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[Cancer Research 65, 6120-6129, July 15, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Epidermal Growth Factor–Induced Signaling in Breast Cancer Cells Results in Selective Target Gene Activation by Orphan Nuclear Receptor Estrogen-Related Receptor {alpha}

Janelle B. Barry1 and Vincent Giguère1,2

1 Molecular Oncology Group, McGill University Health Centre and 2 Departments of Biochemistry, Medicine, and Oncology, McGill University, Montreal, Quebec, Canada

Requests for reprints: Vincent Giguère, Molecular Oncology Group, McGill University Health Centre, Room H5-21, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1. Phone: 514-843-1406; Fax: 514-843-1478; E-mail: vincent.giguere{at}mcgill.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The orphan nuclear hormone receptor estrogen-related receptor {alpha} (ERR{alpha}, NR3B1) is a constitutive transcription factor that is structurally and functionally related to the classic estrogen receptors. ERR{alpha} can recognize both the estrogen response element and its own binding site (ERRE) in either dimeric or monomeric forms. ERR{alpha} is also a phosphoprotein whose expression in human breast tumors correlates with that of the receptor tyrosine kinase ErbB2, suggesting that its transcriptional activity could be regulated by signaling cascades. Here, we investigated growth factor regulation of ERR{alpha} function and found that it is phosphorylated in MCF-7 breast cancer cells in response to epidermal growth factor (EGF), an event that enhances its DNA binding. Interestingly, treatment with alkaline phosphatase shifts ERR{alpha} from a dimeric to a monomeric DNA-binding factor, and only the dimeric form interacts with the coactivator PGC-1{alpha}. In vitro, the DNA-binding domain of ERR{alpha} is selectively phosphorylated by protein kinase C{delta} (PKC{delta}), which increases its DNA-binding activity, whereas expression of constitutively active PKC{delta} enhances TFF1 promoter activity via the ERRE. However, whereas treatment of MCF-7 cells with the phorbol ester phorbol-12-myristate 13-acetate also enhances ERR{alpha} activation of the TFF1 promoter reporter, it does not affect ERR{alpha} activity on its own promoter. In agreement, chromatin immunoprecipitation analysis shows that ERR{alpha} and RNA polymerase II are preferentially recruited to the TFF1 promoter after EGF treatment, whereas recruitment of these factors to its own promoter is not affected. These results reveal a mechanism through which growth factor signaling can selectively activate ERR{alpha} target genes in breast cancer cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The estrogen-related receptor {alpha} (ERR{alpha}, NR3B1) is an orphan member of the nuclear receptor superfamily identified on the basis of its structural similarity to the estrogen receptor {alpha} (ER{alpha}, NR3A1; ref. 1). ERR{alpha} is functionally similar to ER{alpha} in that it can bind the inverted repeat estrogen response element (ERE) sequence in target gene promoters, although ERR{alpha} preferentially binds a nine-nucleotide extended half site sequence, the ERRE (2–4). In contrast to the ligand-dependent activation of ER{alpha}, ERR{alpha} is constitutively active and does not respond to estradiol or other natural estrogens (1, 5), although its activity can be inhibited by the synthetic estrogen diethylstilbestrol (6, 7). The constitutive ERR{alpha} can interact with and be modulated by members of the SRC and PGC-1 families of coactivators (6, 8–14). The function of ERR{alpha} as a transcriptional activator seems to be cell type and promoter specific. This specificity is proposed to be dependent on factors including relative coactivator levels, interactions with other nuclear receptors, and the presence of appropriate activating stimuli or the context of the response element (2, 9, 15–17). ERR{alpha} activates the promoter of its own gene, ESRRA, thereby providing positive regulation of its own expression (13). Other known ERR{alpha} target genes are involved in regulation of mitochondrial biogenesis and energy metabolism (2, 14, 18, 19). ERR{alpha} also activates the TFF1 (also known as pS2) and aromatase genes, which are implicated in breast cancer (17, 20).

ERR{alpha} is widely expressed in normal tissues (2) and RNA expression studies show the presence of ERR{alpha} in a range of breast cancer cell lines (17). It has also been shown that ERR{alpha} is an unfavorable breast cancer biomarker as its presence in tumor samples associates with an increased risk of disease recurrence or adverse clinical outcome (21). In addition, ERR{alpha} mRNA levels in primary breast tumors correlate both with ER{alpha}-negative tumor status, a predictor of poor prognosis, and with expression of ErbB2, an indicator of aggressive tumor behavior (22). ErbB2 is a receptor tyrosine kinase that is overexpressed or amplified in 15% to 30% of malignant breast cancers. ErbB2, in combination with the ErbB family members epidermal growth factor receptor (EGFR), ErbB3 and ErbB4, transmits signals from EGF and EGF-like growth factor ligands to activate a complex array of downstream signaling pathways including the phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase pathways. Such activation leads to phosphorylation of multiple downstream targets and ultimately to regulation of transcription through phosphorylation of specific transcription factors (reviewed in ref. 23).

Nuclear receptors are phosphoproteins and their activity can be modulated by growth factor signaling (24, 25). Phosphorylation occurs most often in the AF-1, AF-2, or DNA-binding domains, and is mediated by kinases downstream of various cytokine and growth factor signaling pathways. Phosphorylation regulates receptor functions, including DNA binding, transactivation, and interaction with coactivators, and can result in both activation and inhibition of receptor transcriptional activity (reviewed in refs. 24, 25). Modulation of ERR{alpha} by phosphorylation may provide a way to regulate its constitutive activity in the absence of a known ligand and may facilitate understanding the apparent promoter specificity of its transcriptional effects. Given the correlation between overexpression of ERR{alpha} and ErbB2 in breast cancer cells, the transcriptional activity of ERR{alpha} may thus be influenced by EGFR/ErbB2 signaling pathways.

Protein kinase C{delta} (PKC{delta}) is a member of the novel subfamily of PKC serine/threonine kinases. It is activated by diacylglycerol and phorbol esters and is phosphorylated by various signaling pathways, including EGFR (26). PKC{delta} is ubiquitously expressed, and once activated it functions in a cell- and tissue-specific manner, probably because of differential phosphorylation events. Several reports indicate that PKC{delta} is a negative regulator of proliferation in certain cell types, and studies from null mice show that PKC{delta} is not required for proliferation of normal cells (27). In contrast, PKC{delta} is thought to play a proliferative role in transformed cells. It is a prosurvival factor and is important in maintaining cell migration in breast cancer cell lines (28, 29). PKC{delta} may also play a role in breast cancer progression as its expression in breast tumor lines correlates with their metastatic potential (30). In addition, enhanced expression of PKC{delta} leads to increased anchorage-independent growth of highly metastatic cells (31). The potential role of PKC{delta} in breast cancer progression thus makes it an interesting target in the investigation of EGF signaling in breast cancer cells.

In this study, we investigated a possible relationship between phosphorylation and ERR{alpha} function through EGF signaling in breast cancer cells. We found that ERR{alpha} is phosphorylated in response to EGF, which enhances receptor DNA binding in vivo. Sequence scanning of ERR{alpha} indicated the presence of potential PKC{delta} phosphorylation sites in the DNA-binding domain. We showed that PKC{delta} does indeed phosphorylate ERR{alpha} in the DNA-binding domain, and enhances its DNA binding in vitro. In addition, PKC{delta} signaling augments ERR{alpha} DNA binding and enhances ERR{alpha} transactivation in MCF-7 cells. Moreover, we found via chromatin immunoprecipitation that EGF enhances ERR{alpha} target promoter occupancy in a gene-specific manner. Our results suggest that ERR{alpha} phosphorylation provides a mechanism to enhance receptor DNA-binding and transactivation, events that contribute to the selective activation of target genes in breast cancer cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. Human recombinant EGF and purified recombinant PKC{delta} were from Upstate Biotechnology (Waltham, MA). The ERR{alpha} polyclonal antibody has been described previously (13). The RNA polymerase II and HA polyclonal antibodies were from Upstate Biotechnology and the HA monoclonal antibody was from Covance Research Products (Princeton, NJ). The PKC{delta} and ER{alpha} antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA) and the phospho-PKC{delta} (Thr505) antibody was from Cell Signaling Technology (Beverly, MA). Phorbol-12-myristate 13-acetate (PMA) was from Calbiochem (San Diego, CA).

In vivo phosphorylation. All cells were plated in 10 cm dishes. COS-1 cells were transiently transfected for 16 hours with 3 µg pCMXhERR{alpha} then incubated in serum- and phosphate-free DMEM for 24 hours, followed by addition of 1 mCi [32P]orthophosphate for 6 hours. MCF-7 cells stably transfected with pOPRShERR{alpha}3 were incubated in serum- and phosphate-free DMEM for 24 hours then induced with 1 mmol/L isopropyl-L-thio-B-D-galactopyranoside (IPTG) and labeled with 1 mCi [32P]orthophosphate for 6 hours. Cells were treated with 100 ng/mL EGF for the final 30 minutes of the 6-hour incubation. MCF-7 cells were incubated in serum- and phosphate-free DMEM for 24 hours, labeled with 1 mCi of [32P]orthophosphate for 6 hours and treated with 100 ng/mL EGF for the final 20 minutes of the 6-hour incubation. Cells were harvested in modified radioimmunoprecipitation assay buffer (RIPA) buffer and lysates immunoprecipitated with anti-ERR{alpha} or anti-HA polyclonal antibodies. Reactions were electrophoresed, transferred to polyvinylidene difluoride (PVDF) membrane and detected by autoradiography or Western blotting using the anti-ERR{alpha} or anti-HA monoclonal antibody.

Electromobility shift assays. Electromobility shift assays (EMSA) were done as described (32) using consensus ERRE 5'-TCGACGCTTTCAAGGTCATATCCG-3' and ERE 5'-TCGACAAAGTCAGGTCACAGTGACCTGATCAAG-3' probes. MCF-7 cells were grown in serum-free medium for 24 hours before treatment with 100 ng/mL EGF for 20 minutes. Nuclear extracts were prepared (33) and 10 µg extract was used per EMSA reaction. For EMSA using acid phosphatase and for PGC-1{alpha} supershift experiments, COS-1 cells were transiently transfected with 5 µg pCMXhERR{alpha} for 16 hours and nuclear extracts were prepared. Two micrograms of extract were incubated with acid phosphatase at 25°C for 1 hour before the EMSA reaction. For PGC-1{alpha} supershift experiments, 2 µg of extract were combined with 1, 2, or 3 µg of purified GST-PGC-1{alpha}. This construct was prepared by PCR cloning the cDNA encoding residues 1 to 250 of PGC-1{alpha} from pcDNA3/HA-hPGC-1 (34) into pGEX-2T. EMSA with recombinant PKC{delta} was done using equivalent amounts of purified GST-O or GST-ERR{alpha}DBD fusion proteins. Samples were incubated with 25 ng purified recombinant PKC{delta} according to the manufacturer's instructions at 30°C for 30 minutes before the EMSA reaction. For EMSA following PMA treatment, MCF-7 cells were grown in serum-free medium for 24 hours before addition of 100 nmol/L PMA for 5, 10, 20, 30, 40, or 60 minutes. Ten micrograms of nuclear extract were used per EMSA reaction. Four microliters of the ERR{alpha} antibody were used per reaction in supershift experiments.

Phosphorylation site prediction. The ERR{alpha} amino acid sequence was scanned for prediction of consensus phosphorylation sites using the following programs: Scansite (http://scansite.mit.edu), Netphos 2.0 (http://www.cbs.dtu.dk/services/NetPhos/), and Macvector Version 7.2 (Accelrys, Inc., San Diego, CA).

In vitro kinase assays. In vitro kinase assays were done using equivalent amounts of GST-O, GST-hERR{alpha}-A/B, DNA-binding domain, or ligand-binding domain fusion proteins bound to glutathione Sepharose. Reactions were prepared with 25 ng purified recombinant PKC{delta} and 10 µCi [{gamma}32P]ATP according to the manufacturer's instructions, and incubated at 30°C for 30 minutes with frequent mixing. Reactions were washed twice in ADBIII according to the manufacturer's instructions then subjected to PAGE. Gels were stained with Coomassie blue, dried, and exposed for 32P incorporation.

Cell culture and transfections. MCF-7 cells were maintained in DMEM without phenol red, supplemented with 10% fetal bovine serum (FBS). All transfections were done using FuGene 6 (Roche Diagnostics, Indianapolis, IN). Cells were plated in 12-well plates using DMEM without phenol red with 10% charcoal-stripped FBS. For PKC-CAT experiments, cells were transfected with 100 ng pHACE-PKC{delta}CAT or pHACE-PKC{delta}CAT-KR (provided by Dr. J. Soh, Inha University, Incheon, Korea), along with 400 ng TFF1Luc reporter and 100 ng pCMVßgal transfection efficiency control plasmid. Cells were transfected for 24 hours before harvesting for Luciferase and ß-galactosidase activity. For PMA experiments, cells were transfected with 400 ng TFF1Luc, TFF1Luc{Delta}ERE, TFF1Luc{Delta}ERRE, TFF1{Delta}ERE/ERRELuc (17) or pGL3ESRRA reporter (13), and 100 ng pCMVßgal transfection efficiency control plasmid. Cells were transfected for 24 hours then treated with 100 nmol/L PMA for a further 8 hours before harvesting for Luciferase and ß-galactosidase activity.

Immunoprecipitation and Western blotting. MCF-7 cells were grown in DMEM without phenol red with 10% charcoal-stripped FBS. Media was changed to serum-free DMEM for 24 hours followed by treatment with 100 ng/mL EGF or 100 nmol/L PMA for 1, 5, 15, 30, 45, or 60 minutes immediately before harvesting. Whole-cell extracts were prepared using modified RIPA buffer and 100 µg extract was electrophoresed then transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk powder in TBST and blotted overnight with the phospho-PKC{delta} (Thr505) antibody in 5% bovine serum albumin/TBST. Blots were stripped in 10 mmol/L Tris/150 mmol/L NaCl (pH 2.3) for 30 minutes at room temperature before reblotting with the PKC{delta} antibody. Western blot analysis for HA-ERR{alpha} and ERR{alpha} was done using PVDF membranes and either the anti-HA monoclonal or anti-hERR{alpha} polyclonal antibody, incubated in 5% skim milk powder/PBST, and blotted overnight.

Chromatin immunoprecipitation assay. Chromatin immunoprecipitation assays were done as previously described (35). Briefly, MCF-7 cells were cultured in DMEM containing 10% charcoal-stripped FBS for 2 days, followed by serum starvation for 24 hours before treatment with 200 ng/mL EGF for 20 minutes. Cells were cross-linked for 10 minutes at room temperature, collected, centrifuged, and the pellets resuspended in lysis buffer [1% SDS, 10 mmol/L EDTA, 50 mmol/L Tris-HCl (pH 8.1)] supplemented with protease inhibitor cocktail (Roche Diagnostics). The lysate was sonicated seven times for 8 seconds and centrifuged. Soluble chromatin was diluted 10-fold in chromatin immunoprecipitation buffer [1% Triton X-100, 2 mmol/L EDTA, 150 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 8.1)] and immunoprecipitated using either the hERR{alpha} polyclonal antibody or preimmune serum or the hER{alpha} antibody. Chromatin for immunoprecipitation with the RNA polymerase II antibody was diluted 2.5-fold in chromatin immunoprecipitation buffer [1% Triton X-100, 2 mmol/L EDTA, 500 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 8.1)]. Overnight immunoprecipitation was followed by addition of 40 µg salmon sperm DNA/protein A agarose (Upstate Biotechnology) for 2 hours. Precipitates were washed for 10 minutes each in Buffer I [0.1% SDS, 1% Triton X-100, 2 mmol/L EDTA, 20 mmol/L Tris-HCl (pH 8.1), 150 mmol/L NaCl], Buffer II [0.1% SDS, 1% Triton X-100, 2 mmol/L EDTA, 20 mmol/L Tris-HCl (pH 8.1), 500 mmol/L NaCl] and Buffer III [0.25 mol/L LiCl, 1% NP40, 1% Na deoxycholate, 1 mmol/L EDTA, 10 mmol/L Tris-HCl (pH 8.1)], followed by washing in TE and elution in 1% SDS, 0.1 mol/L NaHCO3 buffer at 65°C for at least 4 hours. Isolated fragments were purified using the QIAquick spin kit (Qiagen, Valencia, CA). Quantitative PCR was done using the Lightcycler and Fast Start DNA Master SYBG Green 1 (Roche Diagnostics). The primers for the TFF1 promoter were 5'-GGCCATCTCTCACTATGAATCACTTCTGC-3' and 5'-GGCAGGCTCTGTTTGCTT-AAAGAGCG-3', which encompass the ERRE. Primers for the ESRRA promoter were 5'-CCATCCGAGTGGAATTTGAG-TCCTAAAG-3' and 5'-GAACCGTAGACCCAG-TAGCCCCACAGAG-3', which amplifies the region covering the ERRE element. Enrichment of these regions was compared with a negative control segment located 4 kb upstream of the either the TFF1 or ESSRA promoters, using the following sets of primers: TFF1 promoter 5'-CTGGGCAATGCGAGGAGAGTCAAGACTG-3' and 5'-GGGAGGGAGGAGTTTGGAGGAAGTGG-3' and ESRRA promoter 5'-GTGGCCC-ACAGGTGTCGCTCAAGTCTTC-3' and 5'-GGATGCAGTGTCCTTCTCCCCCAG-ATTG-3'. PCR products were visualized after migration on a 2% agarose gel.

Quantitative reverse transcription-PCR. MCF-7 cells were grown in DMEM without phenol red with 10% charcoal-stripped FBS, serum starved for 24 hours, and treated with 100 ng/mL EGF for 8 hours. RNA was extracted from control and treated cells using the RNeasy kit (Qiagen). cDNA synthesis was done using Transcriptor Reverse Transcriptase (Roche Applied Science) according to the manufacturer's instructions. Quantitative PCR was done using the Lightcycler and Fast Start DNA Master SYBG Green 1. Primers used to amplify the TFF1 transcript were 5'-ATGGCCACCATGGAGAACAAGG-3' and 5'-CTAAAATTCACACTCCTCTTCTGG-3', for ESRRA were 5'-CGAGAGGAGTATGTTCTA-3' and 5'-CCCGCCCGCCGCCGCTCAGCA-3', and for the RPLP control were 5'-TGGAGAAACTGCTGCCTCATA-3' and 5'-CCCTGGAGATTTTAGTGGTGA-3'. TFF1 and ESRRA transcript levels were corrected for expression of the RPLP control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Estrogen-related receptor {alpha} is phosphorylated in response to epidermal growth factor, which enhances its DNA binding. To investigate whether ERR{alpha} can be modified by phosphorylation, COS-1 cells were metabolically labeled and ectopic ERR{alpha} immunoprecipitated using a specific ERR{alpha} antibody. Phosphorylated ERR{alpha} was detected in cells transfected with the ERR{alpha} construct but not from control-transfected cells (Fig. 1A). MCF-7 human breast cancer cells stably transfected with an IPTG-inducible HA-ERR{alpha} construct were also metabolically labeled and lysates immunoprecipitated with the HA tag antibody (Fig. 1B). HA-ERR{alpha} was phosphorylated, and the level of phosphorylation increased after treatment of cells with EGF, while Western blot analysis indicated that total receptor levels remained constant (Fig. 1B, bottom). To investigate phosphorylation of the endogenous receptor, lysates from metabolically labeled MCF-7 cells were immunoprecipitated using the ERR{alpha} antibody. Phosphorylated ERR{alpha} was detected and the extent of phosphorylation was also enhanced in response to EGF treatment, while total receptor levels were constant (Fig. 1C).



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  		Figure 1. ERR{alpha} is a phosphoprotein. A, metabolic labeling of COS-1 cells. Cells were transiently transfected with constructs expressing hERR{alpha} or negative control and metabolically labeled with 32P. Cell lysates were immunoprecipitated with the ERR{alpha} antibody, transferred, and exposed to film. B, metabolic labeling of MCF-7 cells. MCF-7 stably transfected with an IPTG-inducible HA-ERR{alpha} construct were induced and labeled with 32P then treated with EGF for 30 minutes. Cell lysates were immunoprecipitated with the anti-HA polyclonal antibody; electrophoresed products were transferred and exposed to film before blotting with the anti-HA monoclonal antibody. C, MCF-7 cells were metabolically labeled with 32P, treated with EGF for 20 minutes, and lysates immunoprecipitated with the ERR{alpha} antibody to detect endogenous ERR{alpha} phosphorylation. Equivalent ERR{alpha} expression in MCF-7 samples was confirmed by Western blotting. ctl, whole cell extract of transiently transfected ERR{alpha} in COS-1 cells; *, nonspecific band. Three replicates were done for each metabolic labeling experiment.

 
Using programs that predict consensus phosphorylation sites (see Materials and Methods for details), the presence of high probability consensus sites were found predominantly in the DNA-binding domain of ERR{alpha}. Hence, we assessed potential effects of endogenous ERR{alpha} phosphorylation on DNA binding. MCF-7 cells were treated with EGF and the resulting nuclear extracts subjected to EMSA. ERR{alpha} binding to a consensus ERRE-containing probe was enhanced after EGF treatment, as illustrated by the representative autoradiogram in Fig. 2A. Western blot analysis confirmed that EGF-treated and untreated nuclear extracts expressed ERR{alpha} equivalently (Fig. 2A, top right). Densitometric analysis of binding for three independent experiments indicated that EGF treatment of MCF-7 cells enhanced ERR{alpha} DNA binding by ~50% over that of untreated cells (Fig. 2A, bottom right). The effects of phosphorylation on ERR{alpha} DNA binding were also assessed using phosphatase treatment (Fig. 2B). Transfected ERR{alpha} is basally phosphorylated (Fig. 1A) so nuclear extracts prepared from transfected cells were treated with acid phosphatase to remove any phosphorylation before EMSA. Ectopic ERR{alpha} binds to the consensus ERRE as a monomer and a dimer, whereas it binds to an ERE element only as a dimer (Fig. 2B). Interestingly, dephosphorylation of the nuclear extracts converted ERR{alpha} from a primarily dimeric to a monomeric DNA-binding protein on the ERRE element (Fig. 2B, left). In addition, ERR{alpha} dimeric DNA binding on the ERE element was decreased after phosphatase treatment (Fig. 2B, right). Together, these experiments indicate that phosphorylation of both endogenous and transiently transfected ERR{alpha} has a positive effect on its DNA binding as a dimer. It has been suggested that ERR{alpha} transactivation function may be dependent on dimeric DNA binding, indicating that dimerization is required for functional interaction with coactivator proteins (36). To further assess the significance of ERR{alpha} coactivator interactions and dimer functionality, EMSA was done using transiently transfected ERR{alpha} nuclear extract in the presence of the coactivator PGC-1{alpha} (Fig. 2C). Incubation of ERR{alpha} with PGC-1{alpha} produced a supershift of the shifted dimer band, while the mobility of the monomer band was unchanged (Fig. 2C). Together, these data indicate that phosphorylation enhances ERR{alpha} DNA binding as a dimer, which, because of its interaction with PGC-1{alpha}, is likely to be the transcriptionally competent form of the receptor.



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  		Figure 2. ERR{alpha} phosphorylation enhances DNA binding. A, EMSA of MCF-7 nuclear extracts on a consensus ERRE probe, after treatment with EGF or control for 20 minutes. The supershift reaction was done using the ERR{alpha} antibody (Ab). EGF-treated and untreated extracts were also subjected to PAGE and blotted with the ERR{alpha} antibody to confirm equivalent expression. Shifted ERR{alpha} bands from three separate EMSA experiments as represented in the autoradiogram were analyzed densitometrically. Columns, mean percentage binding for control and EGF-treated samples; bars, SD. B, EMSA of transfected ERR{alpha} or negative control nuclear extract on ERRE and ERE probes after treatment of extracts with acid phosphatase (AcP). Supershift reactions were done using the ERR{alpha} antibody. D and M denote the positions of dimer and monomer bands, respectively. C, EMSA of transfected ERR{alpha} nuclear extract on the ERRE probe. ERR{alpha} was supershifted using increasing amounts of a purified GST fusion of PGC-1{alpha}. S denotes supershifted band, D and M indicate ERR{alpha} dimer and monomer, respectively, and NS denotes a nonspecific band.

 
Estrogen-related receptor {alpha} is phosphorylated in the DNA-binding domain by protein kinase C{delta}, which enhances its DNA binding and transcriptional activity. Protein sequence analysis of ERR{alpha} revealed the presence of three high probability consensus phosphorylation sites for PKC{delta} in the DNA-binding domain of the receptor. Thus, PKC{delta} was tested for its ability to phosphorylate equivalent amounts of glutathione S-transferase (GST) fusion constructs of the ERR{alpha} A/B domain, DNA-binding domain, or ligand-binding domain (Fig. 3A, top). Purified recombinant PKC{delta} phosphorylated ERR{alpha} in the DNA-binding domain (Fig. 3A, middle) and a control peptide (Fig. 3A, bottom), but not in the A/B domain or ligand-binding domain. In EMSA experiments, binding of GST-ERR{alpha}DBD to an ERRE element was enhanced in the presence of purified recombinant PKC{delta} (Fig. 3B). Given the effects of PKC{delta} on ERR{alpha} in vitro, we investigated its effects on ERR{alpha} transactivation function by transient transfection with constitutively active (PKC-CAT) or kinase dead (PKC-CAT-KR) mutants of PKC{delta} (37). PKC-CAT enhanced the basal activity of endogenous ERR{alpha} on the TFF1 promoter 2-fold, while the inactive PKC-CAT-KR had no effect (Fig. 3C). PKC{delta} can thus phosphorylate ERR{alpha} in the DNA-binding domain, which results in enhanced receptor DNA-binding and transcriptional activity.



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  		Figure 3. PKC{delta} phosphorylates ERR{alpha} in the DNA-binding domain and enhances DNA binding. A, in vitro kinase assay using GST fusions of the ERR{alpha} A/B domain (GST-AB), DNA-binding domain (GST-DBD), or ligand-binding domain (GST-LBD), plus GST alone negative control or positive control peptide. Samples were incubated with purified recombinant PKC{delta} in vitro. After washing, proteins were electrophoresed and stained with Coomassie blue (top) and then exposed to film (bottom) to detect 32P incorporation. Stars in the bottom panel correspond to the location of Coomassie-stained bands in the top panel. B, equivalent amounts of GST-ERR{alpha}DBD or GST control were incubated with purified recombinant PKC{delta} then subjected to EMSA on a consensus ERRE probe. C, transient transfection of MCF-7 cells with constructs expressing constitutively active (PKC-CAT) or a kinase dead mutant (PKC-CAT-KR) of PKC{delta}, and the TFF1LUC reporter.

 
Given that ERR{alpha} phosphorylation and DNA binding are enhanced by EGF in MCF-7 cells, and that PKC{delta} can phosphorylate and enhance the DNA binding of ERR{alpha} in vitro, we next investigated a link between ERR{alpha} and PKC{delta} signaling in MCF-7 cells. PKC{delta} is reportedly phosphorylated in response to EGF in fibroblastic cells and keratinocytes (38–40), so to investigate whether such activation also occurs in breast cancer cells, Western blot analysis was done after treatment of cells with EGF over a 1-hour time course (Fig. 4A). PKC{delta} phosphorylation was enhanced after 15 to 30 minutes of EGF treatment, and the signal was maintained for at least 60 minutes, while total PKC{delta} levels remained constant. The activation time of PKC{delta} also corresponded with the time (20 minutes) at which ERR{alpha} DNA binding was enhanced after EGF treatment (Fig. 2A). To confirm that PKC{delta} was also activated in MCF-7 cells after treatment with the PKC activator PMA, Western blot analysis was done after a 1-hour time course (Fig. 4B). PKC{delta} phosphorylation increased steadily from 1 to 60 minutes of treatment, thus confirming its activation, while total PKC{delta} levels remained constant.



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  		Figure 4. PKC{delta} signaling regulates ERR{alpha} DNA binding in MCF-7 cells. A, Western blot analysis of MCF-7 cell extracts after treatment with EGF over a 1-hour time course. Membranes were blotted with the phospho-Thr505–specific PKC{delta} antibody then with the total PKC{delta} antibody. B, Western blot analysis of MCF-7 cell extracts after treatment with PMA over a 1-hour time course. Membranes were blotted as in (A). C, EMSA of MCF-7 nuclear extracts after treatment with PMA over a 1-hour time course on the consensus ERRE probe. The supershift was done using the ERR{alpha} antibody. Shifted bands were analyzed densitometrically and graphed as percentage increase binding over untreated control. Results are representative of three individual EMSAs, each of which gave at least a doubling of ERR{alpha} DNA binding after 10 minutes of PMA treatment.

 
Because PKC{delta} is activated by both EGF and PMA in MCF-7 cells, and EGF stimulates ERR{alpha} DNA binding in these cells, we did an EMSA experiment to assess the effects of PMA on ERR{alpha} DNA binding over a 1-hour time course (Fig. 4C). ERR{alpha} binding to the ERRE was enhanced to double that of the untreated control after 10 minutes PMA treatment, and this increase was maintained for the length of the time course. Maintenance of enhanced ERR{alpha} binding was consistent with the progressive activation of PKC{delta} from 1 to 60 minutes of PMA treatment. In summary, PKC{delta} is activated in MCF-7 cells by both EGF and PMA, and this activation produces a concomitant enhancement of ERR{alpha} DNA binding.

Estrogen-related receptor {alpha} activation by protein kinase C{delta} signaling induces selective target promoter activation. The effects of PKC{delta} signaling on ERR{alpha} function were further examined using transient transfection experiments in MCF-7 cells. Cells were transfected with reporter constructs containing the promoter of either the breast cancer marker TFF1 or the ERR{alpha} gene, ESRRA. Cells were then treated with PMA to activate endogenous PKC{delta} (Fig. 5A). PMA treatment greatly enhanced the activity of endogenous ERR{alpha} on the TFF1 promoter (Fig. 5A, left), whereas in contrast it did not change its activity on the ESRRA promoter (Fig. 5A, right). The TFF1 promoter contains both ERRE and ERE elements (17); thus, to further investigate PMA-induced ERR{alpha} transactivation on this promoter, transfections were done using constructs with either or both of these elements deleted (Fig. 5B). Deletion of the ERE did not change the PMA-induced activation, while deletion of the ERRE or both elements together inhibited the response by ~40%. These data indicate that the effects of PMA on the TFF1 promoter are partly through enhanced ERR{alpha} binding to the ERRE, but not through the binding of ERR{alpha} or ER{alpha} to the ERE. The partial response on the TFF1{Delta}ERE/ERRE construct likely relates to the observation that PMA can activate proteins other than PKC depending on the functional concentration of the compound in distinct cellular systems (26). Such activation could result in the binding of other proteins to the TFF1 promoter through sites distinct from the ERRE and ERE.



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  		Figure 5. ERR{alpha} activation by PKC{delta} selectively induces target promoter activation. A, MCF-7 cells were transiently transfected with either the TFF1LUC or pGL3ESRRA reporter then treated with PMA for 8 hours before harvesting. B, MCF-7 cells were transiently transfected with the TFF1LUC, TFF1{Delta}ERELUC, TFF1{Delta}ERRELUC, or TFF1{Delta}ERE/ERRELUC reporter. The deletion constructs are the same as TFF1LUC except that they lack either the ERE or ERRE or both binding sequences. Cells were treated with PMA as in (A). In all transfections, the measured Luciferase activity was corrected using the ß-galactosidase transfection efficiency control. All results are representative of at least three independent transfections done in duplicate.

 
To further investigate the apparent selectivity in target gene activation by ERR{alpha} after phosphorylation, chromatin immunoprecipitation experiments were done on the TFF1 and ESRRA promoters. MCF-7 cells were treated with EGF and chromatin was immunoprecipitated with the ERR{alpha} antibody. PCR analysis was done using primers encompassing either the ERRE region of the TFF1 promoter or a negative control region 4 kb upstream (Fig. 6A). ERR{alpha} recruitment to the ERRE region of the TFF1 promoter was enhanced after treatment with EGF compared with control (Fig. 6A, top), but it was not recruited to the upstream negative control region. Quantitative real-time PCR analysis (Fig. 6A, graph on top) showed that EGF produced a 5-fold enhancement of ERR{alpha} recruitment to the TFF1 promoter compared with control. Similarly, recruitment of RNA polymerase II to the TFF1 promoter ERRE region was augmented after EGF treatment compared with control (Fig. 6A, middle). Quantitative analysis (Fig. 6A, graph on middle) showed that EGF produced a 3- to 4-fold enrichment of RNA polymerase II at the ERRE region. PCR analysis after ERR{alpha} immunoprecipitation was next done using primers that cover the ERRE region of the ESRRA promoter or a negative control region 4 kb upstream (Fig. 6B). In contrast to the TFF1 promoter, ERR{alpha} recruitment to the ESRRA promoter did not change after EGF treatment compared with control (Fig. 6B, top). There was also no recruitment of ERR{alpha} to the negative control region of the ESRRA promoter, confirming the specificity of the chromatin immunoprecipitation. Quantitative real-time PCR analysis (Fig. 6B, graph on top) confirmed the steady-state of ERR{alpha} recruitment to the ESRRA promoter in that the fold enrichment was the same without or with EGF. In agreement with this observation, recruitment of RNA polymerase II to the ERRE region was altered only slightly by EGF, as estimated both by electrophoretic and quantitative analysis (Fig. 6B, bottom). Finally, chromatin immunoprecipitation analysis was used to confirm that EGF does not affect recruitment of ER{alpha} to the TFF1 promoter. EGF-treated chromatin was immunoprecipitated with the ER{alpha} antibody and PCR done using the TFF1 promoter primers, as the ERE is located only 124 bp upstream of the ERRE. ER{alpha} recruitment to the TFF1 promoter was unchanged by EGF treatment compared with control (Fig. 6A, bottom). These data indicate that EGF enhances recruitment of both ERR{alpha} and RNA polymerase II to the TFF1 promoter, but not to the ESRRA promoter, and confirm that the EGF-induced effects on the TFF1 promoter are specifically through ERR{alpha}, and are not influenced by ER{alpha}. The presence of more RNA polymerase II at the TFF1 promoter suggests that the TFF1 gene is activated for transcription after EGF treatment, while the ESRRA gene is not. Quantitative reverse transcription-PCR (RT-PCR) analysis (Fig. 6C) was used to show that TFF1 transcript levels were increased 2.3-fold after EGF treatment compared with untreated cells, whereas levels of ESRRA were not significantly affected. Together, these data indicate that EGF selectively up-regulates ERR{alpha} recruitment to the TFF1 promoter, leading to enhanced transcription of this gene.



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  		Figure 6. EGF signaling differentially regulates ERR{alpha} target promoter recruitment in vivo. Chromatin immunoprecipitation of MCF-7 cells treated with EGF for 20 minutes or untreated control. A, schematic of the TFF1 promoter indicates the location of primers for positive ERRE or 4 kb upstream negative control amplicons. Chromatin was immunoprecipitated with the ERR{alpha}, RNA polymerase II, or ER{alpha} antibodies (+) or preimmune serum (–) followed by PCR analysis with TFF1 promoter ERRE (top) or negative control (bottom) primers. i, input. Graph, quantitative RT-PCR analysis of control or EGF-treated chromatin using TFF1 ERRE primers. B, schematic of the ESRRA promoter indicates the location of primers for positive ERRE or 4 kb upstream negative control amplicons. Chromatin was immunoprecipitated with the ERR{alpha} or RNA polymerase II antibodies (+) or preimmune serum (–) followed by PCR analysis with ESRRA promoter ERRE (top) or negative control (bottom) primers. Graph, quantitative RT-PCR analysis of chromatin using ESRRA ERRE primers. C, RT-PCR analysis of TFF1 and ESRRA expression in untreated (–) or EGF-treated MCF-7 cells. Expression levels of both genes were corrected for that of the RPLP control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Phosphorylation of nuclear receptors is an important mechanism for regulation of their transcriptional activity. In the case of classic steroid receptors, phosphorylation provides ligand-independent regulation in addition to their hormone-dependent activation, while for orphan nuclear receptors it provides regulation in the absence of cognate ligand. In this report, we investigated a link between ERR{alpha} phosphorylation and its transcriptional function in breast cancer cells. ERR{alpha} was found to be phosphorylated in vivo in response to EGF signaling, which enhanced receptor DNA binding. In addition, ERR{alpha} was phosphorylated in the DNA-binding domain in vitro by PKC{delta}, an event that also enhanced its DNA binding. PKC{delta} signaling selectively enhanced ERR{alpha} transactivation function in MCF-7 cells, stimulating ERR{alpha}-dependent activation of the TFF1 promoter, but not affecting receptor activity on its own promoter. The selective promoter activation by ERR{alpha} was confirmed using chromatin immunoprecipitation experiments. EGF stimulation resulted in enhanced occupation of the TFF1 promoter by ERR{alpha} and RNA polymerase II, and led to enhanced TFF1 transcription. In contrast, neither ERR{alpha} nor RNA polymerase II recruitment to the ESRRA promoter was changed after EGF treatment. We have hence identified a signaling pathway by which ERR{alpha} is phosphorylated and provides promoter-specific activation of target genes.

The function of several nuclear hormone receptors is regulated by phosphorylation in the DNA-binding domain, leading to both positive and negative effects on receptor DNA binding. Phosphorylation of the HNF4 (NR2A1) DNA-binding domain enhances DNA binding (41), while phosphorylation of both NGF1-B (NR4A1) and T3R{alpha}1 (NR1A1) in the DNA-binding domain by PKA inhibits DNA binding (42, 43). PKC family members directly phosphorylate vitamin D receptor (VDR, NR1I1), retinoic acid receptor {alpha} (RAR{alpha}, NR1B1), and COUP-TF1 (NR2F1) in the DNA-binding domain, with distinct transcriptional outcomes. VDR phosphorylation by PKCß inhibits binding to the VDRE, reportedly because of a receptor conformation change in the region adjacent to the phosphorylation site, as well as through overall altered tertiary structure of the receptor (44). RAR{alpha} is phosphorylated by PKC{alpha} and PKC{gamma} in the extended carboxyl-terminal region of the DNA-binding domain, which inhibits dimerization with the retinoid X receptors, potentially by producing a direct conformational constraint in the dimerization interface or through a conformational change in the receptor DNA-binding domain and ligand-binding domain that reorientates the dimerization interface (45). COUP-TF1 phosphorylation by PKC in the DNA-binding domain enhances its DNA binding but does not change receptor dimerization, so the increased DNA binding is thought to be via direct phosphorylation-induced steric or electrostatic changes leading to enhanced receptor affinity for DNA (46). In this study, we have shown that EGF-induced ERR{alpha} phosphorylation enhances its DNA binding, and phosphatase treatment of the receptor suggests that this occurs through enhanced dimer binding (Fig. 2). A distinctive feature of our findings is that phosphorylation of the DNA-binding domain of ERR{alpha} seems to change not only the DNA-binding capacity of the receptor, but also how the protein interacts with DNA, as a monomer or as a dimer, suggesting a mechanism for selective gene regulation by a receptor possessing the ability to bind DNA in both forms.

We have shown that PKC{delta} directly phosphorylates ERR{alpha} in vitro and increases its DNA binding (Fig. 3), and that PKC{delta} activation augments ERR{alpha} transactivation of the TFF1 promoter in breast cancer cells (Fig. 5). Transfections with TFF1 promoter deletion constructs indicate the effects of PMA on TFF1 activation are specifically mediated through ERR{alpha} and not ER{alpha}. While it is possible that the effects on the ERRE could be mediated via ER{alpha}, this is unlikely as it preferentially binds the inverted repeat ERE element and its activation through the ERRE consensus sequence is promoter specific (47). Evidence suggests that PKC{delta} could be an in vivo regulator of the EGF-induced effects on ERR{alpha} phosphorylation and transcriptional activity. PKC{delta} is activated via EGFR/ErbB2 signaling, an effect that is blocked by EGFR inhibitors (38–40). We have also shown that PKC{delta} is activated in MCF-7 cells by EGF (Fig. 4). In addition, activated PKC{delta} localizes to the nucleus (48) and can associate with nuclear receptors in vivo. PKC{delta} has previously been shown to associate with ligand-activated RAR{alpha} nuclear complexes that bind to retinoic acid response elements (49). Together, these data indicate that PKC{delta} has the potential to be an in vivo kinase for ERR{alpha}, acting downstream of the EGF signal to provide phosphorylation and promoter-specific activation of ERR{alpha}.

Chromatin immunoprecipitation experiments showed that EGF signaling enhanced ERR{alpha} enrichment on the TFF1 promoter, but not on the ESRRA promoter (Fig. 6), indicating that receptor phosphorylation status selectively regulates promoter occupancy. This selectivity likely involves structural and conformational constraints of both the receptor and target gene promoter. As discussed above, ERR{alpha} likely undergoes phosphorylation-induced conformational changes that affect the tertiary structure of the DNA-binding domain itself, as well as the receptor as a whole. In addition, the individual promoter structure will likely be dictated by both the sequence in the immediate vicinity of the ERRE elements and by the nature of the flanking regions. The specific conformation of phosphorylated ERR{alpha} in combination with distinct promoter structural features likely contributes to the preferred affinity of the receptor for the TFF1 promoter over the ESRRA promoter. The distinct conformation of the TFF1-bound ERR{alpha} may also selectively enhance coactivator recruitment, thus dictating the extent of activation of the specific promoter. The selective enhancement of RNA polymerase II occupancy on the TFF1 promoter, and increased TFF1 RNA expression after EGF treatment (Fig. 6) provide evidence for such selective promoter activation.

A potential role for ERR{alpha} in breast cancer is becoming more evident, as its expression correlates with an aggressive tumor phenotype in paralleling ErbB2 overexpression (22). In addition, its presence in tumor samples associates with an increased risk of disease recurrence or adverse clinical outcome (21). There is also some evidence to suggest that PKC{delta} could be important in tumor cells with an aggressive phenotype, as its expression correlates with breast tumor line metastatic potential (30). It could be hypothesized (see model Fig. 7) that enhanced EGF/ErbB2 signaling in tumor cells activates PKC{delta} and promotes its nuclear localization. This may lead to ERR{alpha} phosphorylation, which would promote its dimeric DNA binding and subsequent activation of distinct target promoters. Such events may provide a mechanism to selectively modulate ERR{alpha} target genes involved in breast cancer. The phosphorylation-induced selective promoter activation by ERR{alpha} may have important downstream transcriptional consequences and, therefore, it will be of interest to identify the full range of genes up-regulated by EGF-induced activation of ERR{alpha} in MCF-7 cells to help understand the potential role played by this receptor in breast cancer cells.



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  		Figure 7. Schematic model of ERR{alpha} phosphorylation-induced selective target gene activation. In this model, the EGF signal is transmitted through the EGFR/ErbB2 receptor tyrosine kinases, leading to activation of downstream signaling pathways and phosphorylation of PKC{delta}. PKC{delta} activation enhances its nuclear localization and promotes phosphorylation of ERR{alpha}. This event produces a conformational change in the receptor and selectively enhances ERR{alpha} dimeric DNA binding, resulting in promoter-specific activation of ERR{alpha} target genes.

 

   Acknowledgments
 
Grant support: Canadian Institutes of Health Research operating grant.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Yoshimitsu Kiriyama for cell lines and PCR primers, Dr. Jae-Won Soh for providing us with PKC{delta} constructs, and Geneviève Deblois and Josée Laganière for assistance with the chromatin immunoprecipitation assay.


   Footnotes
 
3 Y. Kiriyama and V. Giguère, unpublished data. Back

Received 3/21/04. Revised 5/ 3/05. Accepted 5/ 9/05.


   References
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 Materials and Methods
 Results
 Discussion
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