to double-stranded DNA and influence gene transcription (20,21,22). For example, we have shown that vitamin D resistance in NWPs is associated with overabundance of an hnRNPA-like suppressor of transcription referred to as the vitamin D response element-binding protein (VDRE-BP) (16,23,24). Characterization of a human equivalent of VDRE-BP has shown that this is also associated with resistance to vitamin D, leading to classical symptoms of rickets (15). We have used this clinical case study to clone the cDNA for human VDRE-BP, which has near sequence identity to hnRNPC1/C2 (13). Importantly, although this peptide was overexpressed in a rachitic patient, it was also involved in vitamin D-mediated gene regulation in cells from a normal control subject, indicating that in humans, VDRE-BP/hnRNPC1/C2 is part of the normal transcriptional machinery associated with this particular steroid hormone receptor. NWP cells were also used to isolate another member of the hnRNPC-like subfamily that binds to EREs as an ERE-binding protein (ERE-BP). In a similar fashion to the VDRE-BP, ERE-BP is able to compete with the liganded ERα for promoter occupancy and suppress E2-induced transcription (17,18). Transgenic animal models have confirmed that tissue-specific overexpression of ERE-BP results in estrogen resistance in vivo (17).

Studies of cells from NWP showed that in addition to hnRNPs, a second class of proteins is also linked to steroid hormone resistance. Collectively termed intracellular binding proteins, these factors are members of the heat-shock protein (hsp) chaperone family that fulfill diverse cellular functions (25,26,27,28,29). Two intracellular vitamin D-binding proteins (IDBPs) identified in NWPs were shown to correspond to hsp70 and its constitutive equivalent, heat-shock cognate protein 70 (hsc70) (30,31,32,33,34). The potent ability of hsp70 and hsc70 to enhance vitamin D-mediated transcription and metabolism suggests that the IDBPs are part of a compensatory mechanism that counteracts the effects of the dominant-negative-acting VDRE-BP (31,34). By contrast, the intracellular estrogen-binding protein (IEBP), hsp27, functions as a suppressor of ERE-mediated transcription (12,35,36). Studies to date have focused primarily on the consequences of enhanced expression of IDBP and IEBP and their respective response element-binding proteins in NWP cells. However, we have postulated that the coordinated actions of cytosolic ligand-binding hsps as well as the hnRNP-like response element-binding proteins are also a crucial component of normal steroid hormone signaling in humans. To test this hypothesis, we have investigated whether the repressive effects of IEBP on ERα-mediated transcription are due entirely to its ability to compete for E2 binding with ERα. Data suggest that IEBP also influences E2 signaling via direct protein-protein interactions with ERα, and this in turn plays a pivotal role in determining the subcellular localization and DNA-binding potential of these proteins. Based on these observations, we propose a novel mechanism for orchestrating the complex spatiotemporal kinetics of E2-ERα signaling.

RESULTS

Hsp27 Is an IEBP

We have shown previously that estrogen resistance in NWP cells is associated with over-abundance of an intracellular protein capable of binding naturally occurring as well as synthetic estrogens such as E2 and estren, respectively (35). Purification of this NWP IEBP revealed 87% identity to the amino acid sequence of human hsp27, with variation between the two sequences restricted to the N terminus (Fig. 1A). Western blot analyses confirmed that antiserum to human hsp27 recognized IEBP from NWP cells as well as recombinant human hsp27 (Fig. 1B, upper panel). Immunoblot analysis demonstrated that hsp27 was constitutively expressed and heat-shock inducible in ERα+ MCF-7 human breast cancer cells (Fig. 1B, lower panel). The role of hsp27 as a human IEBP was confirmed in binding studies using recombinant human hsp27. Binding assays using competitors to displace bound radiolabeled E2 showed that both E2 and estren bound to recombinant human hsp27 with a high affinity (Kd = 0.25 and 0.1 nm for E2 and estren, respectively), similar to that observed for E2 binding to IEBP (35). This binding was distinct from that observed with the intracellular ERα, as illustrated by the lack of IEBP binding to tamoxifen, a known synthetic antagonist/agonist of ERα (Fig. 1C). More studies were carried out to assess the capacity for E2 binding in cytosol from estrogen-responsive, ERα-positive cells. Analysis of human MCF-7 cells showed relatively low levels of cytosolic, non-ERα binding of E2, compared with NWP cells (Fig. 1D). However, this capacity was elevated to NWP cell levels by transfection of MCF-7 cells with a full-length cDNA to IEBP. By contrast, cDNAs encoding the N-terminal (amino acids 1–100) or C-terminal (amino acids 101–207) halves of the IEBP protein (see Fig. 1A) showed much lower levels of E2 binding, indicating that the binding motif was localized to a sequence that straddled the junction of the two IEBP peptides (Fig. 1D). Immunohistochemistry showed that in MCF-7 cells, hsp27 is located primarily in the cytoplasm (Fig. 1E). The levels of detectable protein were enhanced after transfection of cDNA for IEBP but with patterns of distribution similar to those observed in parental MCF-7 cells. These data indicated that in human cells, hsp27 can function as a high-affinity cytosolic binding site for E2 in a similar fashion to the IEBP characterized in NWP cells.

Figure 1.

Figure 1

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Hsp27 Is a Human IEBP

A, NWP IEBP shares homology with human hsp27. A also delineates the deduced amino acid sequences for the N-terminal and C-terminal IEBP cDNAs used to transfect MCF-7 cells in D. B, Upper panel, Western blot comparison of IEBP derived from NWP B95-8 cells and recombinant human hsp27 using antiserum to hsp27; lower panel, Western blot analysis of heat-shock stimulation of IEBP expression in human breast cancer MCF-7 cells using antiserum to hsp27. C, Recombinant human hsp27 binds ER ligands. Dose-response displacement of bound [3H]E2 using excess unlabeled E2, estren (E) or tamoxifen (Tam). D, Increased cytosolic binding of E2 in MCF-7 cells transfected with IEBP. Basal cytoplasmic binding of E2 was observed in parental MCF-7 cells, but this was increased in cells transiently transfected with full-length cDNA to IEBP. Cells transfected with the N-terminal (N-term) or C-terminal (C-term) cDNA fragments for IEBP (see A) showed lower levels of E2 binding. E, Immunolocalization of hsp27 in MCF-7 human breast cancer cells. Parental MCF-7 cells showed predominantly cytosolic expression of IEBP/hsp27, and this was increased in MCF-7 cells transiently transfected with full-length cDNA to IEBP. *, Statistically different from parental MCF-7 cells, P < 0.01.

IEBP/hsp27 and ERE-BP Inhibit ER-ERE-Mediated Transactivation

As shown in Fig. 1, hsp27 has the potential to compete with ERα for binding of E2 in human cells, with concomitant modulation of functional responses to estrogen. In previous studies, we have shown that overexpression of cDNA for the NWP IEBP (human hsp27 homolog) suppresses E2-mediated transactivation in a similar fashion to that observed with cDNA for ERE-BP (human hnRNPC-like homolog), although the latter acts by competing with ERα for binding to ERE (14,35). To determine whether or not endogenous hsp27 and/or ERE-BP are involved in normal responses to E2 in ER+ cells, RNA interference technology (short hairpin RNA [shRNA]) was used to inhibit expression of hsp27 or ERE-BP in MCF-7 cells (Fig. 2, A and B). This enhanced ERE-mediated transactivation (2- and 1.4-fold, respectively, compared with MCF-7 cells treated with E2 and control scrambled shRNA). The amplification of transactivation after abrogation of hsp27 or ERE-BP expression contrasted the suppression of ERE-mediated transactivation that accompanied transfection of sense expression constructs for either IEBP/hsp27 or ERE-BP (Fig. 2, A and B). These data confirmed that 1) the suppressive effects of hsp27 on E2 signaling in ER+ human cells are the same as those induced by IEBP in NWP cells, and 2) both hsp27 and ERE-BP are involved in normal E2 responses in MCF-7 cells.

Figure 2.

Figure 2

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ERE-BP and IEBP/hsp27 Modulate ERE-Mediated Transactivation in Human Breast Cancer Cells

MCF-7 ER+ human breast cancer cells were transfected with either an expression construct for IEBP (A) or ERE-BP (B) or inhibitory shRNAs for hsp27 or ERE-BP (A and B, respectively) with or without cotransfection of an estrogen-responsive luciferase plasmid (23). Vector-only transfectants were used as controls (C), and cells were cultured in the absence (black bars) or presence (gray bars) of 10 nm E2. Data are the mean ± sd of triplicate determinations of luciferase activity. *, Statistically different from empty vector (C) equivalent, P < 0.001.

Hsp27 and ERE-BP Act to Dysregulate Normal Interactions between ERα and Chromatin

The ability of IEBP/hsp27 to suppress E2-induced transcription may involve one of the following mechanisms: 1) competition for binding of E2, 2) dysregulation of ERα-chromatin interaction, 3) interaction with ERα and consequent modulation of receptor function, or 4) interaction with ERE-BP. Data in Fig. 1 confirmed that the first of these mechanisms is a potential consequence of IEBP/hsp27 action. To address the second putative effect of IEBP/hsp27, chromatin immunoprecipitation (ChIP) assays were carried out using an ERE fragment of the E2-responsive cathepsin gene promoter as a DNA target (Fig. 3). Data from wild-type MCF-7 cells showed that occupancy of the ERE by ERα occurred 15 min after treatment with E2. At the same time, ERE-BP was displaced from occupancy of the ERE highlighting a reciprocal relationship between these two DNA-binding proteins. Overexpression of IEBP produced a mirror image of the ChIP profile observed in wild-type MCF-7; ERα was detected only on the ERE in the absence of its ligand E2, and the receptor was displaced after treatment with E2, enabling the ERE-BP to then occupy the ERE. Overexpression of ERE-BP itself produced a similar pattern of ERE occupancy to that observed for the IEBP transfectants, suggesting a common mode of chromatin interaction for these two interacting proteins.

Figure 3.

Figure 3

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Dysregulation of Normal ERα-Chromatin Interaction by IEBP/hsp27 and ERE-BP

Nuclear chromatin from parental MCF-7 cells and stable transfectant variants of MCF-7 overexpressing IEBP/hsp27 (IEBP) or ERE-BP was used in ChIP analyses. Cells were treated with or without E2 (10 nm) for 15 min. Occupancy of the cathepsin D gene promoter by ERα, ERE-BP, and IgG (negative control) was determined by PCR amplification of the estrogen-responsive DNA region of the cathepsin D promoter (−295 to −54 bp).

IEBP/hsp27 and ERE-BP Modulate Proliferation and Estrogen Responsiveness of MCF-7 Cells

To investigate the extent to which the effects of IEBP/hsp27 and ERE-BP described in Figs. 1–3 impact on endogenous cellular estrogen responses, additional studies were carried out to assess the proliferation of MCF-7 cells transfected with cDNAs for these proteins (Fig. 4). Data shown in Fig. 4A indicate that stable overexpression of either IEBP/hsp27 or ERE-BP in MCF-7 cells resulted in a lower rate of proliferation in these cells over a 20-h logarithmic growth period. Additional experiments using 20-h cultures of transfectant variants of MCF-7 cells showed that E2 (1 and 10 nm) stimulated the proliferation of plasmid-only control MCF-7 cells, but this effect was significantly diminished in cells transfected with IEBP/hsp27 or ERE-BP (Fig. 4B). Finally, in MCF-7 transient transfection studies, IEBP/hsp27 or ERE-BP abrogated the suppressive effect of E2 (10 nm, 20 h) on expression of mRNA for the cyclin D-kinase inhibitor p21 (Fig. 4C), underlining the ability of IEBP/hsp27 and ERE-BP to disrupt endogenous pro-proliferative responses to E2 in ER+ MCF-7 cells.

Figure 4.

Figure 4

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IEBP/hsp27 and ERE-BP Inhibit Proliferation of Human MCF-7 Cells and Dysregulate the Pro-Proliferative Effects of Estradiol

Stable variants of the MCF-7 cell line transfected with either empty vector (MCF-7, ▪) or plasmids containing expression constructs for IEBP/hsp27 (MCF-7+IEBP, ○) or ERE-BP (MCF-7+ERE-BP, ▴) were seeded at similar density in 96-well plates and then assessed for the following: A, proliferation at 4, 8, and 20, with proliferation data shown as mean RLU ± sd (n = 3) compared with wells containing no cells, ***, statistically different from equivalent plasmid-only MCF-7 cells, P < 0.001. B, proliferation after treatment with either vehicle (0.1% ethanol) or E2 (1 or 10 nm) for 20 h, with data shown as the mean percent change in proliferation (± SD, n = 3) relative to vehicle-treated controls for each transfectant cell variant, ***, statistically different from equivalent plasmid-only MCF-7 cells, P < 0.001. C, effect of E2 (10 nm, 20 h) on the expression of mRNA for p21 in MCF-7 cells transiently transfected with vector alone, IEBP/hsp27, or ERE-BP, with data shown as the mean fold change in p21 mRNA expression for each transfectant cell line relative to vehicle (0.1% ethanol)-treated plasmid-only MCF-7 cells. Statistical analyses were carried out using raw ΔCt values for p21 (see MATERIALS AND METHODS); ***, statistically different from vehicle-treated equivalent transfectant cell, P < 0.001.

IEBP/hsp27 Interacts with ERα

The fact that hsp27 exhibits E2 binding kinetics similar to ERα (Fig. 1C) underlines the potential for this hsp to act as an alternative binding site for estrogens. However, in previous studies using NWP cells, we showed that IEBP co-immunoprecipitates with ERα (35). Here we extended those observations to human cells. Fluorescent microscopy of ER+ MCF-7 cells showed that in the absence of E2, hsp27 and ERα were found predominantly in the cytoplasm, with the localization of both proteins shifting to the nucleus after treatment with E2 (Fig. 5A). Additional studies were therefore carried out in human MCF-7 cells to determine whether hsp27 physically interacts with ERα and whether that interaction also involves ERE-BP.

Figure 5.

Figure 5

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Interaction between ERα and IEBP/hsp27 in Estrogen-Responsive Human Cells

A, Human hsp27 colocalizes with ERα. Fluorescent immunohistochemical analysis of protein for ERα and hsp27 in human MCF-7 breast cancer cells treated with vehicle (0.1% ethanol) or 10 nm E2 for 45 min. In the absence of E2, ERα (red fluorescence) was localized primarily in the cytoplasm, whereas hsp27 (green fluorescence) was detectable primarily in the cytoplasm, with weaker expression in the nucleus. Colocalization (yellow) occurs in the cytoplasm. In the presence of E2, both ERα and hsp27 were strongly colocalized in the nucleus (all magnifications, ×200). B, Direct interaction between ERα and hsp27. Extracts from ER+ human breast cancer MCF-7 cells and stable transfectant variants of MCF-7 overexpressing IEBP/hsp27 (IEBP) or ERE-BP were subjected to pull-down analysis using a GST-ERα conjugate as bait. The presence of IEBP/hsp27 or ERE-BP in the resulting proteins was then analyzed using specific antisera to either human hsp27 or NWP ERE-BP. In each case, cells were treated with either vehicle (−) or 10 nm E2 (+) for 24 h before extraction of cellular proteins.

Glutathione S-transferase (GST) pull-down analyses in MCF-7 cells showed that ERα was able to bind hsp27 but not ERE-BP (Fig. 5B, left columns). Overexpression of IEBP in MCF-7 cells enhanced the GST-ERα complex detected by antiserum to hsp27 but with the same pattern of ERα-hsp27 interaction observed in wild-type MCF-7 cells (Fig. 5B, center column). By contrast, overexpression of ERE-BP appeared to have little effect on ERα-hsp27 interaction, with the hsp27 antibody pull-down bands being very similar to those detected in wild-type MCF-7 cells in the presence or absence of E2 (Fig. 5B, right column). GST pull-down showed no interaction between ERα and ERE-BP in wild-type MCF-7 cells (Fig. 5B, left column) despite the fact that antiserum to NWP ERE-BP readily detected its human homolog (hnRNPC-like protein) in MCF-7 cells (see Fig. 6C). However, overexpression of ERE-BP or IEBP produced ERα-GST pull-down products that were detectable with antibody to ERE-BP (Fig. 5B, center and right columns). The lack of these pull-down products in wild-type MCF-7 cells suggests that ERα is able to interact with ERE-BP indirectly by binding to IEBP/hsp27, which, in turn, can bind to ERE-BP. To investigate this proposed mode of interaction, additional studies were designed to explore the interaction between IEBP/hsp27 and ERE-BP.

Figure 6.

Figure 6

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Interaction between IEBP/hsp27 and ERE-BP in Estrogen-Responsive Human Cell

A, Co-immunoprecipitation of IEBP/hsp27 with ERE-BP. Extracts from MCF-7 cells were immunoprecipitated with antibodies to either hsp27 or ERE-BP and the resulting proteins analyzed by gel separation and Western blot analysis using the antibody to ERE-BP. B, Yeast two-hybrid analysis of the ERE-BP interaction with IEBP/hsp27. AH109 yeast cells were cotransformed with a Gal4-DB-ERE-BP fusion protein plasmid and a Gal4-AD-hsp27 fusion protein plasmid, and colonies were growth selected using -Leu/-Trp/-His/SD-medium containing either vehicle or E2 (10 nm). C, Fluorescence immunohistochemical analysis of ERE-BP-IEBP/hsp27 subcellular localization in ER+ MCF-7 human breast cancer cells using antibody to human hsp27 (red fluorescence) or ERE-BP (green fluorescence). IEBP/hsp27 shows predominant cytosolic localization, whereas ERE-BP shows predominant nuclear localization, with some colocalization (yellow) in the nucleus. All magnifications ×200. D, Cytosolic and nuclear protein extracts from parental MCF-7 cells and transfectant variants of MCF-7 overexpressing IEBP/hsp27 (IEBP) or ERE-BP were subjected to pull-down analysis using a GST-ERE-BP conjugate as bait. The resulting ERE-BP-associated proteins were probed using antiserum to human IEBP/hsp27.

IEBP/hsp27 Interacts with ERE-BP

Co-immunoprecipitation studies (Fig. 6A) indicated that hsp27 does indeed interact directly with ERE-BP in MCF-7 cells. The capacity for protein-protein interaction was confirmed by yeast two-hybrid analyses (Fig. 6B). AH109 yeast were cotransformed with full-length cDNA for ERE-BP and the DNA-binding domain of GAL4 as one fusion protein and full-length IEBP with the GAL4 activation domain as another. Yeast colonies were selected on synthetic dextrose-His/-Trp/-Leu medium supplemented with either vehicle or E2 (10 nm) to assess whether any interaction between the two proteins was ligand dependent. Yeast containing the full-length ERE-BP and IEBP insertions grew in the presence or absence of E2, confirming the potential for interaction between the two proteins and further suggesting that this association is estrogen independent (Fig. 6B). The hsp27-ERE-BP interaction was further studied by fluorescent immunohistochemical analysis of parental MCF-7 cells (Fig. 6C). Hsp27 was detected principally in the cytoplasm and ERE-BP principally in the nucleus, but there was a small amount of colocalization in both subcellular compartments (Fig. 6C). To investigate this further, additional GST pull-down analyses were carried out using cytosolic and nuclear extracts from MCF-7 cells as well as MCF-7 cells transfected with cDNA to either IEBP or ERE-BP (Fig. 6D). In the absence of E2, hsp27 was detected in ERE-BP pull-down products from both cytosolic and nuclear fractions, whereas pull-down of hsp27 occurred exclusively in the nucleus after treatment with E2 (Fig. 6D, left columns). Cells transfected with IEBP or ERE-BP showed very different patterns of hsp27-ERE-BP complex formation relative to each other and parental MCF-7 cells. Overexpression of IEBP resulted in ERE-BP pull-down of hsp27 that was restricted to the cytoplasm in the absence of E2 but which was detectable in both cytoplasm and nucleus after treatment with E2 (Fig. 6D, center panels). By contrast, in ERE-BP transfectant cells, pull-down of hsp27 occurred exclusively in the nucleus in the absence of E2, but in the cytoplasm after treatment with the hormone (Fig. 6D, right panels).

DISCUSSION

Nuclear receptors are transcription factors that require multiple protein-protein interactions to regulate gene expression (1). Studies to date have focused on three key classes of proteins that are known to influence steroid hormone receptor-mediated responses beyond simple variations in the expression of receptor protein: 1) chaperones that are involved in receptor protein folding, trafficking, and recycling (37,38,39,40); 2) coactivator and corepressor proteins that define the activation or repressive state of steroid hormone receptors (8,9,10,11); and 3) remodeling complexes that repress or facilitate transcription via alterations in chromatin conformation and interactions with coactivator and corepressor proteins (8,9,10,41). In a series of recent studies, we identified two additional mechanisms involving proteins with established cellular functions that are also crucial to steroid hormone signaling. The first of these occurs at the gene promoter level, where we described an alternative function for hnRNPs as steroid hormone response element-binding proteins (15,16,17,18). Using cells from steroid hormone-resistant NWPs, we have characterized hnRNP response element-binding proteins for estrogen (ERE-BP) and vitamin D (VDRE-BP), showing that the human homolog of the latter is hnRNPC1/C2 (13). When overexpressed in steroid hormone-responsive Old World primate, cells ERE-BP and VDRE-BP act to suppress transcriptional regulation by E2 and vitamin D, respectively (16,18). ChIP analysis of the human VDRE-BP has shown that this response involves aberrant association between hnRNPC1/C2 and the VDRE, with concomitant dysregulation of the temporal organization of VDR-chromatin interaction (13).

The second novel mechanism that we have shown to impact on steroid hormone receptor signaling involves an alternative function for hsps as IDBPs (31) and IEBPs (36). As with the response element-binding proteins, IDBP and IEBP were initially identified in NWP cells (23), but human homologs have been cloned for both proteins, revealing almost complete identity between IDBP and hsc70 (31) and IEBP and hsp27 (35). In contrast to VDRE-BP and ERE-BP, the precise mode of action of hsc70 and hsp27, with respect to steroid hormone binding and function remains unclear. On the one hand, IDBP/hsc70 potentiates vitamin D metabolism and function, supporting the idea that it acts as a conduit for the intracellular trafficking of vitamin D metabolites (31,34). Conversely, IEBP/hsp27 suppresses E2-mediated transactivation, suggesting that it acts as an intracellular decoy for estrogens (35). The aim of the current study was to more clearly define the function of IEBP/hsp27 as a pivotal component of ERα-mediated transcription beyond its established actions in NWPs. Specifically, we have characterized the interaction between IEBP/hsp27 and the four principal factors involved in E2 signaling in estrogen-responsive human cells: 1) ligand, 2) ERα, 3) ERE-BP, and 4) ERE. Data presented here show that IEBP/hsp27 plays a pivotal role in orchestrating the integration of these factors, and we propose a mechanism by which this may underpin both normal and dysregulated responses to estrogen.

An association between hsp27 and estrogens has been recognized for many years, particularly in the context of defining the ER responsiveness of different types and grades of tumors (42,43,44,45). More recently, we have postulated that hsp27 is the human homolog of the IEBP isolated from NWPs that confers estrogen resistance in this group of mammals (36). Other groups have also reported suppression of E2-induced transcription by hsp27, but in this case, it was suggested that the hsp acts as an ER corepressor, and interacts with ERβ rather than ERα (46). However, it should recognized that the studies in question used ER− HeLa cells subjected to cotransfection of ERα or ERβ together with hsp27 and, as such, may not faithfully reflect normal physiological interactions. Indeed, in data presented here, we have confirmed that IEBP/hsp27 colocalizes with ERα and binds to the receptor in GST pull-down analyses using material from human cells with endogenous ERα expression.

Initial observations led us to propose that the suppressive effects of IEBP/hsp27 on estrogen could be explained by IEBP/hsp27 acting as a competitor for E2 binding to ERα, thereby squelching ERα-ERE interaction and associated transcription (35). However, several lines of evidence indicate that this is unlikely to be the exclusive function of IEBP/hsp27. First, although cytosolic, non-ER binding of E2 is relatively low in MCF-7 cells, shRNA inhibition of IEBP/hsp27 produced a dramatic rise in E2-induced transactivation (see Figs. 1D and 2C). Second, in unpublished studies, we have shown that, like E2, the ERα agonist tamoxifen induces ERE promoter-reporter activity in MCF-7, and this is attenuated by overexpression of IEBP (data not shown). Because tamoxifen does not bind effectively to IEBP/hsp27 (see Fig. 1C), we can conclude that IEBP/hsp27 effects on responses to this compound involve a nonbinding function of the protein. Finally, the fact that IDBP/hsc70 acts as a cytosolic binding site for vitamin D and yet potentiates rather than inhibits the actions of this steroid hormone (31) lends further weight to the idea that IEBP/hsp27 exerts effects beyond simple competition for steroid hormone binding.

Consistent with data from Old World primate cells (35), we showed that IEBP/hsp27 not only binds E2 but also exhibits protein-protein interaction with ERα in human cells (Fig. 5). Other hsps are known to be involved in modulating the ligand binding and subsequent intracellular distribution of steroid hormone receptors (47,48). Here we have shown for the first time that exposure to E2 enhances nuclear translocation of both ERα and IEBP/hsp27. Although these two proteins have the potential to interact with each other, it remains uncertain whether they enter the nucleus as a protein-protein complex. Likewise, the function of IEBP/hsp27 once it has entered the nucleus is also unclear. We have shown previously that IEBP/hsp27 does not interact with ERE or interfere with ERα binding to the ERE (35). However, by demonstrating that IEBP/hsp27 interacts with ERE-BP (Fig. 6), we have identified a potential indirect mechanism by which IEBP/hsp27 may influence ERα function. Specifically, it appears that by acting as a partner for ERE-BP, IEBP/hsp27 can modulate ERα-mediated transcription indirectly at the level of gene promoter occupancy. The underlying premise for this is that E2-induced transcription involves a wave-like pattern of ERE occupancy by liganded ERα in target gene promoters (see Fig. 3). This appears to involve a reciprocal relationship between ERα and ERE-BP in binding to the ERE, with the latter occupying the ERE in the absence of ligand but then being displaced by ERα after ligand binding. In view of the fact that a similar reciprocal relationship has been described previously for the VDRE-BP and VDR (13), we hypothesize that response element-binding proteins play a key role in controlling the spatiotemporal kinetics of promoter occupancy by steroid hormone receptors. Based on data presented here, we can further speculate on a role for IEBP/hsp27 in this process by modulating the localization and function of ERE-BP through protein-protein interaction.

Collectively, these observations have allowed us to hypothesize a potential mechanism that would incorporate the multifunctionality of IEBP/hsp27 described above, with the initiation of ERE occupancy by liganded ERα (Fig. 7). In this model, the IEBP/hsp27-ERE-BP complex plays a pivotal role as protein-protein repository, which, in the absence of ligand, is in equilibrium between cytosolic and nuclear compartments of estrogen-responsive cells (Fig. 7A). Estrogens such as E2 will bind to both ERα and IEBP/hsp27 (Fig. 7B). The net response to E2 treatment is the nuclear translocation of both ERα and IEBP/hsp27 (Fig. 7C), and this coincides with the displacement of ERE-BP binding from the ERE. The mechanism by which ERE-BP vacates its DNA binding site remains unclear and may involve simple competition with an alternative binder, namely liganded ERα. Alternatively, nuclear translocation of IEBP/hsp27 may favor its association with ERE-BP and thus indirectly shift the latter away from the ERE. In either case, the overall effect will be to allow occupancy of the ERE by ERα, thereby facilitating the recruitment of coactivators and other proteins required to initiate transcription. This model is also consistent with data from cells transfected with IEBP/hsp27 or ERE-BP, in which the altered subcellular localization of these proteins (see Fig. 8) appears to lead to dysregulation of ERE occupancy by ERα either in the absence or presence of E2 (Fig. 3), with concomitant effects on the estrogen responsiveness of ER+ cells (Fig. 4).

Figure 7.

Figure 7

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Schematic Representation of the Spatiotemporal Interactions among ERα, IEBP/hsp27, and ERE-BP during Normal E2-Mediated Transcription in ER+ Cells

A, In the absence of E2, IEBP/hsp27 (IEBP) interacts with ERα (both ellipses) in the cytosol (C), whereas ERE-BP interacts with ERE in the nucleus (N) (ellipse). Reservoirs of IEBP/hsp27 and ERE-BP are also present in cytosolic and nuclear compartments as a protein-protein complex (squares). B, E2 added to this system is able to bind to both ERα and IEBP/hsp27. C, Liganded ERα competes with ERE-BP for occupancy of the ERE, and displacement of the ERE-BP from the ERE is sustained by a shift in the subcellular distribution of unliganded hsp27 to form an enhanced hsp27-ERE-BP in the nucleus (shown as arrows next to squares). D, In the absence of ERE-BP, ERα is able to form a homodimer and initiate transcription.

Figure 8.

Figure 8

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Schematic Representation of the Spatiotemporal Interactions among ERα, IEBP/hsp27, and ERE-BP in ER+ Cells after Overexpression of Either IEBP or ERE-BP

In the absence of E2, overexpression of IEBP in ER+ human cells (left panel) results in 1) increased abundance of IEBP/hsp27 in cytoplasm (C), 2) formation of an ERα-IEBP/hsp27-ERE-BP triple-protein complex in the cytoplasm, and 3) a shift in localization of the IEBP/hsp27-ERE-BP complex to the cytoplasm. The resulting dysregulation of ERE-BP distribution enables illicit occupancy of the ERE by unliganded ERα, with concomitant absence of transcription. In the presence of E2, 1) overabundant IEBP/hsp27 acts as a decoy for E2, preventing normal ligand binding to ERα; 2) binding of E2 to IEBP/hsp27 disrupts the ERα-IEBP/hsp27-ERE-BP triple-protein complex; 3) the resulting free ERE-BP reassociates with free (i.e. non-ERα-bound) IEBP/hsp27, thereby shifting the equilibrium of the IEBP/hsp27-ERE-BP complex to the cytoplasm. The resulting inappropriate occupancy of ERE by ERE-BP suppresses transcription despite the presence of E2. Overexpression of ERE-BP in the absence of E2 (right panel) shifts distribution of the IEBP/hsp27-ERE-BP complex to the nucleus (N) while inducing formation of an ERα-IEBP/hsp27-ERE-BP triple-protein complex in the cytoplasm. This prevents ERE-BP from its normal association with ERE and enables illicit occupancy of the ERE by unliganded ERα similar to that observed with overexpression of IEBP. Addition of E2 to this system once again disrupts the cytoplasmic ERα-IEBP/hsp27-ERE-BP triple-protein complex to produce free cytosolic ERE-BP. This, in turn, shifts the equilibrium of the IEBP/hsp27-ERE-BP complex to the cytoplasm at the expense of free ERE-BP in the nucleus, which is then able to occupy the ERE and prevent transcription.

Data presented here provide further evidence that intracellular steroid binding and response element-binding proteins are not simply phenomena associated with resistance to steroid hormones in NWP. However, several key questions remain to be answered. It is unclear how IEBP/hsp27 and ERE-BP interact and whether this involves the ATP-binding catalytic domain commonly used by hsps to initiate protein-protein chaperone interactions (49,50). It also remains to be determined whether ERE-BP functions simply to prevent occupancy of the ERE by unliganded ERα or whether it may play an active role in orchestrating other aspects of ERα-mediated transcription such as the recruitment of coactivators or the remodeling of chromatin (51). Additional experiments are also required to determine the extent to which IEBP/hsp27 and/or ERE-BP can modulate nonclassical estrogen pathways, including interaction with the activator protein-1 signaling system. Finally, in seeking to establish the generalizability of the intracellular and response element-binding proteins for other members of the steroid hormone receptor superfamily, it will be important to clarify the contrasting effects of IEBP/hsp27 and IDBP/hsc70. Both proteins act as decoys for their respective steroid hormones but exert opposing effects on transcription. Based on data presented here, we postulate that the transcriptional function of intracellular steroid binding proteins is not due primarily to their binding potential but rather their ability to interact with response element-binding proteins. Future studies will thus aim to characterize the same pattern of protein-protein interactions described in this study for other steroid hormones such as vitamin D.

MATERIALS AND METHODS

Analysis of E2 Binding and ERα Expression

The specific ligand binding capacity of affinity-purified cell extracts and recombinant human hsp27 (Stressgen, Inc., Victoria, British Columbia, Canada) was measured by competitive protein binding assay as described previously (36). Western blot analysis of denatured cell extracts was carried out using previously described methods (16), in combination with monoclonal anti-human hsp27 or anti-human ER antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA) or polyclonal anti-ERE-BP antibody, with horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence detection (Amersham Pharmacia Biotech, Piscataway, NJ). For immunofluorescence analyses, specifically bound antibody was visualized using fluorescein thiocyanate- and /or Texas Red-conjugated fluorescent secondary antibodies (Santa Cruz) using an Olympus BX41 microscope.

Transient and Stable Transfection of IEBP and ERE-BP cDNAs and shRNAs

For stable transfection, cells were incubated with 5.0 μg pcDNA3.1/v5-His-TOPO IEBP or ERE-BP plasmid in lipo TAXI solution for 5 h, followed by an equal volume of 20% fetal calf serum-supplemented medium. After incubating overnight, cells were split (1:10 ratio) and incubated with fresh medium containing 500 μg/ml G418 sulfate selective antibiotic (Life Technology, Grand Island, NY). Medium was replaced every 3–4 d until stable colonies formed. Single colonies were picked and subjected to further G418 selection. For transient transfection, cells were grown to 80–90% confluence in 12-well plates. Each well received 0.8 μg ERE-BP or IEBP expression plasmid or hsp27 or ERE-BP shRNA and Opti-MEM medium containing 4.0 μl Lipofectamine 2000 per 100 ml medium and incubated overnight. Medium was replaced and treatments (vehicle or 100 nm E2) added. After an additional 24 h at 37 C, cells were lysed, and luciferase and β-galactosidase activities were measured (Promega, Madison, WI).

Yeast Two-Hybrid Screening

The full-length ERE-BP cDNA was amplified using the oligonucleotides 5′-GCCGAATTCACTATGTCGGAGGAGCAGTTCGGCGG-3′ and 5′-CCCGGATCCTCAGAGGGACCCACCACCGTCATACTTC-3′. The ERE-BP cDNA was cloned into the EcoRI and BamHI site of GAL4 DNA-binding domain vector (GAL4 DNA-BD/ERE-BP). 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/ERE-BP was cotransformed with the GAL4 DNA-AD/hsp27 plasmid using Yeast Transformation System 2 kit (Clontech, Palo Alto, CA) according to manufacturer’s instructions.

GST Pull-Down Assay

GST fusion proteins for ERα, IEBP, or ERE-BP were expressed in Escherichia coli strain DH5 and purified with glutathione-Sepharose beads according to the manufacturer’s instructions (Pharmacia Biotech, Piscataway, NJ). Protein extracts were applied to GST beads, incubated for 1 h, washed repeatedly (five times) with PBS buffer containing 5 mm dithiothreitol (DTT) and 1 mm phenylmethylsulfonyl fluoride (PMSF), resuspended in 2× SDS sample buffer, and boiled for 5 min. Denatured proteins were resolved on 4–20% SDS-PAGE gel, transferred to a nitrocellulose membrane, probed with appropriate antibody (Santa Cruz), and visualized by enhanced chemiluminescence.

Protein Co-Immunoprecipitation

Extracts from MCF-7 cells prepared in the same manner as GST pull-down assays were incubated overnight with antihuman hsp27 antibody (Santa Cruz) or polyclonal anti-ERE-BP antibody. Protein-antibody complexes were precipitated by addition of protein G resin (Sigma-Aldrich, St. Louis, MO) and a 2-h incubation on ice. Beads were washed five times with PBS. Bound material was separated on 10% SDS-PAGE gels, transferred to polyvinylidene difluoride membrane, and probed with anti-ERE-BP antibody.

ChIP Assays

ChIP assays were performed as described previously (13). Briefly, after E2 treatment, cells were washed twice with PBS and cross-linked with 1% formaldehyde at 37 C for 10 min. After quenching of cross-linking with 1.25 m glycine, cells were harvested and rinsed with PBS, and the resulting pellets were resuspended in 1 ml cell lysis buffer [5 mm Pipes (pH 8.0), 85 mm KCl, 0.5% Nonidet P-40, 1 mm DTT, 0.25 mm PMSF, and 1 μg/ml each of pepstatin, leupeptin, and aprotinin]. Nuclei were collected and resuspended in 500 μl nuclear lysis buffer [50 mm Tris-HCl (pH 8.1), 10 mm EDTA, 1% SDS, 1 mm DTT, 2.5 mm PMSF, and 1 μg/ml each of pepstatin, leupeptin, and aprotinin; all from Sigma]. The resulting chromatin samples were sonicated to yield sheared DNA fragments of sizes 300-1000 bp. For each immunoprecipitation, sheared chromatin was diluted with immunoprecipitation dilution buffer [0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA, 16.7 mm Tis-HCl (pH 8.1), and 167 mm NaCl]. Chromatin was collected and incubated at 4 C overnight with 5 μg anti-VDR-9A7 (Affinity Bioreagents Inc., Golden, CO) and anti-hnRNP antibodies (Santa Cruz). Rabbit IgG was used as a negative control. The immune complexes were precipitated with 60 μl protein A-Sepharose beads (source) at 4 C for 1 h and then subjected to serial 1-ml washes of the following: immunoprecipitation dilution buffer, TSE-500 [0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl (pH 8.1), 500 mm NaCl], and LiCl/detergent buffer [100 mm Tris-HCl (pH 8.1), 500 mm LiCl, 1% Nonidet P-40, and 1% deoxycholic acid in Tris-EDTA buffer). Antibody-protein-DNA immunocomplexes were then eluted with 1% SDS in 50 mm NaHCO3. Formaldehyde cross-linking was reversed by heating at 65 C overnight with the addition of 5 m NaCl to a final concentration of 200 mm. All samples were then digested at 45 C for 1 h with 20 μg proteinase K and the resulting DNA amplified by PCR using primer sequences in the cathepsin D promoter, −295 to −54 bp relative to the start site of transcription.

Cell Proliferation Assays

Proliferation data for transfectant variants of MCF-7 cells were obtained using the ViaLight Plus assay kit (Lonza Rockland, Inc., Rockland, ME) according to the manufacturer’s instructions. Data are reported as mean relative luminescence units (RLU) ± sd for n = 3 proliferation cultures.

Real-Time RT-PCR Analyses

Total RNA was extracted from MCF-7 cells and transfectant variants using an RNeasy Kit (QIAGEN, Valencia, CA). RT was carried out using 2 μg RNA, and cDNA was generated using the QuantiTect Reverse Transcriptase Kit according to the manufacturer’s instructions (QIAGEN). RT-PCR was performed using the ABI Prism 7700 Sequence Detection System with SYBR Green PCR master mix reagents (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol. GAPDH was used to normalize target gene expression. The sequences for each primer set were as follows: p21, 5′-GGCGGCAGACCAGCATGACAGATT-3′ (forward) and 5′-GCAGGGGGCGGCCAGGGTAT-3′(reverse), and GAPDH, 5′-TCAACGACCACTTTGTCAAGCT-3′ (forward) and 5′-AGCCAAATTCGTTGTCATACCA-3′ (reverse). Data were obtained as Ct values (cycle number at which PCR plots cross a calculated threshold line) and used to determine ΔCt values (Ct of target gene − Ct of housekeeping gene GAPDH). These values were then used to generate mean ΔCt values ± sd for each treatment, which were employed in statistical comparisons. Visual representation of data was carried out by converting ΔCt values to fold change data relative to ΔCt values for untreated MCF-7 cells using the equation 2ΔΔCt.

Statistics

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

Acknowledgments

We are grateful to Gloria Kiel for help in preparation of the manuscript.

Footnotes

This work was supported by National Institutes of Health Grant RO1DK055843.

Disclosure Summary: The authors have no conflict of interest to declare.

First Published Online December 20, 2007

Abbreviations: ChIP, Chromatin immunoprecipitation; DNA-BD, DNA-binding domain; DTT, dithiothreitol; E2, 17β-estradiol; ERα, estrogen receptor-α; ERE, estrogen response element; ERE-BP, ERE-binding protein; GST, glutathione S-transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; hsc, heat-shock cognate protein; hsp, heat-shock protein; IDBP, intracellular vitamin D-binding protein; IEBP, intracellular E2-binding protein; NWP, New World primate; PMSF, phenylmethylsulfonyl fluoride; RLU, relative luminescence unit; shRNA, short hairpin RNA; VDRE-BP, vitamin D response element-binding protein.

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