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Endocrinology, doi:10.1210/en.2003-0432
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Endocrinology Vol. 144, No. 11 4894-4904
Copyright © 2003 by The Endocrine Society

Estrogen Stimulates Estrogen-Related Receptor {alpha} Gene Expression through Conserved Hormone Response Elements

Dianxin Liu, Zhiping Zhang, Wesley Gladwell and Christina T. Teng

Gene Regulation Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709

Address all correspondence and requests for reprints to: Christina T. Teng, 111 Alexander Drive, P.O. Box 12233, MD E201, Research Triangle Park, North Carolina 27709. E-mail: Teng{at}niehs.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The estrogen-related receptor {alpha} gene encodes a nuclear receptor protein, ERR{alpha}, whose structure is closely related to the estrogen receptors. ERR{alpha} modulates estrogen receptor (ER)-mediated signaling pathways both positively and negatively. It is selectively expressed in a variety of cell types during development and in adult tissues. We have previously shown that estrogen stimulates the expression of the ERR{alpha} gene in mouse uterus. In this study, we found that the ERR{alpha} gene is stimulated by estrogen in mouse uterus and heart but not in liver. Estrogen also stimulates the expression of ERR{alpha} in the human breast and endometrial cell lines. The human ERR{alpha} gene promoter contains multiple Sp1 binding sites, and the Sp1 protein is required for the promoter activity. The major estrogen response is mediated by a 34-bp DNA element that contains multiple steroid hormone response element half-sites (MHREs) that are conserved between the human and mouse ERR{alpha} gene promoters. Mutations made at a single or multiple sites of the MHREs abolished the ER-mediated transcription of the element in transient transfection experiments. By chromatin immunoprecipitation assay, we demonstrated the interaction between ER{alpha} and MHREs of the endogenous ERR{alpha} gene promoter in MCF-7 cells. Estrogen treatment further enhanced the association of ER{alpha} and MHREs in vivo. The present study demonstrated that the ERR{alpha} gene is a downstream target of ER{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS PLAY A PIVOTAL role in the proper development and physiological function of both female and male reproductive organs (1, 2). Recently, estrogens have been found to be involved in a much broader spectrum of biological functions than previously had been recognized (1, 2, 3, 4). Estrogen regulates its target genes through estrogen receptors (ERs) by genomic and nongenomic pathways (5, 6, 7). ER-dependent estrogen action is transduced by ER{alpha} and the recently discovered ERß subtype (8, 9, 10, 11, 12, 13). ERs are structurally organized into functional domains consisting of a highly conserved DNA binding domain flanked by an activation domain 1 (AF1) in the N-terminal region and a ligand-binding domain and activation domain 2 (AF2) in the C-terminal region (7, 14). The function of AF1 is not well understood (15); however, the AF2 function has been extensively studied (10, 16, 17). Upon ligand binding, ERs transactivate the target gene by binding to a short DNA sequence termed an estrogen response element (ERE) that is composed of two AGGTCA motifs arranged in a palindrome structure separated by three base pairs (review) (18). In concert with other transcription factors and coactivators or corepressors, the ERs regulate the target gene activity in a ligand-, cell type-, response element-, and promoter context-specific manner (10, 11, 12, 13, 19, 20).

The estrogen-related receptor (ERR) subfamily contains three closely related members ({alpha}, ß, and {gamma}) (21, 22). The genes for ERR{alpha} and ß were identified during a search for genes related to the ER{alpha} (22). ERRs display significant homology to the ER{alpha} at the DNA binding domain (68% similarity of amino acids) and less homology with either N- or C-terminal regions. ERR{alpha} was first discovered to bind the extended hormone response element half-site, TCAAGGTCATC of the human lactoferrin gene (23) that is also the steroid factor 1 (SF1) binding element (24, 25), and later it was found to bind a variety of EREs as monomer or homodimer (23, 26, 27, 28, 29). In the absence of exogenous ligand, ERR{alpha} has been shown to interact with some of the same coactivators that interact with ER{alpha} (27, 28, 30, 31). Based on the nature of ERR{alpha} binding and the interaction with coactivators, it has the potential to regulate genes that are the target for either SF1 or liganded ER{alpha}. Recently, it was shown that ERR{alpha} activity can be inhibited upon binding to the organochlorine pesticides such as toxaphene and chlordane (32) and diethylstilbestrol (DES) at high concentrations (10–100 µM concentration level) (33). These observations offer additional layers of regulatory complexity for ERR{alpha} to modulate the estrogen signaling pathway (34).

Other than modulation of ER-mediated transcriptional activity, ERR{alpha} plays an active role in bone morphogenesis such as regulation of the osteopontin gene, bone resorption, and osteoprogenitor cell proliferation and differentiation (35, 36). ERR{alpha} regulates the transcriptional activity of human lactoferrin gene (23, 28), human medium-chain acyl coenzyme A dehydrogenase gene (37, 38, 39), thyroid receptor {alpha} gene (40), aromatase gene (41), osteopontin gene (42), and small heterodimer partner orphan nuclear receptor (43). These findings imply that ERR{alpha} modulates activities of a number of genes and consequently, different biological functions.

ERR{alpha} is expressed early in the embryo and in selected fetal and adult tissues (44, 45, 46, 47). However, regulation of its expression in various tissues and during development is largely unexplored. Because ERR{alpha} functions as constitutive activator in the cell (27, 28, 30), its regulated expression could be a preliminary step in the regulation of its function. Our laboratory has previously shown that estrogen stimulates ERR{alpha} expression in the mouse uterus (47). In this report, we demonstrated that the expression of ERR{alpha} gene in the mouse uterus and heart is stimulated by estrogen. Furthermore, we established the estrogen responsiveness of the endogenous ERR{alpha} gene in human uterine and mammary gland cell lines. We characterized the multiple hormone response element half-sites (MHREs) of the ERR{alpha} responsible for estrogen-stimulated activity in both human cell lines and mouse tissues. By chromatin immunoprecipitation (ChIP) assay, we showed that ER{alpha} indeed interacts with the MHREs of the ERR{alpha} gene in vivo and the exposure to estradiol-17ß (E2) enhances their interaction. Regulation of ERR{alpha} expression by ER{alpha} provides a foundation for understanding how ERR{alpha} modulates the estrogen response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female CD-1 mice (21-d-old) were obtained from Charles River Laboratories (Wilmington, MA). They were housed and fed in the National Institute of Environmental Health Sciences (NIEHS) animal facility, and handled according to the Guidelines of the Animal Care and Use Committee. The mice were divided into three groups: control, injected with vehicle (0.2 ml); 1x DES, one injection of DES in corn oil (0.2 ml) at 300 µg/kg of body weight and killed 24 h later; 3x DES, three injections every 24 h and killed on d 4. Each experimental group contains three mice, and the experiments were repeated. Tissues were removed and placed on dry ice immediately after the animals were killed and stored at -70 C until use.

Plasmids and oligos
Full-length ERß cDNA in pcDNA3 expression vector was a gift from K. Korach (Laboratory of Reproductive and Developmental Toxicology, NIEHS). ER{alpha} expression vector (pSG-HEGO) was a gift from P. Chambon (Centre National de la Recherche Scientifique, Strasbourg, France) and the pPacSp1 was from R. Tjian (University of California Berkeley). The 0.8-kb ERR{alpha} promoter (-811/+12) was amplified by PCR with the 1.3-chloramphenicol acetyltransferase (CAT) reporter (46) as the template and the forward primer 5'-CGGGTACCCGTGGAATTTGAGTCCTA-3' (-811/-794) containing the KpnI site (underlined) and the reverse primer with the NheI site (underlined) 5'-GCGCTAGCCCTACTCCGCTTCCTCCAGC-3' (-8/+12). The PCR product was cloned into CAT-B (basic 3) reporter at the KpnI/NheI sites as the 0.8-CAT. The 0.6-CAT (-593/+12) was constructed with an identical template and the reverse primer of the 0.8-CAT and a different forward primer, 5'-CGGGTACCTATTGCATGTTTTAAGTCGGC-3' (-593/-573) containing the KpnI site (underlined). Sequence of the human ERR{alpha} multihormone response element half-sites (MHREs, -712/-649) consists of a 23-bp nucleotides (A, 5'-GTGACCTTCATTCGGTCACCGCA-3') twice and a shorter 11-bp (B, 5'-GTGACCTTGAG-3') once (A,A,B). The mouse ERR{alpha} MHREs is identical with the human sequence except the A region appears only once (A, B). The wild-type and mutant oligos containing the A and B regions and its complementary strand were synthesized (Sigma Genosys, The Woodlands, TX). The top strand contains NheI site, whereas the bottom strand has the XhoI site. To make the double-stranded oligos, we first placed an equal amount of the complementary oligos in TE [10 mM Tris-HCl and 1 mM EDTA (pH 8.0)] buffer, left in boiling water for 10 min and then slowly cooled to room temperature. The double-stranded oligos were then cloned into the simian virus 40 (SV40)-CAT reporter at the NheI/XhoI sites. The reporters are: A,A,B-CAT (wild-type human MHERs); A,B-CAT (wild-type mouse MHERs); A-CAT (B sequence deleted); Am1,B-CAT (the 5' CC in A was mutated into AA); Am2,B-CAT (the GG in A was mutated into AA); A,Bm-CAT (the CC in B was mutated into AA); Am1,m2,Bm-CAT (contains the above three mutations). The sequences of all constructs were verified by automated sequencing (NIEHS, sequencing core laboratory).

Oligo primers used in standard RT-PCR to detect mRNAs are as follows: the forward primer (exon 6) 5'-AGATGTCAGTACTGCAGAGCGT-3' and reverse primer (exon 7) 5'-GCTTCATACTCCAGCAGG-3' to detect the level of ERR{alpha} mRNA. The primer pairs for human ß-actin are as follows: the forward primer (exon 5) 5'-GACAGGATGCAGAAGGAGATCAC-3' and the reverse primer (exon 6) 5'-GCTGATCCACATCTGCTGGAA-3', which encodes a 144-bp product. The primer pairs for ER{alpha} are as follows: the forward primer (exon 1) 5'-TGCCCTACTACCTGGAGAACGA-3' and the reverse primer (exon 2) 5'-GCCATACTTCCCTTGTCATTGG-3', which encodes a 142-bp product. The primer pairs for ERß are as follows: the forward primer (exon 1) 5'-GGTGTGAAGCAAGATCGCTAGA-3' and reverse primer (exon 2) 5'-GTGAGCATCCCTCTTTGAACCT-3', which encodes a 122 bp product.

Oligo primers used in real-time PCR and the amplicon size are as follows: human ERR{alpha} forward primer 5'-GGCCCTTGCCAATTCAGA-3' (exon 6) and the reverse primer 5'-GGCCTCGTGCAGAGCTTCT-3' (exon 7) with a 79-bp amplicon; mouse ERR{alpha} forward primer 5'-TTCGGCGACTGCAAGCTC-3' (exon 6) and reverse primer 5'-CACAGCCTCAGCATCTTCAATG-3' (exon 7) with a 107-bp amplicon; mouse lactoferrin forward primer 5'-GAGATGTGGCTTTTACCAGAGGA-3' (exon 6) and reverse primer 5'-CTGGGCAGAGCAGCTTGTACT-3' (exon 7) with a 87-bp amplicon; human ß-actin primer pair has been described above; mouse ß-actin forward primer 5'-GACAGGATGCAGAAGGAGATTAC-3' (exon 5) and reverse primer identical with human ß-actin reverse primer (exon 6) with a 144-bp amplicon; mouse mitochondrial ribosomal protein L32 (MRPL32) forward primer 5'-AGAGGTGCTGGGAGCTGCTA-3' and reverse primer 5'-GATGGATGGTCTCTGGACGG-3' with a 101-bp amplicon.

Oligo primers used in ChIP to detect the human ERR{alpha} MHREs region by PCR are as follows: the forward primer 5'-GTCAGTGCAGGACAGCCCGCGAG-3' (-758 /-734) and the reverse primer 5'-GATAGGGCCCGGACGGAGAAAGC-3' (-649/-627).

Cell culture and transient transfection
Human endometrial carcinoma (HEC)-1B [American Type Culture Collection (ATCC) (Manassas, VA) no. HTB-113] was maintained in 90% Eagle’s MEM (E-MEM) and 10% fetal bovine serum (FBS) at 37 C. MCF-7 (ATCC no. HTB-22) was cultured in 90% E-MEM, 10% FBS, and 10 µg/ml bovine insulin. Schneider’s Drosophila line 2 (SL2, ATCC, CRL-1963) was maintained in 90% Schneider’s Drosophila medium and 10% FBS. Cells were transferred into phenol red-free medium containing 10% dextran-coated charcoal-stripped FBS (CS-FBS) for at least 24 h before transfection and 7 d before real-time PCR and ChIP assay studies. The amount of DES, E2 and ICI 182,780 (Astra Zeneca, Wilmington, DE) is indicated for each experiment. The transient transfection experiments in HEC-1B cells and SL2 insect cells were performed with QIAGEN effectene transfection system (QIAGEN, Valencia, CA). A DNA mixture consisting of 300 ng of reporter plasmids, 100 ng of internal control, 50 ng of expression vector (ER{alpha} or ERß), or empty vector and carrier DNA (to make the final DNA concentration of 500 ng/well in a six-well plate) was transfected into HEC-1B cells whereas total 750 ng/well of DNA consists of 650 ng reporter, 100 ng pPacSp1, or empty vector was transfected into SL2 cells. Twenty-four hours after transfection of HEC-1B cells, 10 nM DES was added for another 24 h. CAT reporter activities were measured and normalized with the ß-galactosidase (PCH110) activities as previously described (23). To measure the effect of ER{alpha} and ERß on the endogenous ERR{alpha} expression, 2 µg of either ER{alpha} or ERß expression vectors were transfected into HEC-1B cells. The transfected cells were either treated with 10 nM of DES or vehicle for 24 h. The total RNA was extracted from the cells according to the protocol of RNeasy Mini Kit (QIAGEN). RT-PCR was performed according to the instruction of Titan One Tube RT-PCR system (Roche Diagnostics Corp., Indianapolis, IN) with 200 ng of total RNA from each sample. We performed 25 cycles of PCR at 94 for 45 sec, 58 for 45 sec and 68 for 45 sec to ensure that the PCR is not oversaturated.

Real-time PCR
The total RNA extracted from MCF-7 cells and the mouse tissues were copied into cDNA in a final volume of 20 µl containing 1x PCR buffer II, 5.5 mM MgCl2, 2 mM deoxy-NTP, 8 U of ribonuclease inhibitor, 2.5 µM random hexamers, 25 U of murine leukemia virus reverse transcriptase, and 1 µg total RNA according to the instructions of Applied Biosystems (Foster City, CA). PCR primers were designed with the Primer Express software package that accompanies the Applied Biosystems Model 7700 sequence detector (Perkin-Elmer Life Sciences, Foster City, CA). We performed basic local alignment and search tool searches to confirm the gene specificity of the nucleotide sequences chosen as primers. To avoid amplification of contaminating genomic DNA, one of the two primers was placed at the junction between two exons or in a different exon. Primer sets were further analyzed by PCR for a single band on agarose gel. All PCRs were carried out using an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) in 50 µl reaction mixture containing 150 ng (for ERR{alpha} and lactoferrin) or 50 ng (for ß-actin or MRPL32) cDNA template, 2.9 mM MgCl2, 1x SYBR-green buffer, 1 mM each deoxy-NTP, 0.2 µM of each forward and reverse primer, and 1.25 U Amplitaq Gold (Applied Biosystems). The PCR conditions were set up as follows: after incubation at 50 C for 2 min and denaturing at 95 C for 10 min, 40 cycles of 95 C for 30 sec, 60 C for 1 min. Quantitation was accomplished by measuring the incorporation of the fluorescent dye SYBR-green. All PCRs were performed in duplicate, and the results were averaged. The averaged threshold cycle (CT) values of the real-time PCR were used in subsequent calculations. To quantify transcripts of the genes precisely, we monitored ß-actin gene expression (for MCF-7 samples) or MRPL32 (for mouse tissue samples) transcripts as the internal quantitative control. Each sample was normalized by quantitating against its ß-actin or MRPL32 transcript contents. The relative difference between E2 or DES induction was determined using the {Delta}{Delta}CT method as outlined in the Applied Biosystems protocol and the results are presented as fold increases.

Western blotting
The ER{alpha} or ERß transfected HEC-1B cells were treated with 10 nM DES or vehicle as described above. Nuclear protein was extracted according to the protocol of TransFactor Extraction Kit (CLONTECH, Palo Alto, CA). Protein concentration was determined (Pierce, Madison, WI; bicinchoninic acid method) and the total of 30 µg protein was resolved by 12% NuPAGE gel. After electrophoresis, the proteins were blotted onto PVDF membrane (NOVEX, San Diego, CA). Western blotting was carried out by specific antibody to ER{alpha} (ER AB-10, NEOMarkers, Fremont, CA) or ERß (CO1531, mouse monoclonal antibody, a gift from Dr. G. K. Greene, University of Chicago) and ECL detection system (Amersham Biosciences, Piscataway, NJ). Blots were stripped according to the instructions of NOVEX and reprobed with specific antibody to ß-actin (mouse monoclonal antibody, SIGMARBI) as control.

ChIP
The ChIP was performed according to the instructions of ChIP Assay Kit (Upstate Biotechnology, Lake Placid, NY) with slight modifications. MCF-7 cells that express endogenous ER{alpha} were grown in phenol red-free E-MEM supplemented with 10% CS-FBS and 10 µg/ml of insulin for 7 d. E2 (100 nM) or vehicle was added to the cells for 45 min followed by cross-linking the protein and DNA with 1% formaldehyde at 4 C overnight. The cells were washed with ice-cold PBS twice and permeablized in sodium dodecyl sulfate lysis buffer containing the protease inhibitor cocktail (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin and 1 µg/ml pepstatin A). Chromatin was sheared by sonication until the average length of DNA was 200 and 1000 bp as evaluated by agarose gel electrophoresis. The sheared chromatin was diluted in ChIP dilution buffer of the ChIP assay kit. An aliquot of the chromatin solution was reserved as an input control for the PCR. Eight microliters of specific ER{alpha} (ER Ab-10) or Ets-1 (Santa Cruz Biotechnology, Santa Cruz, CA) antibody (200 µg/ml) was added to the chromatin solution and incubated overnight at 4 C on a rotator. At the end of incubation, 60 µl of salmon sperm DNA/protein A agarose slurry was added, incubated for 1 h at 4 C with continuous rotation and the antibody/DNA complex on the agarose beads was collected by centrifugation. The beads were washed in the order of low salt immune complex buffer, high salt immune complex buffer, LiCl immune complex buffer, and the TE buffer provided by the supplier. The beads were suspended in elution buffer (1% sodium dodecyl sulfate and 0.1 M NaHCO3) and the precipitated protein/DNA complexes were eluted from the antibodies/beads. The resulting protein/DNA complexes were subjected to cross-link reversal in 5 M NaCl at 65 C for 4 h followed by the addition of 0.5 M EDTA, 1 M Tris-HCl, and 10 µg/ml proteinase K (Pierce) at 45 C for 1 h. DNA was purified by phenol/chloroform extraction and ethanol precipitation. The PCR condition was: 94 C for 30 sec, 55 C for 30 sec, and 72 C for 30 sec for 30 cycles.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Estrogen stimulates ERR{alpha} expression in vivo
We have previously shown that estrogen stimulates the transcription and translation of the ERR{alpha} gene in mouse uterus (47). To validate the earlier observation and to investigate whether the transcription of ERR{alpha} is also up-regulated in other estrogen target tissues such as liver and heart, we treated the 21-d-old immature mouse with one or three doses of DES at 300 µg/kg every 24 h. Total RNAs prepared from the uterus, liver, and heart of six mice were first reverse transcribed and then subjected to real-time PCR for quantitation of the mRNA (Fig. 1Go). As expected, ERR{alpha} mRNA (lanes 1–3) in the uterus was induced almost 3-fold in 24 h after DES injection (Fig. 1AGo, lane 2) and a 9-fold increase was found after multiple injections (lane 3). The results demonstrated that ERR{alpha} gene is sensitive to estrogen stimulation in immature mouse uterus and the level of sensitivity to estrogen was much lower than the estrogen-responsive lactoferrin gene (48, 49) (lanes 4–6) in the uterus (compare lanes 3 and 6). Two other estrogen-responsive tissues, liver and heart, were also examined. Interestingly, ERR{alpha} gene was not estrogen responsive in the liver (Fig. 1BGo, lanes 1–3), whereas it was in the heart (Fig. 1CGo, lanes 1–3); the opposite was found for lactoferrin gene (compare Fig. 1BGo, lanes 4–6 with Fig. 1CGo, lanes 4–6). The ß-actin mRNA was generally considered as constant after estrogen treatment and used to normalize the tested gene activity. However, we found that estrogen has a moderate effect on the expression of ß-actin gene in the uterus (Fig. 1AGo, lanes 7–9), whereas the mouse mitochondria ribosomal protein L32 mRNA (MRPL32) remains constant. Therefore, the MRPL32 mRNA level was used to normalize the tested gene products.



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  		FIG. 1. Estrogen stimulates mouse ERR{alpha} expression in selected tissues. Total RNAs of estrogen or vehicle-treated immature mouse uterus (A), liver (B), and heart (C) were isolated. The mRNA corresponding ERR{alpha} (1 2 3 ), lactoferrin (4 5 6 ), and ß-actin (7 8 9 ) were quantitated by real-time PCR as described in Materials and Methods. The mRNA levels of the tested genes were normalized with MRPL32 mRNA. The normalized ERR{alpha}, lactoferrin, and ß-actin mRNA from vehicle-treated mice were set as 1; and the estrogen-treated mice presented as fold of induction ± SD. Vehicle-treated mice are shown in lanes 1, 4, and 7; 1x DES at 300 µg/kg dose are shown in lanes 2, 5, and 8; 3x DES at 300 µg/kg dose every 24 h three times are shown in lanes 3, 6, and 9.

 
To investigate the estrogen responsiveness of human ERR{alpha} gene, we used human endometrial carcinoma HEC-1B cells transiently transfected with ER{alpha} or ERß expression constructs. HEC-1B cells do not express ER{alpha} or ERß (50), which we also verified by RT-PCR. Twenty-four hours after transfection, the cells were treated with 10 nM DES for another 24 h and the total RNA prepared. After introducing the ER{alpha} expression plasmid, the endogenous ERR{alpha} mRNA of the HEC-1B cells was slightly increased and a significant induction of ERR{alpha} mRNA occurred while also treating the cells with DES, whereas the ß-actin mRNA remained constant in the cells with or without estrogen treatment (Fig. 2AGo, top panel). The bar graph of ß-actin normalized ERR{alpha} mRNA clearly demonstrated the ER{alpha}-mediated estrogen response (Fig. 2AGo, bottom panel). The ERR{alpha} gene was not responsive to ERß-mediated estrogen action in the HEC-1B cells (Fig. 2BGo, top panel), and the inability of ERß to induce endogenous ERR{alpha} gene expression was shown by the bar graph normalized with ß-actin mRNA (Fig. 2BGo, bottom panel). To demonstrate that both ER{alpha} and ERß expression constructs indeed expressed in the HEC-1B cells after transfection, we prepared nuclear extracts from the transfected HEC-1B cells and performed Western blotting with specific antibody to ER{alpha} or ERß (Fig. 2CGo). Polypeptides with the predicted size of ER{alpha} (Fig. 2CGo, lanes 2 and 3) and ERß (Fig. 2CGo, lanes 5 and 6) were detected in HEC-1B nuclear extracts after transfection with the respective expression vectors. ERß in the transfected cells was weakly detected by the ERß antibody in Western blotting. To demonstrate that both ER{alpha} and ERß expression vectors were transcribed equally in HEC-1B cells, we measured the mRNA level of ER{alpha} and ERß by RT-PCR (Fig. 2DGo). As expected, both ER mRNAs were detected at a similar level (compare lanes 2 and 3 with lanes 5 and 6). The results established that both human and mouse ERR{alpha} are estrogen responsive in vivo and the ER{alpha} is the receptor primarily mediating the response.



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  		FIG. 2. Estrogen stimulates human ERR{alpha} expression in human HEC-1B cells. The human endometrial carcinoma HEC-1B cells were transfected with expression vector of ER{alpha} or ERß and treated with DES (10 nM) as described in Materials and Methods. A, ER{alpha}-mediated estrogen effect. Cells were transfected with ER{alpha} expression vector, treated with vehicle or DES and the total RNAs were extracted from the cells. The mRNAs for ERR{alpha} and ß-actin were determined by nonsaturating RT-PCR (25 cycles). The upper panels are the agarose gel image of RT-PCR products of ERR{alpha} and ß-actin. The gel picture was scanned by densitometry. The ERR{alpha} level was normalized with ß-actin and the number from untransfected and treated with vehicle cells was set as 1. The ER{alpha}-mediated estrogen effect was presented as relative fold induction and presented as bar graph at the lower panel. B, ERß-mediated estrogen effect. Identical experiments as described in A except ERß expression vector was transfected. C, Western blot. The expression of the transfected ER{alpha} and ERß expression vector in HEC-1B cells was examined by Western blotting with specific antibody to either ER{alpha} or ERß. The cells were transfected and treated as described in Materials and Methods. The nuclear protein extracts were prepared from the above experiments (A and B). Total of 30 µg protein from each cell extract was subjected to Western blotting with specific antibody to ER{alpha} (left panel) and ERß (right panel). The positive ER{alpha} (66 kDa) and ERß (54 kDa) band is indicated by arrows. After probing with ERs antibodies the filter was stripped and reprobed with specific antibody to ß-actin (43 kDa). D, RT-PCR. The total RNAs from the above experiments (A and B) were subjected to nonsaturating RT-PCR to measure the relative transcription of ER{alpha} and ERß expression vector. The primer pairs used in PCR are indicated on top. The PCR product size for ER{alpha} (142 bp) and ERß (122 bp) are indicated.

 
Sp1 is important for the TATAless and GC-rich human ERR{alpha} promoter activity
The human ERR{alpha} gene promoter has been identified (46). The promoter lacks the CAAT and TATA boxes and contains high levels of Gs and Cs (-600/+1, 70%). There are 11 consensus Sp1 binding sites in the 600 bp of the promoter region. Further upstream from this region, a 57-bp sequence that is composed of MHREs arranged with variable orientations and spacing configurations was identified. To examine whether Sp1 is required for the ERR{alpha} promoter activity, we constructed two reporters, one which contains the 11 Sp1 sites was designated as 0.6-CAT, whereas a longer one that contains both the Sp1 sites and the MHREs was designated as 0.8-CAT (Fig. 3AGo). The activities of the reporters were tested in the SL2 cells, an Sp1-null environment (51). Both 0.6- and 0.8-CAT reporters showed very little activity when the pPac0 empty vector was coexpressed; however, a robust reporter activity was seen after coexpression of the pPacSp1 expression vector with either 0.6-CAT or 0.8-CAT (Fig. 3BGo). The Sp1-stimulated activity on either 0.6-CAT or 0.8-CAT was comparable (compare lanes 2 and 4), suggesting that the Sp1 is involved in the basal promoter activity of ERR{alpha}. Nonetheless, which Sp1 sites are crucial for the ERR{alpha} promoter activity is unclear at the present time.



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  		FIG. 3. The human ERR{alpha} gene promoter requires Sp1 for its activity. A, Diagram of human ERR{alpha} gene promoter-reporter constructs 0.8-CAT (-811/+12), which includes the multiple hormone response element half-sites (MHREs) and the GC-rich promoter with 11 Sp1 binding sites. The 0.6-CAT (-593/+12) contains the GC-rich promoter with 11 Sp1 binding sites only. B, Sp1 is required for ERR{alpha} promoter activity. The insect SL2 cells were transfected with the 0.8-CAT or 0.6-CAT reporter together with either the pPac0 empty vector or the pPacSp1 expression vector. The experiments were repeated three times with duplicated samples and presented as the relative CAT activity ± SD.

 
Estrogen stimulates human ERR{alpha} promoter reporter activity in HEC-1B cells
To define the region that is responsible for estrogen action, 0.6-CAT and 0.8-CAT were tested in HEC-1B cells with overexpression of either ER{alpha} or ERß and in the presence or absence of 10 nM DES (Fig. 4AGo). We found that both ER{alpha} (lane 3) and ERß (lane 5) in the presence of DES stimulate the 0.6-CAT reporter activities severalfold compared with the promoter activity of the control cells (lane 1), even though there is no obvious estrogen response element present. Nonetheless, the ER{alpha}-mediated estrogen response of 0.6-CAT was moderate when compared with the 0.8-CAT (compare lanes 3 and 8). Inclusion of MHREs in the 0.8-CAT reporter rendered this reporter extremely responsive to ER{alpha}-mediated estrogen action. Interestingly, ERß elicited similar activation to 0.6-CAT and 0.8-CAT in the presence of DES (compare lanes 5 and 10), suggesting that the ERß-mediated estrogen action comes mainly from the 600-bp GC rich promoter region, whereas an additional estrogen response element that resides within the MHREs is the target for ER{alpha}. The estrogen response of the 0.8-CAT was dependent on both ER{alpha} expression level and DES concentration (data not shown). Although liganded ERß activated the ERR{alpha} promoter reporter activity in transiently transfected HEC-1B cells (Fig. 4AGo), it did not stimulate the expression of endogenous ERR{alpha} gene to the level detected by RT-PCR (Fig. 2BGo). This observation is consistent with the report that ER{alpha} is a much stronger activator than ERß in comparison of many estrogen response genes (5). The chromatin structure of the endogenous ERR{alpha} gene and the transiently transfected gene promoter may also account for this variability. To verify the ER{alpha}-mediated estrogen response, we blocked the estrogen stimulated 0.8-CAT activity by ICI 182,780, a specific inhibitor for ERs (Fig. 4BGo), thus confirming the ER{alpha}-mediated transactivation of ERR{alpha} gene.



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  		FIG. 4. The human ERR{alpha} gene promoter is estrogen responsive. A, ER{alpha}- or ERß-mediated estrogen response. The 0.6-CAT or 0.8-CAT (50 ng) reporters and 100 ng of ER{alpha} or ERß expression vectors were cotransfected into HEC-1B cells for 24 h and treated with 10 nM DES for another 24 h. CAT activities were normalized with ß-galactosidase activities and the CAT activity of control cells (treated with vehicle and no ER expression vectors) was set as 1, and the data presented as fold induction ± SD from three independent experiments with duplicated samples. B, ICI 182,780 blocked the ER{alpha}-mediated estrogen response. After transfection, cells were treated with vehicle, with 10 nM DES or DES plus 1 µM ICI 182,780.

 
The MHREs of ERR{alpha} gene is evolutionary conserved and is responsible for ER{alpha}-mediated activity
Because both human and mouse ERR{alpha} genes are estrogen responsive in selected tissues or cells, it is possible that a conserved estrogen response element is present in the gene and a similar estrogen signaling pathway is operative in both species. Searching the mouse genomic sequence database, we found a perfect match of 34 bp of the mouse ERR{alpha} gene to the human ERR{alpha} gene MHREs (Fig. 5AGo). Sequence within the MHREs of the human ERR{alpha} can be divided into groups designated as A (23 bp) and B (11 bp). The human has duplicated A and one B that covers 57 bp region (Fig. 5AGo, A,A,B), whereas the mouse has one A and one B that extends to 34 bp (Fig. 5AGo, A,B). The "A" region composed of two AGGTCA motifs arranged as tail-tail orientation with five spaces in between and the "B" region has one reversed AGGTCA motif (Fig. 5AGo, underlined). These AGGTCA motifs do not constitute typical estrogen response elements.



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  		FIG. 5. A conserved hormone response element of the ERR{alpha} gene mediates the estrogen action. A, Sequence alignment of the MHREs between human and mouse ERR{alpha} gene. Within the MHREs, multiple ERE half-sites (underlined) were arranged in different orientation and spacing. The 23-bp sequence which contains two ERE half-sites was marked as A, and the 11-bp sequence that contains one ERE half-site was marked as B. Single or multiple mutations made at the ERE half-sites within the MHREs in A and B regions were indicated by bolded lowercase (aa). B, The number of ERE half-sites determines the strength of estrogen response. SV40-CAT reporters containing various regions of MHREs and the mutant constructs were cotransfected with ER{alpha} expression vector into the HEC-1B cells. The transfected cells were treated with either vehicle or 10 nM DES for 24 h. CAT activities were measured and normalized with ß-galactosidase activities. The experiments were repeated three times with duplicated samples, and the data are presented as ±SD. The fold of DES response is determined by dividing the activity from ER{alpha} and DES-treated samples with the ER{alpha} sample of each individual construct and the result is indicated on top of the bar graph (lanes 3, 6, 9, 12, 15, 18, 21, and 24).

 
To test the strength of these motifs in estrogen response, we cloned the MHREs and its shorter versions into SV40-CAT promoter and measured the ER{alpha}-mediated effect on the reporter activities (Fig. 5BGo). The result showed that the short sequence such as A alone did not respond to estrogen (lanes 10–12), even though it consists of two AGGTCA motifs. The increase of A (lane 12) reporter activity in response to DES was similar to the SV40-CAT vector activity in the presence of DES (lane 3). To achieve a strong estrogen response, the ERR{alpha} gene requires both A and B regions such as in the mouse gene (lane 9), and with duplicated motifs such as in the human gene a robust estrogenic effect was observed (lane 6). To verify the finding from the deletion experiments that both A and B regions are involved in estrogen response, we generated single or multiple AGGTCA motifs mutation within the conserved A and B regions of human and mouse ERR{alpha} genes (Fig. 5AGo). Our results demonstrated that the mutation of any one of the three AGGTCA motifs within the A and B region has significant impact on the estrogen response (Fig. 5BGo, lanes 15, 18, and 21). The estrogen response through MHREs was abolished when all three AGGTCA motifs were mutated (lane 24). Again, the small estrogen response seen in Am1, m2, Bm-SV40-CAT (lane 24) in the presence of DES was similar to the SV40-CAT (lane 3) vector.

In vivo binding of ER{alpha} to the MHREs of ERR{alpha} promoter
We have shown earlier that the endogenous ERR{alpha} in HEC-1B cells was estrogen responsive when ER{alpha} expression vectors were transiently transfected into the cells (Fig. 2AGo). It is also important to be able to demonstrate that the endogenous ER{alpha} in the presence of natural estrogen can stimulate the expression of endogenous ERR{alpha} and the ER{alpha} indeed bind to the MHREs of the ERR{alpha} promoter in vivo. The MCF-7 cells contain both ERR{alpha} and ER{alpha} but lack of ERß (52) is an ideal cell line for the study. We treated the MCF-7 cells with 10 nM E2 for 24 h and the total RNA was prepared from the sample. The ERR{alpha} mRNA in the untreated and treated samples were quantitated by real-time PCR and normalized with ß-actin mRNA. ERR{alpha} mRNA levels increased more than 3-fold after E2 treatment (Fig. 6AGo). With ChIP assay, we found that the MHREs region of the ERR{alpha} gene in MCF-7 cells was immunoprecipitated by the ER{alpha} antibody in the absence of added estrogen (Fig. 6BGo, upper panel, lane 2). Nonetheless, the ER{alpha} immunoprecipitated with the MHREs chromatin fragment was enhanced after addition of E2 (lane 5). The nonimmune serum and the nonspecific immune serum could not immunoprecipitate the MHREs chromatin fragment of the ERR{alpha} gene (lanes 3 and 4, respectively). The association of ER{alpha} with MHREs was more evident by the densitometry scanning of the gel (Fig. 6BGo, lower panel), and the increased association of ER{alpha} and MHREs after estrogen treatment was clearly shown (lower panel, compare ER{alpha} alone and ER{alpha} plus E2). It was interesting to find that the ER{alpha} was associated with the ERR{alpha} promoter/enhancer region in the absence of exogenous hormone. Because the cells were cultured in CS-FBS for 7 d before performing the experiments and because no activity was detected from a transfected ERE-luciferase (ERE-Luc) reporter (data not shown), it is unlikely that the CS-FBS contains residue estrogens. The present results provide in vivo evidence that the ERR{alpha} gene is an end target of the ER{alpha}-mediated signaling pathway in MCF-7 cells.



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  		FIG. 6. ER{alpha} interacts with MHREs of ERR{alpha} in vivo and stimulates ERR{alpha} transcription in the presence of natural estrogen. A, ERR{alpha} mRNA in MCF-7 cells was induced by E2 (10 nM) through endogenous ER{alpha}. After the cells were exposed to the hormone for 24 h, total RNA from untreated and treated cells were extracted and the real-time PCRs were performed to quantitate the mRNA of the ERR{alpha}. The mRNA value from vehicle-treated cells was set as 1, and the data from hormone-treated cells presented as fold induction ± SD. The experiments were repeated three times with duplicated samples each time. B, ChIP for the presence of ER{alpha} on the MHREs of the ERR{alpha} gene promoter. The MCF-7 cells were cultured in phenol red-free E-MEM supplemented with CS-FBS and insulin for 7 d. The cells were treated with 100 nM of E2 or vehicle for 45 min, and the protein and DNA were cross-linked with formaldehyde in 4 C overnight. Chromatin preparation and ChIP assay were described in Materials and Methods. Antibodies used to precipitate the chromatin were indicated, and the region containing the MHREs was marked by arrow. Top panel, Agarose gel analysis of the PCR products. Lower panel, Densitometry scanning of the bands from the agarose gel.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ER-dependent estrogen action is a complicated process that not only involves ligands that activate the ERs, a number of different cofactors, the context of target promoter (12, 13, 53, 54), but also the closely related ERR orphan receptors that modulate the estrogen response (28, 31, 34). Physiological ligands for the ERRs have not yet been found and some reports have demonstrated that these receptors function as constitutive activators in cells (21, 27, 30, 55). Because the ERRs bind similar response elements within target genes and interact with the same coactivators as ERs, the location of ERRs in the estrogen target cells and the regulation of their expression in those cells is essential to better understand the role of ERRs in the estrogen response. In this report, we demonstrated that the mouse ERR{alpha} gene is estrogen responsive in selective estrogen target tissues and the human ERR{alpha} is also estrogen responsive in endometrial and mammary gland cell lines. These observations are supported by the identification of estrogen-responsive MHREs in both human and mouse ERR{alpha} genes. Finally, we showed an in vivo interaction between ER{alpha} and the MHREs of ERR{alpha} promoter.

ERs and ERR{alpha} were found in many tissues (1, 46, 47) and in some tissues both receptors were coexpressed. We examined the functional relationship of ER{alpha} and ERR{alpha} in three estrogen target tissues, liver, heart, and uterus of the mouse that also express abundant ERR{alpha} mRNA and protein. We found that ERR{alpha} was estrogen responsive in heart and uterus but not in liver, whereas the estrogen-responsive lactoferrin gene was up-regulated by estrogen in uterus and liver but not in heart (Fig. 1Go). The chromatin structure of the ERR{alpha} gene in those tissues may play an important role in estrogen responsiveness and is an area currently under investigation. Both in vivo and in vitro studies suggested that ER{alpha} is the primary receptor to regulate ERR{alpha} expression under the influence of estrogen (Figs. 2Go and 4AGo). The ERR{alpha} is highly expressed in heart (46, 47), and the discovery of its estrogen responsiveness in the heart (Fig. 1CGo) suggests that ERR{alpha} may play some role in the ability of estrogen to afford some protection against heart disease (56, 57).

The promoter from the human ERR{alpha} gene consists of 70% of Gs and Cs within a 600-bp region and 11 Sp1 binding sites (46), whereas the corresponding region for ERR{alpha} gene in mouse is 61% Gs and Cs with only four Sp1 binding sites (genomic database). In addition, whereas the human promoter lacks TATA and CAAT elements, the mouse promoter has a candidate TATA element 23 bp upstream from the transcription initiation site. Based on the organization of the human ERR{alpha} gene promoter, the Sp1 transcription factor could be important for the promoter activity. This was confirmed by the transfection experiments conducted in the insect SL2 cells (Fig. 3BGo). Although we have not shown the actual binding of Sp1 protein to the Sp1 sites of ERR{alpha} gene promoter, the sequence of these putative Sp1 binding sites matches the consensus sequence of Sp1 binding element. It is well documented that estrogen stimulates GC-rich promoter through protein-protein interaction between the Sp1 and ERs in which Sp1 binds the DNA but not the ERs (58). Furthermore, ER{alpha} and ERß subtypes differentially transactivate GC-rich promoter in a cell type-, ligand-, and promoter context-dependent manner (54). Our current study supported these reports. The GC-rich human ERR{alpha} promoter indeed mediates estrogen action via both ER{alpha} and ERß with ER{alpha} yielding stronger response (Fig. 4AGo, 0.6-CAT). Despite the moderate estrogen response of the promoter, ER{alpha}-induced transcription of ERR{alpha} gene was predominantly dependent on a MHREs. It is not known whether the Sp1 site 7 bp upstream to the MHREs (Fig. 3AGo, Sp1 12) plays any role in estrogen response. Nonetheless, the MHREs (AAB element) conferred a robust estrogen response in the transfection studies (Fig. 5BGo), suggesting that the MHREs is the major estrogen response element in ERR{alpha} gene. This conclusion is also supported by the ChIP assay (Fig. 6Go) that demonstrated the MHREs is the target site of ER{alpha}. Detection of ER{alpha} binding to the MHREs of endogenous ERR{alpha} in MCF-7 without estrogen suggests that the MHREs adopts an open chromatin structure and the chromatin remodeling is not a prerequisite for estrogen response of this gene (59). Nonetheless, addition of estrogen further enhances the ER{alpha} binding to MHREs and stimulates the transcriptional activity of the ERR{alpha} gene. We could not exclude the possibility that a small amount of estrogen is present in the charcoal-stripped serum even though there was no detectable activity of transfected ERE-Luc reporters in these cells without addition of exogenous estrogen (data not shown).

Mutation and deletion analyses revealed that the estrogen response requires at least three of the AGGTCA motifs within the MHREs such as those found in the mouse ERR{alpha} gene. Widely spaced multiple AGGTCA motifs that serve as promiscuous enhancers for different classes of nuclear receptors have been reported (60) and the natural genes that confer hormone actions through these types of elements were documented (61, 62, 63, 64). The presence of promiscuous nuclear receptor binding elements such as the MHREs in ERR{alpha} indicates that the gene may respond to a number of distinct nuclear receptor regulated signaling pathways. Indeed, ERR{alpha} may play multiple roles in regulating different aspects of cellular physiology. For example, in addition to modulating ER{alpha}-mediated response, ERR{alpha} is coexpressed with the proliferator-activated receptor coactivator-1{alpha}, a key regulator of cellular energy metabolism in tissues that metabolize fatty acids and it controls the expression of medium-chain acyl coenzyme A dehydrogenase gene (37, 38, 39, 65), suggesting that ERR{alpha} may be important in regulating the balance of cellular energy. These biological activities are regulated by many nuclear receptors including the orphan receptors (66). It is possible that ERR{alpha} not only modulates estrogen action, it also plays a balancing role for other nuclear receptor-mediated pathways.


   Acknowledgments
 
We thank Drs. T. Sueyoshi and C. Weinberger for critically reading the manuscripts, and K. Korach, S. Hewitt [National Institute of Environmental Health Sciences (NIEHS)], P. Chambon (Centre National de la Recherche Scientifique), G. K. Greene (University of Chicago) and R. Tjian (Uuniversity of California Berkeley) for providing reagents for this study. Sequencing provided by the NIEHS sequencing core laboratory is greatly appreciated. L. Moore edited the manuscript.


   Footnotes
 
Abbreviations: AF1 or AF2, Activation domain 1 or 2; CAT, chloramphenicol acetyltransferase; ChIP, chromatin immunoprecipitation assay; CS-FBS, charcoal-stripped FBS; CT, threshold cycle; DES, diethylstilbestrol; E2, estradiol-17ß; E-MEM, Eagle’s MEM; ERE, estrogen response element; ERE-Luc, ERE luciferase; ERR, estrogen related receptor; FBS, fetal bovine serum; HEC, human endometrial carcinoma; MHRE, multiple steroid hormone response element half-sites; MRPL32, mitochondrial ribosomal protein L32; SF1, steroid factor 1; SV40, simian virus 40; SL2, Schneider’s Drosophila line 2.

Received April 8, 2003.

Accepted for publication July 16, 2003.


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