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Molecular Endocrinology 20 (9): 1982-1995
Copyright © 2006 by The Endocrine Society

Protein Disulfide Isomerase Serves as a Molecular Chaperone to Maintain Estrogen Receptor {alpha} Structure and Function

Jennifer R. Schultz-Norton, W. Hayes McDonald, John R. Yates and Ann M. Nardulli

Department of Molecular and Integrative Physiology (J.R.S.-N., A.M.N.), University of Illinois, Urbana, Illinois 61801; and Department of Cell Biology (H.M. and J.R.Y.), The Scripps Institute, La Jolla, California 92037

Address all correspondence and requests for reprints to: Ann M. Nardulli, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801. E-mail: anardull{at}life.uiuc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The effects of the steroid hormone 17ß-estradiol are mediated through its interaction with the nuclear estrogen receptor (ER). Upon binding 17ß-estradiol, the ER initiates changes in gene expression through its interaction with specific DNA sequences, estrogen response elements (EREs), and recruits coregulatory proteins that influence gene expression. To better understand how estrogen-responsive genes are regulated, we have isolated and identified proteins associated with ER{alpha} when it is bound to the consensus ERE. One of these proteins, protein disulfide isomerase (PDI), has two distinct functions: acting as a molecular chaperone to maintain properly folded proteins and regulating the redox state of proteins by catalyzing the thiol-disulfide exchange reaction through two thioredoxin-like domains. Using a battery of biochemical and molecular techniques, we have demonstrated that PDI colocalizes with ER{alpha} in MCF-7 nuclei, alters ER{alpha} conformation, enhances the ER{alpha}-ERE interaction in the absence and presence of an oxidizing agent, influences the ability of ER{alpha} to mediate changes in gene expression, and associates with promoter regions of two endogenous estrogen-responsive genes. Our studies suggest that PDI plays a critical role in estrogen responsiveness by functioning as a molecular chaperone and assisting the receptor in differentially regulating target gene expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGEN IS CRITICAL for the growth, development, and maintenance of reproductive tissues (1, 2). The actions of estrogen are mediated by two members of the nuclear receptor superfamily, ER{alpha} and ERß. These receptors have functionally conserved regions including a central DNA binding domain (DBD or C domain; Ref. 3) and a carboxy-terminal ligand binding domain (LBD or region E; Ref. 3). Upon binding hormone, the receptor interacts with its cognate recognition sequence in DNA, the estrogen response element (ERE).

Once bound to an ERE, the DNA-bound receptor recruits transcription factors and regulatory proteins that influence ER{alpha}-mediated gene expression. ER{alpha} interacts with the p160 proteins SRC-1, TIF-2/GRIP-1, and AIB-1/RAC3/pCIP/ACTR (4, 5, 6, 7, 8, 9) and other transcription factors such as cAMP response element binding protein-binding protein and p300 (10, 11). Although the interaction of ER{alpha} with these and other coactivators has been widely reported, the effect of DNA binding on receptor-protein interactions has not generally been considered. We have demonstrated that ERE binding increases the association of ER{alpha} with the DNA repair protein 3-methyladenine DNA glycosylase (12), but decreases the association of the receptor with two proteins that inhibit acetylation of histones and ER{alpha}, template activating factor 1ß and pp32 (13, 14). These studies and others (15, 16) support the idea that interaction of ER{alpha} with DNA can influence coregulatory protein recruitment.

To better understand how ER{alpha}-mediated transcription is regulated, we have isolated and identified novel coregulatory proteins from MCF-7 cells that interact with ER{alpha} when it is bound to the consensus ERE. We have characterized the interaction of ER{alpha} with one of these regulatory proteins, protein disulfide isomerase (PDI), using a variety of molecular and biochemical approaches.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PDI Is Associated with ERE-Bound ER{alpha} in Vitro
To isolate endogenously expressed MCF-7 nuclear proteins that interact with ERE-bound ER{alpha}, a modified gel shift assay was performed. This method, developed in our laboratory, used an agarose gel matrix rather than a traditional polyacrylamide gel to allow resolution of large protein-DNA complexes. When MCF-7 nuclear extracts were combined with ERE-containing oligos, we observed a single protein-DNA complex (Fig. 1AGo, lane 2), which was supershifted by an ER{alpha}-specific antibody (lane 3) indicating that ER{alpha} was present in the complex.


Figure 1
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  		Fig. 1. Isolation, Identification, and Expression of PDI

A, 32P-labeled oligos containing the consensus ERE were incubated without (lane 1) or with nuclear proteins from E2-treated MCF-7 cells in the absence (lane 2) or presence (lane 3) of an ER{alpha}-specific antibody and fractionated on an agarose gel. Results shown are representative of 10 independent experiments. B, Human PDI has four regions, a, b, b', and a', which contain thioredoxin-like domains, but only the a and a' domains contain active thiol-disulfide catalase activity (65 ). The acidic carboxy terminus (c) and the sequence and location of two thiol-disulfide isomerase active sites (CGHC) are indicated. C, Nuclear proteins (10 µg) from U2-OS, MDA-MB-231 (231) and MCF-7 breast cancer cells were fractionated on a denaturing acrylamide gel and subjected to Western blot analysis. ER{alpha}, PDI, Sp1, and GAPDH were detected with antibodies specific for each protein. Results are representative of two (GAPDH and Sp1) or six (ER{alpha} and PDI) independent experiments.

 
For large-scale isolation of proteins associated with the ERE-bound receptor, the binding reactions were combined and fractionated on an agarose gel exactly as described for Fig. 1AGo except that the reaction size was increased 15-fold. An equal amount of MCF-7 nuclear extract alone or in combination with oligos containing a nonspecific DNA sequence were run in parallel. The protein-DNA complexes formed with the ERE-containing oligos or an equal number of control lanes that comigrated with the protein-DNA complex were excised and the proteins were identified by mass spectrometry analysis. One of the proteins identified in the presence, but not in the absence of the ERE-containing oligos, was the 55-kDa protein disulfide isomerase (PDI). PDI acts as a catalase of thiol-disulfide exchange reactions through two active sites (CGHC), one each in the amino (a) and carboxy (a') termini, that catalyze disulfide bond formation, reduction, and isomerization (Fig. 1BGo; and Refs. 17 and 18). In addition to its ability to act as a thiol-disulfide catalase, PDI also acts as a molecular chaperone (19, 20, 21, 22, 23). The b' domain binds peptides 10–15 amino acids in length (24) and a region overlapping the a' and c domains binds larger peptides (25). Interestingly, Landel et al. (26) previously reported that PDI was one of four proteins that copurified with ER{alpha} but did not determine whether PDI alone was capable of interacting with ER{alpha} or influencing its activity.

PDI Is Present in the Endoplasmic Reticulum and in the Nucleus of MCF-7 Cells
PDI has been typically been described as a ubiquitously expressed, endoplasmic reticulum-associated protein (27). However, we had identified it as a protein that was present in our MCF-7 nuclear extracts. Thus, we examined the levels of PDI in cultured cell lines that have previously been used to study estrogen responsiveness (13, 14, 28, 29, 30, 31, 32). Nuclear extracts from U2 osteosarcoma (U2-OS), MDA-MB-231 breast cancer, and MCF-7 breast cancer cells were subjected to Western analysis. As expected, substantial levels of ER{alpha} were observed only in MCF-7 cells (Fig. 1CGo). PDI was detected in the nuclear extracts of all three cell lines, but the level of PDI in MDA-MB-231 cells was less than was observed in U2-OS or MCF-7 cells. Although the levels of ER{alpha} and PDI varied in the cells tested, similar amounts of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Sp1 were seen in all three cell lines.

Fluorescence microscopy was then used to localize the endogenously expressed PDI, ER{alpha}, and the compartment-specific proteins, calnexin and lamin A/C in intact MCF-7 cells. As anticipated, PDI colocalized with the endoplasmic reticulum-specific protein calnexin (Fig. 2AGo). However, using an antibody to the nuclear matrix protein lamin A/C, we also detected colocalization with PDI (Fig. 2BGo). We did, in fact, observe colocalization of PDI in the nucleus with ER{alpha} (Fig. 2CGo). These findings complemented our Western analysis, which demonstrated that PDI was present in MEF-1 nuclear extracts (Fig. 1CGo). When purified PDI or ER{alpha} was preadsorbed to their respective primary antibodies or no primary antibody was included, little or no immunofluorescence was observed (data not shown). Thus, although PDI is more highly expressed in the endoplasmic reticulum, it is also present in the nucleus of MCF-7 cells.


Figure 2
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  		Fig. 2. Localization of PDI

MCF-7 cells were fixed with methanol, permeabilized with acetone, and incubated with antibodies specific to PDI and either calnexin (A), lamin A/C (B), or ER{alpha} (C). The three panels show each protein stained separately as well as together (Merge). Colocalized proteins are blue. Results are representative of multiple fields in three independent experiments.

 
PDI Alters Estrogen-Mediated Transactivation
Given the association of PDI with the ERE-bound ER{alpha} in our agarose gel experiments and the colocalization of ER{alpha} and PDI in the nucleus, we were interested in determining whether PDI might influence estrogen-mediated gene expression. Transient transfections were carried out in MDA-MB-231 cells with an ER{alpha} expression vector, a luciferase reporter plasmid containing two copies of the consensus ERE, and increasing amounts of the PDI expression vector. As seen in Fig. 3AGo, decreases in 17ß-estradiol (E2)-mediated transcription were observed with increasing amounts of a PDI expression vector, although no significant changes in transcription were observed in the absence of hormone. Decreased transcription was also observed in MCF-7 cells, which relied on endogenously expressed, not transfected, ER{alpha} (Fig. 3BGo). Furthermore, PDI had similar effects on ER{alpha}-mediated transcription when MCF-7 cells were transfected with a luciferase reporter plasmid containing a single copy of the ERE (data not shown). Thus, PDI decreased estrogen-dependent transactivation in both mammary cell lines tested.


Figure 3
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  		Fig. 3. Effect of PDI on a Transiently Transfected, Estrogen-Responsive Reporter Plasmid

Transient transfections were performed in human MDA-MB-231 (A) or MCF-7 (B) cells with a reporter plasmid containing two copies of the consensus ERE, an ER{alpha} expression vector (panel A only), and increasing amounts of a PDI expression vector. A parental expression vector lacking the PDI sequence was included as needed to maintain constant DNA levels. 10 nM E2 was included as indicated. Data from at least three independent experiments performed in duplicate were combined and are presented as the mean ± SEM. Significant differences in estrogen responsiveness in the presence and in the absence of PDI are indicated by an asterisk (*, P < 0.05).

 
PDI Influences Endogenous Estrogen-Responsive Gene Expression in MCF-7 Cells
Although overexpression of PDI altered ER{alpha}-mediated transcription, we felt it was important to consider whether the PDI expressed in MCF-7 cells affects expression of endogenous estrogen-responsive genes. To document the effects of endogenously expressed PDI in MCF-7 cells, RNA interference experiments were carried out using control small interfering RNA (siRNA), which was directed against Renilla luciferase, or PDI-specific siRNA. As expected, E2 treatment of MCF-7 cells increased pS2, progesterone receptor (PR), and Bcl-2 mRNA levels when control siRNA was used (Fig. 4AGo). Importantly, the PDI-specific siRNA, which targets exons 2 and 3, effectively reduced PDI mRNA levels. When PDI levels were knocked down, PR and Bcl-2 mRNA levels increased, but pS2 mRNA levels declined suggesting that PDI differentially regulates estrogen-responsive gene expression.


Figure 4
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  		Fig. 4. Effect of PDI on Endogenous Gene Expression

MCF-7 cells were transfected with 50 pmol (A) or 0, 50, or 100 pmol (B) of double-stranded control or PDI-specific siRNA and treated with 10 nM E2 or ethanol vehicle. A, RNA was harvested and cDNA was synthesized. Real-time PCR was performed using primers specific to 36B4, PDI, ER{alpha}, pS2, PR, or Bcl-2 mRNA sequences. Standard curves were derived for each primer set in each experiment. The relative ng equivalent of RNA was obtained for each sample based on the standard curve. Data are reported as the average of two replicates ± SD. Some error bars are too small to be visible. A representative of four independent experiments is shown. B, Cells were lysed and subjected to Western analysis with antibodies specific to PDI, ER{alpha}, CatD, PRA and PRB, or GAPDH. Results are representative of three independent experiments.

 
Because there is not always a good correspondence in mRNA and protein expression, we determined the effect of knocking down PDI on the expression of proteins regulated by E2. In doing so, we would be assured that the functional moiety required for biological activity was present. MCF-7 cells were transfected with no, control, or PDI-specific siRNA and the levels of PDI, ER{alpha}, cathepsin D (CatD), and PR protein were monitored. In the absence of siRNA, PRs A and B (PRA and PRB), and CatD levels increased and the level of ER{alpha} decreased in the presence of 10 nM E2 (Fig. 4BGo, compare lanes 1 and 7 with lanes 4 and 10). When control siRNA was used, similar patterns were detected in protein expression (lanes 1–6).

When PDI-specific siRNA was used, PDI levels were dramatically decreased in the absence (lanes 7–9) and in the presence (lanes 10–12) of E2. The decreased expression of PDI in MCF-7 cells led to an increase in ER{alpha} protein levels in the absence and in the presence of E2 (compare lanes 1–6 with lanes 7–12), but E2-mediated down-regulation of the receptor was still observed. CatD expression was decreased in the absence (lanes 7–9) and in the presence (lanes 10–12) of E2. Although PRA expression increased substantially in the presence, but not in the absence, of E2 (compare lanes 7–9 with 10–12), PRB levels were modestly increased in the absence (lanes 7–9), but not in the presence of E2 (lanes 10–12). Neither siRNA nor E2 affected GAPDH expression. These studies document the importance of PDI in altering the expression of proteins regulated by E2 in MCF-7 cells.

PDI Is Associated with Endogenous Estrogen-Responsive Genes
The ability of PDI to associate with the ERE-bound receptor, colocalize with ER{alpha} in intact MCF-7 nuclei, and influence ER-mediated gene expression suggested that PDI might associate with estrogen-responsive genes to regulate transcription. Thus, chromatin immunoprecipitation (ChIP) assays were performed in MCF-7 cells that had been treated with ethanol vehicle or E2 for 2 or 24 h to examine the association of ER{alpha} and PDI with a region of the pS2 gene containing an imperfect ERE or one of three estrogen-responsive regions of the PR gene containing either Sp1 or activator protein (AP)-1 sites (28, 29, 30, 31, 32, 33). After 2 h of hormone treatment, there was a dramatic increase in the association of ER{alpha} with the pS2 ERE region, which was reduced at 24 h (Fig. 5AGo). Such changes in the association of ER{alpha} with the pS2 ERE region have been attributed to cycling of the receptor on and off chromatin (34, 35). Increases in association of ER{alpha} were also observed with the regions of the PR gene containing the +571 ERE/Sp1 site and the +90 and +745 AP-1 sites after 24 h of estrogen treatment. However, we did not observe changes in ER{alpha} association with a region ~3500 bp upstream of the ERE in the pS2 gene that contained no identifiable ERE, AP-1 or Sp1 sites. Interestingly, a similar pattern of association with these estrogen-responsive genes was observed when the PDI-specific antibody was used (Fig. 5BGo). Taken together with the transfection and siRNA data, these results demonstrate that PDI not only influences estrogen-mediated gene expression, but is also associated with endogenous estrogen-responsive genes.


Figure 5
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  		Fig. 5. Association of PDI with Endogenous, Estrogen-Responsive Genes

Sheared chromatin from MCF-7 cells that had been treated with ethanol vehicle or 10 nM E2 for 2 or 24 h was immunoprecipitated with an ER{alpha}- (A) or PDI- (B) specific antibody. DNA was isolated and real time PCR was performed to monitor the association of ER{alpha} and PDI with the imperfect ERE or upstream control region of the pS2 gene or the +571 ERE/Sp1, +90 AP-1, or +745 AP-1 site in the PR gene. Standard curves were derived for each primer set and the relative copy number for each sample was obtained based on the standard curve. The average of two replicates ± SD is shown and is representative of three independent experiments. Some error bars are too small to be visible.

 
PDI Interacts with ER{alpha}
One way that PDI might influence estrogen-mediated transcription is through interacting with ER{alpha}. To determine whether the receptor interacted with PDI, his-tagged PDI was immobilized, purified, and then incubated with in vitro-translated, 35S-labeled full-length or truncated ER{alpha}. As seen in Fig. 6AGo, full-length ER{alpha} as well as ABC, CD, and DEF were able to bind to PDI in the presence and absence of E2, but AB failed to bind to PDI. To confirm that PDI interacted with ER{alpha}, flag-tagged ER{alpha} (Fig. 6BGo) and CD (Fig. 6CGo) were immobilized, purified, and incubated with in vitro-translated, 35S-labeled PDI. Both full-length ER{alpha} and CD retained PDI thereby confirming the ER{alpha}-PDI interaction.


Figure 6
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  		Fig. 6. Interaction of PDI with ER{alpha}

A, Nickel-affinity resin without (lane 2) or with purified his-tagged PDI (lanes 3 and 4) was combined with in vitro-translated 35S-labeled full-length or truncated ER{alpha} or unprogrammed lysate (UPL). B and C, Flag-affinity resin without (lane 2) or with purified flag-tagged ER{alpha} (B, lanes 3 and 4) or CD (C, lanes 3 and 4) was combined with in vitro-translated 35S-labeled full-length PDI or UPL. E2 was added as indicated. Proteins were separated on a denaturing gel and detected by autoradiography. 10% input was included for reference (lanes 1). Results are representative of at least three independent experiments.

 
PDI Enhances ER{alpha}-ERE Complex Formation
Because PDI influences the association of nuclear factor-{kappa}B and AP-1 proteins with their cognate recognition sequences (36), it seemed plausible that PDI might alter estrogen-mediated transcription by influencing the ability of ER{alpha} to bind to DNA. When gel mobility shift assays were performed with a constant amount of purified ER{alpha} and increasing amounts of purified PDI, there was a dose-dependent increase in protein-DNA complex formation (Fig. 7Go, lanes 2–5). This complex was supershifted by an ER{alpha}-specific antibody (lane 6), but not a PDI-specific antibody (lane 7), demonstrating that PDI significantly enhanced ER{alpha}-DNA complex formation but was not present in this complex. Although this was a somewhat unexpected result given the association of PDI with the ERE-bound receptor in vitro (Fig. 1AGo) and with endogenous estrogen-responsive genes in MCF-7 cells, these combined studies suggest that, although PDI can bind to the free receptor, other proteins may be required to stabilize the association of PDI with the ERE-bound receptor. PDI also enhanced the interaction of a truncated ER{alpha} protein containing only the DBD and hinge region, but was not present in the DBD-DNA complex (data not shown). These findings are consistent with earlier studies in which PDI altered protein-DNA complex formation of both nuclear factor-{kappa}B and AP-1 proteins with their cognate recognition sequences, but did not form a ternary complex with these proteins (36). When PDI was included, but ER{alpha} was omitted from the binding reaction, no protein-DNA complex was observed (lanes 8–9). Thus, PDI enhanced the ER{alpha}-DNA interaction but did not interact with the ERE-containing oligos. Although a minor, higher mobility protein-DNA complex was sometimes present when higher amounts of PDI were included in the binding reaction (lanes 5–9), the inability of ER{alpha}- or PDI-specific antibodies to alter the formation of this complex suggested that it was a nonspecific complex (lanes 6, 7, and 9).


Figure 7
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  		Fig. 7. Effect of PDI on ER{alpha}-ERE Complex Formation

32P-labeled oligos containing the consensus ERE were incubated with 50 fmol (lane 1) or 10 fmol (lanes 2–7) of purified ER{alpha}. Purified PDI (lanes 3–9) and ER{alpha}- (lane 6) or PDI- (lanes 7 and 9) specific antibody (Ab) were added to the binding reaction as indicated. Bound and unbound 32P-labeled oligos were fractionated on a nondenaturing polyacrylamide gel and visualized by autoradiography. Complexes containing ER{alpha} are indicated (ER{alpha}->). Results are representative of four independent experiments.

 
PDI Enhances ER{alpha}-DNA Interactions in the Presence of an Oxidizing Agent
Oxidation of ER{alpha} causes the release of zinc and formation of disulfide bonds thereby disrupting DBD structure (37, 38) and reducing the interaction of ER{alpha} with the ERE (37, 38, 39, 40, 41). To determine whether the isomerase activity of PDI might protect ER{alpha} from oxidation and enhance the receptor-DNA interaction, ER{alpha} was incubated with the thiol oxidizing agent diamide in the absence and in the presence of dithiothreitol (DTT), which reduces disulfide bonds. Although increasing amounts of diamide abolished the ER{alpha}-ERE interaction (Fig. 8AGo, lanes 1–4), addition of DTT restored the ability of the receptor to bind to DNA (lanes 5–7).


Figure 8
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  		Fig. 8. Effect of Oxidizing/Reducing Agents and PDI Mutations on the ER{alpha}-ERE Interaction

A, 32P-labeled oligos containing the consensus ERE were incubated with 50 fmol purified ER{alpha} in the absence (lane 1) or in the presence of increasing (lanes 2–4) or constant (lanes 4–7) amounts of diamide and increasing amounts of DTT (lanes 5–7). B, 32P-labeled oligos containing the consensus ERE were incubated with 50 fmol purified ER{alpha} in the absence (lane 1) or in the presence of 2 mM diamide (lanes 2–5) and increasing amounts of PDI (lanes 3–5). C, 32P-labeled oligos containing the consensus ERE were incubated with 25 fmol purified ER{alpha} and wild type PDI (WT, lane 2), a PDI isomerase mutant ({Delta}C1,2, lane 3), a PDI isoleucine mutant (I272W, lane 4) or a PDI isomerase/isoleucine double mutant ({Delta}C1,2-I272W, lane 5). Complexes were fractionated on nondenaturing polyacrylamide gels and visualized by autoradiography. Complexes containing ER{alpha} are indicated (->). Results are representative of three (A), six (B), or five (C) independent experiments.

 
It has been suggested that proteins with thiol-disulfide catalase activity such as thioredoxin may alter the oxidation state of ER{alpha} and enhance receptor-DNA interactions in the presence of diamide (40). Because PDI contains two thioredoxin-like domains, we determined whether PDI might influence the receptor-DNA interaction after ER{alpha} had been oxidized. Gel mobility shift assays demonstrated that PDI did increase the interaction of ER{alpha} with ERE-containing DNA after diamide treatment (Fig. 8BGo). This enhanced ER{alpha}-ERE interaction was also observed when PDI was added before diamide exposure (data not shown) indicating that PDI has a protective effect on the receptor-DNA interaction. The increase in ER{alpha}-DNA complex formation in the presence of PDI demonstrates that PDI enhances the interaction of ER{alpha} with DNA even in the presence of a potent oxidizing agent.

Eliminating the Isomerase Activity of PDI Does Not Affect ER{alpha}-ERE Complex Formation
PDI has separable isomerase activity, which resides in a and a' domains, and chaperone activity, which involves multiple PDI domains (17, 18). PDI had enhanced ER{alpha}-ERE complex formation after exposure to the oxidizing agent diamide, but it was unclear whether the isomerase or chaperone activity of PDI was responsible for fostering this interaction.

To determine whether the increased ER{alpha}-ERE interaction was due to the isomerase activity of PDI, we used a PDI mutant protein in which cysteine residues in the thioredoxin active sites were replaced by serines to eliminate the isomerase activity of PDI (18). Interestingly, the ability of this mutant PDI to foster the ER{alpha}-ERE interaction was similar to that of wild type PDI (Fig. 8CGo, compare lanes 2 and 3). Furthermore, the isomerase mutant was as effective as wild-type PDI in restoring the receptor-DNA interaction after diamide exposure (data not shown). These findings are consistent with earlier studies in which alkylation inhibited the isomerase activity of PDI but had no effect on its ability to interact with a synthetic peptide (20).

In addition to its isomerase activity, PDI serves as a molecular chaperone and catalyzes the refolding of numerous proteins. However, when an isoleucine in the b' domain of PDI is replaced by a tryptophan, the mutant protein is no longer able to refold {Delta}-somatostatin (42). When we tested this PDI mutant in gel shift experiments, its ability to enhance the ER{alpha}-ERE interaction was similar to that of wild type PDI (Fig. 8CGo, compare lanes 2 and 4). We also saw similar binding with PDI containing both the isomerase and isoleucine mutations (lane 5). Like the isomerase PDI mutant, both the isoleucine and the isoleucine/isomerase PDI double mutants enhanced estrogen-mediated transcription to the same extent as wild-type PDI (data not shown). Taken together, these findings suggest that although the isomerase activity of PDI does not play a major role in the enhanced ER{alpha}-ERE interaction, it may be involved in responding to oxidative stress. Furthermore, if the chaperone activity of PDI is involved in the enhanced ER{alpha}-DNA interaction, a more extended region of PDI must be required.

PDI Alters ER{alpha} Structure
A molecular chaperone is able to interact with and alter/stabilize another protein’s structure. Because of its ability to refold proteins, PDI has been described as a molecular chaperone (22). The stability of the zinc fingers in the ER{alpha} DBD is critical for functional interaction of ER{alpha} with DNA and modulation of estrogen-responsive gene expression. We reasoned that if PDI interacted with the receptor and altered its structure, we might be able to detect perturbations in ER{alpha} structure by monitoring the susceptibility of the receptor to limited protease digestion. When no protease was present, only the full-length 66-kDa ER{alpha} was detected by ER{alpha}-specific antibodies in the absence and in the presence of PDI (Fig. 9Go, A–D, lanes 1 and 2). When the receptor was subjected to limited chymotrypsin digestion and detected with an antibody directed against the amino terminus of ER{alpha}, the digestion patterns were similar in the absence and in the presence of PDI (panel A, lanes 3 and 4). When an antibody to the ER{alpha} hinge region was used for detection, subtle differences in the distribution of the full-length and 15-kDa chymotrypsin digestion product were observed (panel B). The 15-kDa product was more prominent in the absence of PDI (panel B, lane 3) and the full-length ER{alpha} was more prominent in the presence of PDI (panel B, lane 4). Thus, relatively modest differences in digestion patterns were observed with chymotrypsin in the absence and in the presence of PDI.


Figure 9
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  		Fig. 9. Effect of PDI on ER{alpha} Structure

ER{alpha} was incubated without (lanes 1 and 3) or with PDI (lanes 2 and 4), subjected to limited protease cleavage (lanes 3 and 4), fractionated on a denaturing acrylamide gel, and subjected to Western blot analysis using an ER{alpha}-specific antibody. Proteases and antibodies used were chymotrypsin (Chymo) and an antibody that recognizes the amino terminus of ER{alpha} (A), chymotrypsin and an antibody that recognizes the hinge region of ER{alpha} (B), Proteinase K (Prot K) and an antibody that recognizes the amino terminus of ER{alpha} (C), or V8 protease (V8 Prot) and an antibody that recognizes the LBD of ER{alpha} (D). Approximate molecular masses (kDa) of digestion products are indicated. Results are representative of three independent experiments.

 
The somewhat limited digestion of ER{alpha} with chymotrypsin in the presence of PDI might suggest that the interaction of PDI with the receptor had occluded the epitope(s) targeted by this protease so that the digestion process was inhibited by the ER{alpha}-PDI interaction. However, when ER{alpha} was exposed to Proteinase K or V8 protease, more extensive digestion of the receptor was observed in the presence of PDI. When Proteinase K was used, a 55-kDa digestion product was observed (panel C, lane 3), but in the presence of PDI a 50-kDa product was detected (panel C, lane 4). Dramatic differences in digestion profiles were also evident when the receptor was exposed to V8 protease (panel D). In the absence of PDI, 42- and 35-kDa digestion products were more prominent (panel D, lane 3) and in the presence of PDI, a 20-kDa product was more prominent (panel D, lane 4). Thus, PDI did not simply limit the accessibility of the proteases to the receptor, but modified ER{alpha} structure so that the receptor epitopes targeted by Proteinase K and V8 protease were, in fact, more accessible. The differential susceptibility of ER{alpha} to three different proteases in the absence and in the presence of PDI suggests that PDI interacts with ER{alpha} and alters its conformation so that different ER{alpha} surfaces are exposed. Thus, by definition, PDI functions as a molecular chaperone for ER{alpha}.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
During times of oxidative stress, cellular proteins unfold and molecular chaperones are required to restore unfolded proteins to their native conformation (27). One protein that assists in the cellular response to stress is the multifunctional protein PDI. PDI catalyzes the formation, reduction, and isomerization of disulfide bonds, functions as a chaperone to prevent the aggregation of unfolded substrates, and serves as a subunit of prolyl 4-hydroxylase and microsomal triglyceride transferase (43). We have identified additional new functions for PDI. We show that PDI interacts with ER{alpha}, increases the capacity of ER{alpha} to bind to DNA, enhances ER{alpha}-ERE complex formation after exposure to the oxidizing agent diamide, alters ER{alpha} structure, colocalizes with ER{alpha} in MCF-7 nuclei, influences estrogen-regulated gene expression, and associates with endogenous estrogen-responsive genes in MCF-7 cells.

We were initially surprised to find PDI in a complex with the ERE-bound ER{alpha} because PDI has generally been considered to be a protein that resides primarily in the endoplasmic reticulum. One rationale for the high concentration of PDI in the endoplasmic reticulum would be to assist in establishing and reshuffling disulfide bonds during protein synthesis. However, as proteins are distributed to various cellular compartments and cells are exposed to oxidative stress, the isomerase and chaperone activities of PDI could help to maintain the structural integrity of the existing pool of proteins. PDI has been localized to cellular compartments other than the endoplasmic reticulum including the nuclear matrix (43, 44). Our Western analysis and immunofluorescence studies of endogenously expressed PDI in MCF-7 cells support the idea that PDI is present in the nucleus of these cells. Taken together, there is substantial accumulated evidence that PDI is not just confined to the endoplasmic reticulum, but that it is present in the nucleus as well. We believe that the nuclear-localized PDI may help to maintain ER{alpha} structure and function.

An important feature of the ability of PDI to function as an isomerase and a molecular chaperone is its capacity to interact with a variety of proteins (19, 20, 21, 22, 23). The b' domain of PDI binds to peptides 10–15 amino acids in length (24), but a significantly larger portion of PDI is required to interact with full-length proteins (25). Isoleucine 272 is critical for the interaction of PDI with {Delta}-somatostatin (42). However, our studies indicate that replacement of this isoleucine with a tryptophan does not affect the ability of PDI to interact with ER{alpha} or enhance the ER{alpha}-DNA interaction and that a more extended region of PDI must be involved in the ER{alpha}-PDI interaction. Darby et al. (45) have suggested that multiple PDI domains are required to interact with full-length proteins. The multidomain structure of PDI may allow it to interact with and alter the conformation of a wide variety of proteins. Thus, one might predict that PDI would associate with an extended region of ER{alpha}. In fact, we did find that this was the case.

We have demonstrated that exposure of ER{alpha} to the oxidizing agent diamide has a profound effect on the receptor’s ability to bind to DNA and that PDI is able to partially restore the ER{alpha}-ERE interaction after exposure to diamide. PDI levels increase in response to hypoxia in glial cells and in response to oxidizing agents in yeast (46, 47), suggesting that PDI helps to maintain protein structure and function during times of cellular stress. Interestingly, although PDI appears to be ubiquitiously expressed, higher levels of PDI mRNA have been observed in the uterus, pituitary, and liver, three ER{alpha}-positive and estrogen-responsive tissues (48).

The labile nature of the ER{alpha} DBD zinc fingers has been exploited with the use of electrophilic agents that disrupt DBD structure, decrease ER{alpha} binding and estrogen-responsive gene expression, and limit estrogen-dependent proliferation of MCF-7 cells in nude mice (37, 41). Thus, rather than using a hormonal ligand to decrease estrogen action, it is possible to suppress estrogen responsiveness by decreasing ER{alpha} binding to DNA. Liang et al. (39) reported a significant decrease in the ability of ER{alpha} from primary mammary tumors to bind to DNA but found that DNA binding could sometimes be restored after exposure of the receptor to DTT. Thus, oxidation of the receptor could inhibit ER{alpha} binding to DNA, result in hormone insensitivity of mammary tumors, and limit the effectiveness of antiestrogen therapy. Just as DTT restores the ER{alpha}-ERE interaction in vitro, PDI could serve as an endogenously expressed protein in cells to maintain the DNA-binding competence of ER{alpha}. These findings suggest that by regulating the redox state of the receptor, it may be possible to modulate transcription of estrogen-responsive genes and influence the receptor’s ability to bind to DNA. In support of this hypothesis, Hayashi et al. (40) have reported that transcription of an ERE-containing reporter plasmid and the estrogen-responsive pS2 gene is decreased when ZR-75-1 breast cancer cells are exposed to hydrogen peroxide-induced oxidative stress. Interestingly, however, estrogen responsiveness is restored by overexpression of the thiol/disulfide isomerase thioredoxin. Given that PDI contains two thioredoxin-like domains, it is possible that PDI may be able to influence ER{alpha}-mediated transcription in a similar manner.

We have shown that PDI alters transcription of an ERE-containing reporter plasmid in two different cell lines. More importantly, we have demonstrated that native PDI associates with regulatory regions of endogenous estrogen-responsive genes in MCF-7 cells and influences mRNA and protein expression. Taken together with our siRNA studies, we have provided substantial evidence that PDI plays an important role in differentially altering the expression of endogenous estrogen-responsive genes. The multiple cis elements residing in promoters of estrogen-responsive genes and their corresponding trans-acting factors must also participate in the differential responsiveness of individual genes to PDI.

Another nuclear receptor that is subject to oxidative stress is the glucocorticoid receptor. It has been suggested that thioredoxin serves as an auxiliary factor to decrease oxidation of the glucocorticoid receptor, maintain its capacity to bind to DNA, and modulate transcription (49). Thus, ER{alpha} is not the only nuclear receptor affected by oxidative stress. In addition to nuclear receptors, a number of other transcription factors including Sp1 and AP-1 proteins are sensitive to oxidative stress and exhibit decreased DNA binding in the presence of diamide (50). Moreover, decreased transcription has been observed with Sp1-containing promoters when cells are exposed to oxidative stress. One would predict that other nuclear transcription factors would be similarly affected and would rely on proteins such as thioredoxin and PDI to maintain their capacity to bind to DNA and modulate gene expression. Given its interaction with a wide variety of proteins and the effect of oxidation on binding of multiple transcription factors to DNA, PDI could have profound effects on more global gene expression.

Our data support the idea that PDI functions as a molecular chaperone for ER{alpha}. PDI-induced alterations in ER{alpha} structure were detected using three proteases with different specificities. There is also substantial evidence from these studies and others (37, 39, 40) that ER{alpha} is subject to oxidative damage. Although PDI was able to enhance ER{alpha} binding to ERE-containing DNA, it failed to fully restore the ER{alpha}-ERE interaction after diamide treatment. More importantly, a PDI mutant lacking isomerase activity was as effective in enhancing the ER{alpha}-ERE interaction as wild-type PDI. These findings suggest that the chaperone activity of PDI is more important in refolding and maintaining ER{alpha} structure than its isomerase activity but do not rule out the importance of isomerase activity of PDI in responding to oxidative stress.

In addition to facilitating the interaction of ER{alpha} with ERE-containing DNA, PDI alters the accessibility of various ER{alpha} epitopes and could thereby influence the interaction of ER{alpha} with coregulatory proteins. Thus, PDI may help to sustain hormone responsiveness by maintaining/restoring the structural integrity of ER{alpha} so that it can effectively bind to DNA and altering the accessibility of various ER{alpha} epitopes so that different cohorts of accessory proteins and transcription factors are recruited to the DNA-bound receptor. It has been estimated that zinc finger proteins account for as much as 1% of the human genome (51). Through its chaperone and isomerase functions, PDI may not only influence estrogen-responsive gene expression but also have far-ranging effects on transcription by altering and/or stabilizing the structural integrity of other nuclear receptor family members, transcription factors, and regulatory proteins.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Isolation and Identification of PDI Using Agarose Gels and Gel Mobility Shift Assays
Oligos containing the Xenopus laevis vitellogenin A2 DNA sequence (5'-GAT TAA CTG TCC AAA GTC AGG TCA CAG TGA CCT GAT CAA AGT TAA TGT AA-3' and 5'-TTA CAT TAA CTT TGA TCA GGT CAC TGT GAC CTG ACT TTG GAC AGT TAA TC-3') were annealed and end labeled with {gamma}32P-ATP. The radiolabeled ERE-containing oligos were incubated for 15 min at room temperature in 15 mM Tris (pH 7.9), 0.2 mM EDTA, 10% glycerol, 80 mM KCl, 2 µg of poly(deoxyinosine/deoxycytosine), 1 µg salmon sperm DNA, 1 mM MgCl2, and 4 mM DTT in a final volume of 20 µl with 20 µg of nuclear extract prepared from MCF-7 cells, which had been treated with 10 nM E2 for 20 min. Samples were fractionated on a 1% low melt agarose (Bio-Rad, Hercules, CA) gel and visualized by autoradiography.

For large-scale isolation of proteins associated with the ERE-bound receptor, the binding reactions were combined and fractionated on an agarose gel exactly as described above except that the reaction size was increased 15-fold. The radiolabeled DNA and protein complexes were excised from the gel matrix, diluted with an equal volume of water and frozen at –80 C. Frozen agarose was thawed and pelleted. Proteins were precipitated from the supernatant using acetone (80% final concentration), dried, and analyzed by mass spectrometry as previously described (13). Two peptides (ADAPEEEDHVLVLRKS and KSNFAEALAAHKY), which comprised 4.9% of the PDI amino acid sequence, were identified using the SEQUEST database (52). Two controls were used to ensure that PDI was associated with the ER-DNA complex in our agarose gel experiments and did not simply comigrate with the receptor-DNA complex. MCF-7 nuclear extracts were fractionated in the presence and in the absence of annealed, 32P-labeled nonspecific oligos (5'-CTA GAT TAC TTC TCA TGT TAG ACA TAC TCA GAT CTA GAC ATA CTC AGA TC-3' and 5'-GAT CTG AGT ATG TCT AGA TCT GAG TAT GTC TAA CAT GAG AAG TAA TCT AG-3') and run in parallel with the ERE-containing oligo and MCF-7 nuclear extracts. PDI was not present in the absence of oligos or in the presence of the labeled oligo containing a nonspecific sequence, but was present when ERE-containing oligos were used.

For polyacrylamide gel shifts, 10–50 fmol of purified ER{alpha} and 0.5–10 µg of purified wild-type or mutant PDI were incubated in 15 mM Tris (pH 7.9), 0.2 mM EDTA, 10% glycerol, 20 mM KCl, 10 nM E2, and 2 mM DTT for 10 min on ice with 100 ng of poly(deoxyinosine/deoxycytosine) in a final volume of 20 µl. BSA, ovalbumin, and KCl were included as needed to maintain constant protein and salt concentrations. ER{alpha} was expressed and purified to near homogeneity as we have previously described (53). 0.5–2 mM N,N,N',N'-tetramethylazodicarboxamide (diamide) and 1–5 mM DTT were included in binding reactions as indicated and incubated at 4 C for 20 min. For antibody supershift experiments, the PDI-specific monoclonal antibody, MA3–018 (Affinity Bioreagents, Golden, CO) or ER{alpha}-specific monoclonal antibody SC-8002 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added to the binding reaction mixture and incubated for 10 min on ice before addition of DNA. Radiolabeled ERE-containing oligos were added to the binding reactions and incubated for 10 min at room temperature before fractionation on low ionic strength polyacrylamide gels (54) at 4 C with buffer recirculation. Radioactive bands were visualized by autoradiography.

Western Blots
Nuclear extracts were prepared from U2-OS, MDA-MB-231 breast cancer, or MCF-7 breast cancer cells as previously described (55). Ten micrograms of nuclear proteins were fractionated on a 10% sodium dodecyl sulfate polyacrylamide gel, transferred to nitrocellulose (56), and detected with the ER{alpha}-specific rabbit polyclonal antibody sc-543 (Santa Cruz Biotechnologies) or the PDI-specific MA3–018. The blots were probed with a horseradish peroxidase-conjugated secondary antibody and developed using a chemiluminescent detection system as previously described (57).

Expression and Purification of his-Tagged Wild-Type and Mutant PDI Proteins
Bacterial expression vectors for his-tagged wild-type PDI and an isomerase PDI mutant were graciously provided by L. Ruddock and R. Rudolph (University of Oulu, Finland and Martin-Luther Universitat Halle-Wittenberg, Institut Fur Biotechnologie, Germany; Ref. 18). An isoleucine PDI mutant and an isoleucine/isomerase double PDI mutant were synthesized using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) to create an isoleucine to tryptophan mutation at residue 272 alone or in combination with the isomerase mutation. All mutations were verified by DNA sequencing. Purified plasmids were transformed into Escherichia coli BL21DE3pLysS cells, which were then induced with 1 mM IPTG at 37 C for 3 h, chilled on ice for 5 min, and pelleted at 4700 x g for 5 min at 4 C. Cells were lysed with one freeze/thaw cycle. Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidizole) was added to the cell lysate and incubated for 15 min at 4 C. The lysate was centrifuged at 142,000 x g for 30 min at 4 C and the pellet was discarded. The supernatant was diluted with one half volume of lysis buffer and incubated with Ni-NTA agarose beads (QIAGEN, Valencia, CA) with rotation for 1 h at 4 C. The beads were washed three times with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidizole, and 0.5% Triton X-100). His-PDI was eluted with 50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidizole. Protein purity was assessed on Coomassie-stained gels. Protein concentrations were determined using the Bio-Rad protein with BSA as a standard.

Pull-Down Assay Using in Vitro-Translated Proteins
His-tagged PDI was expressed, immobilized on Ni-NTA (QIAGEN), washed as described above, and resuspended in wash buffer. The ER{alpha} expression vectors pBSK-ER{alpha}, pBSK-ER{alpha}(ABC), pBSK-ER{alpha}(AB), and pBSK-ER{alpha}(DEF), kindly provided by Benita Katzenellenbogen (University of Illinois, Urbana, IL; Refs. 58 and 59) and pET21b(+):Flag:hER{alpha}DBD (CD) generously provided by David Shapiro (University of Illinois, Urbana, IL; Ref. 60) were used to synthesize 35S-labeled full-length ER{alpha} and the truncated ER{alpha} proteins ABC, AB, CD, and DEF in vitro. 35S-labeled proteins were synthesized using the TNT T7 Quick Coupled Transcription/Translation system (Promega, Madison, WI) and incubated at 4 C for 45 min with the immobilized his-PDI in wash buffer with or without 10 µM E2. After two washes with wash buffer, proteins were eluted with 50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidizole, separated by SDS-PAGE, and subjected to autoradiography for 1–2 d.

Flag-tagged full-length ER{alpha} and CD was expressed in Sf9 cells and bacteria, respectively, as previously described (53, 61), immobilized on M2-agarose (Sigma, St. Louis, MO), and washed with purification buffer [20 mM Tris (pH 7.5), 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 10% glycerol]. pCDNA3.1(+)PDI, kindly provided by Kari Kivirikko (University of Oulu, Finland; Ref. 62), was used to synthesize 35S-labeled PDI using the TNT T7 Quick Coupled Transcription/Translation system (Promega). 35S-labeled PDI was incubated with immobilized ER{alpha} proteins at 4 C for 45 min in purification buffer with or without 10 µM E2. After three washes with purification buffer, proteins were eluted with 20 mM Tris, 400 mM NaCl, 0.2 mM EDTA, 10% glycerol, 0.1% Nonidet P-40 (NP-40), and 2 mM DTT with 500 µg/ml ovalbumin and 200 µg/ml M2 Peptide (Sigma). Eluted proteins were separated by SDS-PAGE, and subjected to autoradiography for 1–2 d.

Transient Transfections
MDA-MB-231 cells were maintained in L-15 medium supplemented with 10% fetal bovine serum and placed on IMEM with 5% charcoal dextran-treated calf serum (CDCS) 24 h before transfection. Cells were seeded in 24-well plates and transfected with 25 ng CMV5-hER{alpha} (63), 10 ng TK-Renilla (Promega), and 1 µg of a luciferase reporter vector containing two copies of the consensus ERE (2ERE-TK-Luciferase, a gift from B. Katzenellenbogen, University of Illinois, Urbana, IL) without or with 1–10 µg of a PDI expression vector (pCDNA3.1(+)PDI, kindly provided by Kari Kivirikko, University of Oulu, Finland; Ref. 62). MCF-7 cells were maintained in phenol red-containing MEM supplemented with 10% calf serum and placed on phenol red-free MEM with 5% CDCS 24 h before transfection. Cells were seeded in 24-well plates and transfected with 5 ng TK-Renilla and 1 µg of 2ERE-TK-Luciferase without or with 1–5 µg of pCDNA3.1(+)PDI. For all transfection experiments, a parental expression vector lacking the PDI sequence was included to maintain constant DNA concentrations in each well. Cells were transfected using Lipofectin (Invitrogen, Carlsbad, CA) for 8 h, after which they were treated with ethanol vehicle or 10 nM E2 for 24 h. Luciferase activity was quantitated using the Dual Luciferase Assay kit (Promega).

For siRNA experiments, MCF-7 cells were maintained as above and seeded in 12-well plates 24 h before transfection. Cells were transfected with or without 50 or 100 pmol of control (Renilla luciferase) or PDI-specific siRNA oligos (4630 or 110789, respectively; Ambion, Austin, TX) in the absence of antibiotics using siLentFect (Bio-Rad) for 48 h. Phenol red-free MEM with 5% CDCS was added for an additional 24 h, followed by treatment with 10 nM E2 or ethanol vehicle for 24 h. Preliminary time course experiments demonstrated that cells must be exposed to siRNA for at least 72 h to ensure that PDI protein levels are sufficiently reduced. Even after 7 d, the half-life of PDI in rat liver cells (64), some PDI protein was still present in MCF-7 cells (data not shown). RNA was harvested using Trizol (Invitrogen) and processed according to manufacturer’s directions. cDNA was synthesized using the Reverse Transcription System (Promega). Real-time PCR was performed using iQ SYBR Green Supermix and the iCycler PCR thermocycler (Bio-Rad) according to manufacturer’s directions and the following primer sets: 36B4 (5'-GTG TTC GAC AAT GGC AGC AT-3' and 5'-GAC ACC CTC CAG GAA GCG A-3'), PDI (5'-ACG CCA CGG AGG AGT CTG-3' and 5'-TCT TCA GCC AGT TCA CGA TGT C-3'), ER{alpha} (5'-TGC CCT ACT ACC TGG AGA AC-3' and 5'-CCA TAG CCA TAC TTC CCT TGT C-3'), pS2 (5'-GCT GTT TCG ACG ACA CCG TT-3' and 5'-TTC TGG AGG GAC GTC GAT G-3'), PR (5'-GTG CCT ATC CTG CCT CTC AAT C-3' and 5'-CCC GCC GTC GTA ACT TTC G-3'), and Bcl-2 (5'-ATC GCC CTG TGG ATG ACT G-3' and 5'-GCC TCA GCC CAG ACT CAC-3'). Standard curves were derived using cDNA equivalents of 0.02, 0.2, 2, and 20 ng RNA, and were run in duplicate for each primer set during each experiment. The ng equivalents of RNA were determined from the standard curve. The average of two replicates is shown and is representative of four independent experiments. Protein knockdown was monitored by Western blot analysis of whole cell lysates as described above using antibodies to PDI (MA3-018; Affinity Bioreagents), ER{alpha}, Cat D, GAPDH (sc-543, sc-10725, sc-20357, respectively; Santa Cruz Biotechnologies), or PR (RM-9102, LabVision, Fremont, CA).

Immunofluorescence
MCF-7 cells were maintained as described above and seeded in 12-well plates onto poly-L-lysine-treated coverslips in phenol red-free MEM containing 5% CDCS. Cells were fixed with methanol for 10 min at –20 C, permeabilized with acetone for 1 min at –20 C, and washed with PBS (1 mM KH2PO4, 250 mM NaCl, 3 mM Na2HPO4·7H2O) containing 0.5% Triton X-100 before incubation with ER{alpha} (sc-543; Santa Cruz Biotechnologies), lamin A/C (sc-20681, Santa Cruz Biotechnologies), calnexin (sc-11397, Santa Cruz Biotechnologies), or PDI (MA3-018; Affinity Bioreagents) antibody for 1 h at room temperature. Cells were washed three times with PBS containing 0.5% Triton X-100 and incubated with donkey antimouse Texas Red or donkey antirabbit Fluorescein conjugated antibodies (Jackson ImmunoResearch, West Grove, PA) for 30 min. Cells were mounted with Vectashield medium (Vector Laboratories Inc., Burlingame, CA) and visualized on a Zeiss Axioskop (Carl Zeiss Microimaging Inc., Thornwood, NY) with mercury lamp using a x100 oil objective. Images were acquired through a Hamamatsu Photonics (Hamamatsu, Japan) charge-coupled device camera using IPLab imaging software (Scanalytics, Fairfax, VA) and merged using Adobe Photoshop CS2 software (Adobe Systems Inc., San Jose, CA).

ChIP Assays
MCF-7 cells were exposed to ethanol vehicle or 10 nM E2 for 2 or 24 h and ChIP assays were carried out essentially as recommended by Upstate (Waltham, MA) except that pelleted cells were washed three times in lysis buffer [10 mM Tris (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40], resuspended in micrococcal nuclease buffer (lysis buffer with 10 mM CaCl2 and 4% NP-40), and treated with 75 U micrococcal nuclease (USB, Cleveland, OH) for 10 min before sonication. The ER{alpha}-specific antibody sc-8002 (Santa Cruz Biotechnologies) or PDI-specific antibody MA3–018 (Affinity Bioreagents, Golden, CO) was used for immunoprecipitation of protein-DNA complexes. PCR primers flanking the pS2 ERE, +571 ER/Sp1 site of the PR gene, +90 AP-1 site of the PR gene, or +745 AP-1 site of the PR gene were used for semiquantitative PCR using iQ SyBr Green Supermix and the iCycler PCR thermocycler according to manufacturer’s directions (Bio-Rad). The gene regions amplified and the primer sets used were: pS2 upstream control (5'-GTA TGG TGT GGT CTT GGG TTC C-3' and 5'-GGG TTG GAG CGG CTG GAG-3'), pS2 ERE (5'-CCC GTG AGC CAC TGT TGT C-3' and 5'-CCT CCC GCC AGG GTA AAT AC-3'), PR +571 ERE/Sp1 site (5'-CCA AGG GCA GAG CTG ACC AG-3' and 5'-GGG CAG AGG GAG GAG AAA GTG-3'), PR +90 AP-1 (5'-GCG TGT GGG TGG CAT TCT C-3' and 5'-GGC GAC AGT CAT CTC CGA AG-3'), and PR +745 AP-1 (5'-ACC CAC TTT CTC CTC CCT CTG-3' and 5'-CCC TTT GCC TTC AGC TCA GTC-3'). Standard curves using 1000, 5000, 10,000, and 25,000 copies of each gene were run for each primer set during each experiment. Data are reported as the average copy number for two replicates in a single experiment and are representative of three independent experiments.

Protease Digestion
Purified ER{alpha} (150 fmol) was incubated alone or combined with PDI (5 µg) in15 mM Tris (pH 7.9), 0.2 mM EDTA, 10% glycerol, 0.05 mM ZnCl2, 2 mM DTT, 50 mM KCl, and 10 nM E2 on ice for 10 min. Reactions were then subjected to limited protease digestion with 5 ng chymotrypsin (Sigma), 20 ng Proteinase K (Promega), or 2.5 µg Staphylococcus aureus V8 (Worthington Biochemical Corp., Lakewood, NJ) for 10 min at room temperature before fractionation on denaturing polyacrylamide gels. The protease concentrations used have previously been shown to be effective in digesting ER{alpha} (55, 57). Fractionated proteins were transferred to nitrocellulose (56) and subjected to Western blot analysis. Cleaved ER{alpha} was detected using the rabbit polyclonal antibodies sc-544 (Santa Cruz Biotechnologies), LP-1 (Immunological Resource Center, University of Illinois, Urbana, IL), or mouse monoclonal antibodies sc-543 or sc-8005 (Santa Cruz Biotechnologies) and visualized with a horseradish peroxidase-conjugated secondary antibody and a chemiluminescent detection system as previously described (57).


   ACKNOWLEDGMENTS
 
We thank V. Likhite for intitial development of agarase gel methods; C. Curtis, B. Freeman, and C. Benz for advice and helpful discussions; Y. Ziegler for antibody characterization; S. Siechen and A. Chiba for assistance with immunofluorescence studies; and L. Ruddock, K. Kivirikko, R. Rudolph, B. Katzenellenbogen, D. Shapiro, L. Kraus, and J. Kadonaga for plasmids and viral stock.


   FOOTNOTES
 
This work was supported by National Institutes of Health (NIH) Grants DK-53884 and DK-061463 (to A.M.N.) and P41 RR11823 (to J.R.Y.). J.R.S.-N. received predoctoral support from the Susan G. Komen Breast Cancer Foundation Dissertation Fellowship DISS0201937 and postdoctoral support from NIH Reproductive Biology Training Program Grant PHS 5T32 HD07028.

J.R.S.-N., H.M., and A.M.N. have nothing to declare. J.R.Y. has received consulting fees from ThermoElectron and is inventor of U.S. Patent 5,538,897.

First Published Online May 11, 2006

Abbreviations: AP, Activator protein; CatD, cathepsin D; CBCS, charcoal dextran-treated calf serum; ChIP, chromatin immunoprecipitation; DBD, DNA binding domain; DTT, dithiothreitol; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response elements; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LBD, ligand binding domain; NP-40, Nonidet P-40; PDI, protein disulfide isomerase; PR, progesterone receptor; PRA and PRB, PRs A and B; siRNA, small interfering RNA; U2-OS, U2 osteosarcoma.

Received for publication January 5, 2006. Accepted for publication April 27, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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