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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1421
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 3 1830-1844
Copyright © 2005 by The Endocrine Society

Expression and Functional Study of Estrogen Receptor-Related Receptors in Human Prostatic Cells and Tissues

C. P. Cheung, Shan Yu, K. B. Wong, L. W. Chan, Fernand M. M. Lai, Xianghong Wang, Masatomo Suetsugi, Shiuan Chen and Franky L. Chan

Departments of Anatomy (C.P.C., S.Y., F.L.C.), Anatomical and Cellular Pathology (F.M.M.L.), Biochemistry (K.B.W.), and Surgery (L.W.C.), The Chinese University of Hong Kong, Hong Kong, China; Department of Anatomy (X.W.), University of Hong Kong, Hong Kong, China; and Beckman Research Institute of the City of Hope (M.S., S.C.), Duarte, California 91010

Address all correspondence and requests for reprints to: Franky L. Chan, Department of Anatomy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. E-mail: franky-chan{at}cuhk.edu.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen receptor-related receptors (ERRs; {alpha}, ß, {gamma}) are orphan nuclear receptors and constitutively active without binding to estrogen. Like estrogen receptors (ERs), ERRs bind to estrogen receptor elements and estrogen receptor element-related repeats. Growing evidence suggests that ERRs can cross-talk with ERs in different cell types via competition for DNA sites and coactivators. We hypothesize that ERRs might play regulatory roles in normal and neoplastic prostatic cells by sharing similar ER-mediated pathways or acting independently. In this study, we investigated mRNA and protein expression patterns of three ERR members in normal human prostate epithelial cells, established cell lines, cancer xenografts, and prostatic tissues. Additionally, effects of transient transfection of ERRs on prostatic cell proliferation and ER expression were also examined. RT-PCR showed that ERR{alpha} and ERR{gamma} transcripts were detected in most cell lines and xenografts, whereas ERRß was detected in normal epithelial cells and few immortalized cell lines but not in most cancer lines. Similar results were demonstrated in clinical prostatic specimens. Western blottings and immunohistochemistry confirmed similar expression patterns that ERR proteins were detected as nuclear proteins in epithelial cells, whereas their expressions became reduced or undetected in neoplastic prostatic cells. Transient transfection confirmed that ERRs were expressed in prostatic cells as nuclear proteins and transcriptionally active in the absence of estradiol. Transfection results showed that overexpression of ERRs inhibited cell proliferation and repressed ER{alpha} transcription in PC-3 cells. Our study shows that ERRs, which are coexpressed with ERs in prostatic cells, could regulate cell growth and modulate ER-mediated pathways via interference on ER{alpha} transcription in prostatic cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN ADDITION TO ANDROGENS, which play a central role in the normal development and neoplastic growths of prostate gland, estrogens have been suggested for a long time to play synergetic or distinct roles in the same processes (for recent reviews, see Refs. 1, 2, 3). However, the exact functional roles of estrogens and their receptors in normal and neoplastic prostates are still unclear. The effects of estrogens on the prostate are complex and involve both direct and indirect actions, which may be important at different periods of development.

The direct effects of estrogens on the prostate or their involvements in the development of prostate cancer and benign prostatic hyperplasia are believed to be mediated through their cognate receptors, estrogen receptors (ERs). Two distinct forms of ER are present in rodents and human, ER{alpha} and ERß, and they are functionally expressed in the prostate gland (4). ER{alpha} is expressed weakly and localized to the stromal cells as demonstrated by immunohistochemistry and in situ hybridization (5, 6); whereas ERß, which was originally cloned from a rat prostate cDNA library, is mainly expressed in the epithelial cells and at low levels in some stromal cells (4, 7, 8, 9). The distinct expression pattern of ER{alpha} and ERß in different tissue components in prostate has led to a view that estrogens and their receptors play different functional roles in the prostate and could explain why estrogens and their antagonists or selective ER modulators (SERMs) exert different actions on prostatic cells. However, this could be part of the story of estrogen signaling pathways in the prostate. Studies from ER knockout mouse models show that targeted disruption of either ER ({alpha} or ß) or both ERs does not affect the prostatic phenotype and function (10), suggesting that other ER-related receptors, ER isoforms, or alternative signaling pathways could exist and be involved in the prostatic growth and functions.

ER-related receptors (ERRs; also called estrogen-related receptors) belong to the nuclear receptor subfamily 3 (11) and consist of three closely related subtypes/members: {alpha}, ß, and {gamma} (also called ERR1/NR3B1, ERR2/NR3B2, ERR3/NR3B3). All ERR subtypes share significant homology with ERs in their protein structures, particularly in the DNA-binding domain (DBD) and ligand-binding domain, and thus they are named. However, they do not bind to estrogens or any other known physiological ligands and are classified as orphan nuclear receptors. Human ERR{alpha} and ERRß were isolated from kidney and heart cDNA libraries by screening with an ER{alpha}-DBD cDNA probe (12), whereas ERR{gamma} was cloned by PCR from fetal brain and other tissue cDNA libraries (13, 14, 15). Mouse ERR homologs were also identified by cDNA library cloning and yeast two-hybrid screening for proteins interacting with coactivator glucocorticoid receptor interacting protein-1 (16, 17, 18, 19, 20). Similar to ERs, all ERRs can bind to the consensus palindromic estrogen-responsive element (ERE) (16, 19, 21, 22, 23, 24, 25, 26). In addition, ERRs can bind as monomers or homodimers to some ERE-related response elements containing an extended half-site core sequence (TNAAGGTCA; steroidogenic factor-1 response element or ERR response element), which are also recognized by ER{alpha} and steroidogenic factor-1 (SF-1) but not ERß (15, 17, 18, 21, 23, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36). ERRs can constitutively activate transcription of target gene promoters or response element-controlled reporter genes in the absence of exogenous estrogens or ligands (15, 19, 22, 32, 33, 37, 38, 39). In vitro studies show that similar to ERs and other steroid hormone receptors, transactivation of ERRs depends on their interaction with nuclear receptor coactivators, including the p160 family of coactivators (19, 22, 23, 30, 38, 40, 41, 42, 43, 44, 45, 46) and heat shock protein 90 (16), for efficient DNA binding and homodimer formation. In addition, a few in vitro studies show that ERR{alpha} can interact with ER{alpha} to form heterodimer via direct protein-protein interaction (27, 30). Therefore, ERRs could regulate their own target genes independently or modulate the transcription of genes, which are also regulated by ERs or other orphan nuclear receptors via competition for DNA binding sites and coactivators in different cell types (23, 24, 25, 26, 39).

Based on this background, we hypothesize that besides ERs, ERRs could also be involved in the estrogen-signaling pathways in prostatic cells and play certain regulatory roles in normal and neoplastic growth of prostates. In this study, we investigated the expression patterns of the three ERR members in normal human prostate epithelial cells, immortalized and prostate cancer cell lines, cancer xenografts, and prostatic tissues. In addition, their effects on cell growth and transcription of ERs in prostatic cells were also explored by transient transfections.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and cell cultures

Normal human prostate epithelial cells (PrECs) (derived from an 18-yr-old black male; Clonetics, Cambrex, Walkersville, MD), nine immortalized human PrEC lines derived from either normal (RWPE-1, RWPE-2, PWR-1E, PZ-HPV-7, HPr-1, HPr-1AR, PNT1A, PNT2) or hyperplastic (BPH-1) prostates, seven prostate cancer lines (CA-HPV-10, LNCaP, C4–2B, DU 145, PC-3, MDA PCa 2b, CWR22Rv1), and one breast cancer cell line (MCF-7) were used in this study. RWPE-1, RWPE-2, PZ-HPV-7, CA-HPV-10, CWR22Rv1, LNCaP, DU 145, PC-3, MDA PCa 2b and MCF-7 were obtained from American Type Culture Collection (Manassas, VA), whereas PNT1A and PNT2 were obtained from European Collection of Cell Cultures (Salisbury, UK). HPr-1 and HPr-1AR were kindly provided by Professor Y. C. Wong (University of Hong Kong), BPH-1 by Dr. S. Hayward (University of Vanderbilt, Nashville, TN), and C4–2B by Dr. E. T. Keller (University of Michigan, Ann Arbor). PrECs were grown in prostate epithelial basal medium with growth supplements (Clonetics); RWPE-1, RWPE-2, PWR-1E, PZ-HPV-7, HPr-1, HPr-1AR, and CA-HPV-10 in keratinocyte serum-free medium supplemented with 5 ng/ml human recombinant epithelial growth factor and 50 µg/ml bovine pituitary extract; PNT1A, PNT2, and BPH-1 in RPMI 1640 with 10 mM HEPES and 10% fetal bovine serum (FBS); CWR22Rv1 and LNCaP in RPMI 1640 with 4.5 g/liter glucose, 1 mM sodium pyruvate, 10 mM HEPES, and 10% FBS; C4–2B in T-medium (80% DMEM and 20% F12K) with 5 µg/ml insulin, 13.6 pg/ml triiodothyronine, 5 µg/ml apo-transferrin, 0.25 µg/ml biotin, 25 µg/ml adenine, and 10% FBS; DU 145 in MEM with 1 mM sodium pyruvate and 10% FBS; PC-3 in F12K with 10% FBS; MDA PCa 2b in F12K with 25 ng/ml cholera toxin, 10 ng/ml epithelial growth factor, 5 µM phosphoethanolamine, 100 pg/ml hydrocortisone, 45 nM selenious acid, 5 µg/ml insulin, and 20% FBS; MCF-7 in RPMI 1640 with 10 µg/ml insulin and 10% FBS. All cells were routinely grown in media without antibiotics in 60- or 100-mm dishes at 37 C with a 5% CO2 in atmosphere. All culture media and reagents were obtained from Life Technologies Inc. (Invitrogen Corp., Carlsbad, CA), whereas F12K was from Sigma (St. Louis, MO). Total RNA and protein were extracted from cells when they grew to 80–90% confluency.

Prostate cancer xenografts

Human primary prostate cancer xenograft CWR22 (kindly provided by Dr. T. G. Pretlow, Case Western Reserve University, Cincinnati, OH) was propagated and serially transplanted sc at scapular region in male nude mice (BALB/c strain 10 wk old), which were implanted with one 1-cm-long testosterone-filled Silastic tubing (1.67 mm in inner diameter x 3.18 mm in outer diameter; Dow Corning, Midland, MI). Two androgen-independent sublines were developed from the original CWR22 by growing the xenografts in castrated male nude mice (CWR22R-AIM) and intact female mice (CWR22R-AIF), respectively. Some xenografts, which were atrophied after castration, became relapsed after 6–9 months of growth, and the sublines were selected for serial propagation in castrated males and intact female mice. Xenografts were harvested when tumors grew to 1–1.5 cm3 in sizes and frozen at –80 C. All animal protocols were approved by the Animal Experimentation Ethics Committee at the Chinese University of Hong Kong.

Human prostatic tissues

Archival paraffin blocks of normal human prostates from organ donors were used for immunohistochemical study (47). Benign hyperplastic tissues were obtained from patients with benign prostatic hyperplasia by transurethral resection of prostate, whereas neoplastic tissues were obtained by radical prostatectomy or ultrasound-guided needle biopsy from patients with prostate cancer. Surgical specimens were either rapidly frozen in liquid nitrogen and stored at –80 C for RNA or protein extraction or fixed in 4% paraformaldehyde in PBS for histological preparation (47). All human tissues were obtained with informed consent and approval from the Chinese University of Hong Kong Clinical Research Ethics Committee.

RNA extraction, RT-PCR, and Southern blot analyses

Total RNA was extracted from homogenized cultured cells, xenografts, and prostatic tissues using TRIzol reagent (Invitrogen). Approximately 5 µg DNase I-treated RNA samples were reverse transcribed to cDNAs by Muloney murine leukemia virus reverse transcriptase using Oligo(dT) primer (Invitrogen), and 1 µl of cDNA samples was used for PCR. The sequences of oligonucleotide primers used in this study and the conditions of PCR are listed in Table 1Go. The PCR products of ERR subtypes amplified in prostatic cells and tumor xenografts were also analyzed by Southern blottings, using digoxigenin-labeled cDNA probes of ERRs. Semiquantitative RT-PCR analysis of mRNAs of ERRs and ERs in various cell lines was performed. Optimal PCR conditions were predetermined so that amplifications were within the exponential phase of PCR with reference to the amount of input RNA. The PCR products were resolved in 1.2% agarose gel electrophoresis with ethidium bromide. The intensities of fluorescent images captured under UV illumination were quantified by a gel documentation program (FluorChem with AlphaEase FC software, Alpha Innotech Corp., San Leandro, CA). Signal intensities of PCR products of ERRs and ERs were normalized to those of ß-actin as ratios to produce arbitrary units of relative abundance. The mean values obtained from four separate experiments were plotted.


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  		TABLE 1. Sequences of primers for RT-PCR and conditions

 
Generation of antibodies

For protein expression studies, polyclonal antibodies against ERRs were raised in rabbits toward synthetic peptides designed from predicted amino acid sequences of ERRs. A 20-mer peptide NH2-KAEPASPDSPKGSSETETEC-CO2H, which represents amino acids 14–32 of human ERR{alpha} and contains one additional cysteine residue at C terminal, was synthesized and cross-linked via the C-terminal cysteine to keyhole limpet hemocyanin (KLH). Another 15-mer peptide AcNH2-YEDCTSGIMEDSAIK-CONH2 representing amino acids 76–90 of human ERRß was synthesized and cross-linked via the C-terminal lysine to KLH (Peptron Inc., Daejeon, South Korea). Antisera were generated by injecting sc 500 µg peptide-KLH conjugates mixed with Freud’s adjuvant into male New Zealand rabbits at intervals of 3 wk. Sera of the third booster were used. The rabbit antibody against ERR{gamma} was developed and described previously (36).

Generation of polyhistidine-ERR fusion proteins

Plasmid constructs for the expression of N-terminal polyhistidine (His) tagged-ERR fusion proteins were generated by in-frame subcloning the full-length cDNAs of ERR{alpha}/ß/{gamma} [amplified from pSG5-ERRs (49) with full-length cDNA inserts of ERRs by PCR] into a prokaryotic expression vector pRSETA (Invitrogen), which has a 6 x His coding sequence at the 5' end of subcloned sequences. The primers used for PCR of ERRs and subcloning into pRSETA are as follows: 1) ERR{alpha}, 5'-primer with BamHI linker, 5'-AAT CGG ATC C ATG TCC AGC CAG GTG GTG GG-3', 3'-primer with EcoRI linker, 5'-TAC CGA ATT C TCA GTC CAT CAT GGC CTC GA-3'; 2) ERRß, 5'-primer with BamHI linker, 5'-TGC AGG ATC C ATG TCG TCC GAA GAC AGG CA-3', 3'-primer with EcoRI linker, 5'-ATC CGA ATT C TCA CAC CTT GGC CTC CAG CA-3'; and 3) ERR{gamma}, 5'-primer with BamHI linker, 5'-TGC AGG ATC C ATG GAT TCG GTA GAA CTT TG-3', 3'-primer with EcoRI linker, ATC CGA ATT C TCA GAC CTT GGC CTC CAA CA-3'. The translational start and stop codons are italicized. All clones were confirmed by DNA sequence before using for expression in bacteria. The 6 x His-ERR fusion proteins were expressed in C41 Escherichia coli at 25 C for 4 h by induction with 0.2 mM isopropyl ß-D-1-thiogalactopyranoside. The cells were lysed in 2x sodium dodecyl sulfate (SDS) loading buffer [100 mM Tris-HCl (pH 6.8), with 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, and 20% glycerol] with 1 mM phenylmethylsulfonyl fluoride and boiled at 95 C for 10 min before storing at –80 C for subsequent characterization of specificities of ERR antibodies by blotting with an anti-HisG mouse monoclonal antibody (Invitrogen).

Immunohistochemistry

Hydrated paraffin sections of human prostates were treated with 0.5% H2O2 in absolute methanol for 30 min. Sections were heated in 0.1 M Tris-HCl (pH 9.5), containing 5% urea at 95 C for 15 min for antigen retrieval before incubating with rabbit antisera against ERRs diluted (1:200–400) in 0.05 M PBS with 3% BSA. Control sections were incubated in dilution buffer without antisera or antisera preabsorbed with free forms of ERR synthetic peptides. After incubating with a biotinylated goat antirabbit IgG and streptavidin-peroxidase (Jackson Immuno-Research Laboratories Inc., West Grove, PA), the immunoreactive sites were visualized by a glucose oxidase-diaminobenzidine-nickel procedure (47).

Protein extraction and immunoblot analysis

Trypsinized cultured prostatic cells were solubilized in a cold lysis buffer [50 mM Tris-base, 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1% Nonidet P-40, 0.5% SDS (pH 7.4)] for 30 min at 4 C, followed by repeated passages of lysates through 25-G needles for shearing the chromosomal DNA. Supernatants were collected after centrifugation at 11,620 x g at 4 C. Total soluble proteins extracted were qualified by a modified Lowry method using BSA as standard. Protein samples were separated by 10% SDS-PAGE gels and loaded at 50 µg/lane. After electrophoresis, proteins were electrophoretically transblotted onto 0.2-µm polyvinylidene difluoride membranes. Blotted membranes were preblocked in PBS with 1% Tween 20 for 1 h before incubating with the ERR-antibodies diluted (1:500–1000) in PBS with 0.1% Tween 20 and 4% nonfat milk powder, followed by a goat antirabbit IgG conjugated to alkaline phosphatase (Zymed, San Francisco, Ca). The immunoreactive bands were revealed by incubating in a chromogen solution containing 4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indoyl-phosphate.

Construction of plasmids and cell transfections

Green fluorescent protein (GFP)-ERR fusion plasmids were constructed by in-frame subcloning the full-length cDNAs of ERR{alpha}/ß/{gamma} (amplified from pSG5-ERRs with full-length cDNA inserts of ERRs by PCR) into plasmid enhanced GFP (EGFP)-C3 vector (BD Biosciences Clontech, Palo Alto, CA). The primers used for PCR of ERRs and subcloning into pEGFP-C3 are as follows: 1) ERR{alpha}, 5'-primer with HindIII linker, 5'-TAT GAA GCT T ATG TCC AGC CAG GTG GTG GG-3', 3'-primer with BamHI linker, 5'-CAT AGG ATC C TCA GTC CAT CAT GGC CTC GA-3'; 2) ERRß, 5'-primer with XhoI linker, 5'-TAT GCT CGA G ATG TCG TCC GAA GAC AGG CA-3', 3'-primer with BamHI linker, 5'-TAT AGG ATC C TCA CAC CTT GGC CTC CAG CA-3'; and 3) ERR{gamma}, 5'-primer with XhoI linker, 5'-GTC GCT CGA G ATG GAT TCG GTA GAA CTT TG-3', 3'-primer with BamHI linker, 5'-TAT CGG ATC C TCA GAC CTT GGC CTC CAA CA-3'. The translational start and stop codons are italicized. The luciferase reporter plasmids, pGL3-ERE3-Luc and pGL3-SF-13-Luc containing three copies of ERE or SF-1 response elements subcloned into pGL3-promoter vector (Promega, Madison, WI) were constructed as described (48). The expression plasmids, pSG5-ERR{alpha}/ß/{gamma}, were constructed by subcloning the PCR-amplified full-length human ERR ({alpha}, ß, and {gamma}) coding regions into pSG5 plasmid at the EcoRI site (49).

For subcellular localization of GFP-tagged ERRs in transfected prostatic cells, PC-3 cells were seeded at 9 x 104 cells on 25-mm polylysine-coated coverslips with metal rings and grown for 24 h before transfection. PC-3 cells were transiently transfected with 0.5 µg pEGFP-C3-ERR{alpha}/ß/{gamma}, using FuGENE 6 transfection reagent (Roche, Stockholm, Sweden) according to the manufacturer’s protocol. Transfected cells were maintained in culture medium with or without FBS. After 48 h of transfection, cells were either briefly fixed in 4% paraformaldehyde in PBS for 10 min or unfixed. Both fixed or unfixed cells were directly examined under a confocal scanning microscope with a 522-nm emission filter for GFP localization (MRC 1024, Bio-Rad Laboratories, Hercules, CA). Immunofluorescence of ERRs was also performed on fixed transfected cells using rabbit antibodies against ERRs followed by a secondary donkey antirabbit IgG conjugated with Cy3. Stained cells were examined under a confocal microscope using a fluorescein isothiocyanate (522 nm) and rhodamine (585 nm) emission filter for GFP fluorescent signals and ERR immunosignals, respectively.

Luciferase reporter assay

PC-3 cells were seeded at 5 x 104 cells/well in 24-well plates and grown for 24 h in F12K medium with or without 10% FBS before transfection. The cells were cotransfected with 0.125 µg pEGFP-C3-ERR{alpha}/ß/{gamma} (or empty vector pEGFP-C3), 0.125 µg pGL3-ERE3-Luc or pGL3-SF-13-Luc, and 0.0125 µg pRL-cytomegalovirus plasmids using FuGENE 6 transfection reagent in the presence or absence of FBS. After 48 h post transfection, cells were lysed and the luciferase activity in cell lysates was determined using a dual-luciferase reporter assay system (Promega). The luciferase activation activity was normalized to that of the internal control Renilla luciferase activity by pRL-cytomegalovirus (Promega) as relative luciferase unit and the fold of activation of relative luciferase unit, compared with control (cells transfected with pEGFP-C3), was determined. All experiments were performed in triplicate, and data were presented as mean ± SD. One-way ANOVA was performed.

Cell growth assay

PC-3 and PNT2 cells were chosen for study of transient expression of ERRs on proliferation of prostatic cells because both cell lines were easy to be transfected and showed similar ERR and ER expression patterns. Cells were plated at a density of 5 x 104 cells/well in 24-well plates and grown for 24 h before transfection. Cells were transfected with 0.4 µg pSG5-ERR{alpha}/ß/{gamma} or empty plasmid pSG5 using Lipofectamine 2000 and Plus reagents (Invitrogen). After 48 or 72 h post transfection, cells were trypsinized and cell numbers counted with a hemocytometer under an inverted microscope. The transfection efficiency was determined by transfecting the cells with a lacZ-expressing vector pcDNA3.1/His/lacZ followed by X-gal staining. All experiments were performed in quadruplicate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
mRNA expression of ERRs in prostatic cell lines and tissues

The results of RT-PCR showed that ERR{alpha} transcripts were expressed at high to moderate levels in normal PrECs, all immortalized epithelial cell lines (RWPE-1, RWPE-2, PWR-1E, PZ-HPV-7, HPr-1, HPr-1AR, PNT1A, PNT2, BPH-1), prostate cancer cell lines (CA-HPV-10, LNCaP, C4–2B, DU 145, PC-3, MDA PCa 2b, CWR22Rv1), and CWR22 xenografts (CWR22, CWR22R-AIM, CWR22R-AIF) (Fig. 1Go). Similar to ERR{alpha}, ERR{gamma} transcripts were expressed at moderate to high levels in most tested prostatic cell lines and CWR22 xenografts, weakly in PrECs and DU 145, but undetected in PNT1A and MDA PCa 2b. Compared with ERR{alpha} and ERR{gamma}, ERRß exhibited a distinct expression pattern that its transcripts were detected at high levels in PrECs and four immortalized lines (RWPE-1, RWPE-2, PWR-1E, PZ-HPV-7) but weakly or not expressed in most prostate cancer cell lines and CWR22 xenografts (Fig. 1Go). For comparison, the expressions of three ERR subtypes in surgical prostatic samples were also examined by RT-PCR. ERR{alpha} transcripts were detected in all hyperplastic and carcinomatous samples, with stronger signals in hyperplastic than carcinomatous samples. However, ERRß and ERR{gamma} transcripts were variably expressed in hyperplastic tissues and down-regulated or undetected in most carcinomatous samples (Fig. 2Go).



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  		FIG. 1. RT-PCR analysis of ERRs and ERs in normal PrECs, various prostatic cell lines, and CWR22 xenografts. Representative results of RT-PCR (A) and graphs of expression levels (B) relative to ß-actin are shown. Each bar is the mean ± SD of four independent experiments. All prostatic cells and cell lines and CWR22 xenografts show positive expression of ERR{alpha}, with particularly high signals in PrECs, RWPE-1, RWPE-2, PZ-HPV-7, BPH-1, LNCaP, PC-3, and CWR22Rv1. High levels of ERRß transcripts are detected in PrECs and four immortalized cell lines (RWPE-1, RWPE-2, PWR-1E, PZ-HPV-7) but barely detectable or negative in most prostate cancer cell lines and CWR22 xenografts. ERR{gamma} transcripts are expressed at moderate to high levels in all examined cell lines and CWR22 xenografts but weak in PrECs and barely detected in DU 145 and negative in PNT1A and MDA PCa 2a. ER{alpha} transcripts are detected in all immortalized cell lines (high in RWPE-1, RWPE-2, PWR-1E, PZ-HPV-7, PNT2, and CA-HPV-10 but weak in HPr-1AR and PNT1A) but weak in PrECs and negative in most cancer cell lines except PC-3 and CWR22Rv1. ERß transcripts are detected in PrECs, all prostatic cell lines, and CWR22 xenografts. Transcripts of ERR{alpha}, ERR{gamma}, ER{alpha}, and ERß are detected in MCF-7 cells. Controls for RT-PCR include omission of reverse transcriptase (no RT) or RNA sample (no RNA) in reverse transcriptase and absence of cDNA templates in PCR (no cDNA). No signal is detected in controls.

 


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  		FIG. 2. RT-PCR analysis of ERRs in human hyperplastic and carcinomatous prostatic tissues. ERR{alpha} transcripts are consistently detected in all hyperplastic and carcinomatous tissues, with stronger signals in hyperplastic than carcinomatous samples. ERRß signals are detected in some hyperplastic samples but negative in carcinomatous samples. ERR{gamma} signals are also detected in most hyperplastic samples and weakly in one neoplastic sample.

 
We also examined the mRNA expression patterns of steroid hormone receptors [ER{alpha}, ERß, androgen receptor (AR), progesterone receptor (PR), glucocorticoid receptor (GR)], aromatase and four prostatic differentiation markers [prostate-specific antigen (PSA), kallikrein 2/hK2, pS2, osteopontin] in the same prostatic cell lines, and tumor xenografts to demonstrate any correlation between the expressions of ERRs and these differentiation markers. ER{alpha} transcripts were expressed weakly in PrECs, variably in immortalized cell lines (stronger signals in RWPE-1, RWPE-2, PWR-1E, PZ-HPV-7, PNT2 but weakly in HPr-1, HPr-1AR, PNT1A, BPH-1), but undetected in most cancer cell lines and CWR22 xenografts except PC-3 cells (Fig. 1Go). Similarly, ERß transcripts were expressed weakly in PrECs and variably in immortalized and cancer cell lines and CWR22 xenografts (Fig. 1Go). AR transcripts were expressed strongly in PrECs and variably in immortalized lines (positive in PWR-1E, HPr-1AR, PNT2, BPH-1 but negative in others), most cancer lines (except DU 145), and CWR22 xenografts (Fig. 3Go). PR transcripts were expressed at weak levels in LNCaP, C4–2B, and CWR22Rv1 cells. Compared with other receptors, transcripts of GR and aromatase were detected in all prostatic cell lines and tumor xenografts, with GR expressed strongly and aromatase weakly (Fig. 3Go). Three prostatic differentiation markers (PSA, hK2, pS2) exhibited similar expression patterns in prostatic cell lines, with high levels in most cancer cell lines and xenografts but negative in most immortalized cell lines (Fig. 4Go). Compared with PSA and hK2, the estrogen-inducible pS2 was also expressed in two androgen-insensitive DU 145 and PC-3 cells. Osteopontin transcripts were detected in all cell lines and xenografts. The results of gene expression of ERRs and various prostatic markers in prostatic cell lines and CWR22 xenografts are summarized in Table 2Go.



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  		FIG. 3. RT-PCR analysis of AR, PR, GR, and aromatase in PrECs, prostatic cell lines, and CWR22 xenografts. Strong signals of AR (shown as 395 bp) are detected in PrECs, HPr-1AR, LNCaP, C4–2B, MDA PCa 2b, and CWR22 xenografts but negative in most immortalized lines and DU 145. A mutated AR due to tandem duplication of exon 3 is detected in CWR22Rv1 (shown as a band of 512 bp). Weak levels of PR transcripts are shown in LNCaP, C4–2B, and CWR22Rv1. Strong signals of GR are detected in PrECs, all cell lines, and xenografts. Weak levels of aromatase are seen in PrECs, most cell lines, and xenografts. Strong signals of AR, PR, and GR and weak signal of aromatase are detected in MCF-7 cells.

 


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  		FIG. 4. RT-PCR analysis of PSA, hK2, pS2, and osteopontin in PrECs, prostatic cell lines, and CWR22 xenografts. PSA, hK2, and pS2 exhibit almost identical expression patterns in the prostate cancer cell lines and xenografts, with high levels in LNCaP, C4–2B, MDA PCa 2b, CWR22Rv1, and three CWR22 xenografts. In addition, pS2 transcripts are expressed moderately in PC-3 and MCF-7 and weakly in DU 145 and several immortalized lines. No PSA and hK2 transcripts are detected in PrECs and immortalized cell lines. Osteopontin transcripts are detected in all cell lines and xenografts.

 

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  		TABLE 2. Expression profiles of ERRs, hormone receptors, and prostatic markers in prostatic cell lines and xenografts as shown by RT-PCR

 
Protein expression of ERRs in prostatic cell lines and tissues

The specificities of anti-ERR antibodies against ERR cognate peptides conjugated to KLH were evaluated by blotting of the recombinant His-tagged ERR fusion proteins expressed in a bacterial system. The anti-HisG antibody recognized the 6 x His-ERR{alpha} at a size of molecular mass 55 kDa, 6 x His-ERRß at 49 kDa, and 6 x His-ERR{gamma} at 58 kDa (Fig. 5AGo). The ERR antisera also identified the respective His-tagged ERR fusion proteins at the same molecular sizes (Fig. 5AGo). The expression of native ERRs in five selected prostatic cell lines was examined by protein blottings. RWPE-1 was chosen among the immortalized cell lines because it expressed high transcript levels of ERRs and ER{alpha}. Immunoreactive signals of ERR{alpha}, expressed at the size of 53 kDa, were detected in all tested cell lines, with higher levels in RWPE-1, LNCaP, and PC-3 cells. Similarly, immunosignals of ERR{gamma}, identified as a band of 51 kDa, were detected in protein extracts of all tested cell lines. On the other hand, immunosignals of ERRß, expressed at 48 kDa, were detected only in RPWE-1 cells but not cancer cell lines (Fig. 5BGo). Both ERR{alpha} and ERRß antisera exhibited similar immunostaining patterns in normal and premalignant prostates. The nuclei of luminal and basal epithelial cells were intensely reacted with both ERR{alpha} and ERRß antisera (Fig. 6Go, A and E). The location of basal epithelial cells was confirmed by staining with an antibody against high-molecular-weight keratins (clone DE-SQ). The stromal cells also showed positive reaction in their nuclei. The premalignant epithelial cells in high-grade prostatic intraepithelial neoplasia (PIN) lesions remained positively stained in their nuclei but showing a decreased immunoreactivity, compared with normal prostate (Fig. 6Go, B and F). In lesions of well- and poorly differentiated adenocarcinoma, the malignant cells showed variable but reduced staining in their nuclei by both ERR{alpha} and ERRß antisera (Figs. 6Go, C and G). ERR{gamma} antiserum stained mainly and moderately the glandular epithelial cells in their nuclei and cytoplasm, whereas the stromal cells were less reacted in normal prostate (Fig. 6IGo). In high-grade PIN lesions, the dysplastic epithelial cells showed positive ERR{gamma} immunoreactivity mostly in their cytoplasm (Fig. 6JGo), whereas in the adenocarcinoma lesions, the malignant cells were weakly stained or unstained (Fig. 6KGo).



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  		FIG. 5. A, Western blot analysis of bacterial expressed His-tagged ERR fusion proteins. The anti-HisG monoclonal antibody identifies the 6 x His-ERR{alpha}, 6 x His-ERRß, 6 x His-ERR{gamma} with apparent molecular masses of 55, 49, and 65 kDa, respectively (first panel). The three antisera against ERR cognate peptides also recognize specifically the respective 6 x His-ERRs as single bands of the same sizes as revealed by anti-HisG antibody (secondto fourth panels). B, Western blot analysis of native ERRs expressed in selected prostatic cell lines. Immunoreactive signals of ERR{alpha} (expressed at molecular mass of 53 kDa) are detected in whole-cell extracts of all tested cell lines, whereas ERR{gamma} (51 kDa) are detected in RWPE-1, LNCaP, PC-3, and CWR22Rv1 but not DU 145. ERRß (48 kDa) is detected only in RWPE-1.

 


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  		FIG. 6. Immunohistochemistry of ERRs in normal, dysplastic, and carcinomatous human prostates. A–D, ERR{alpha}. A, The glandular epithelial cells in the peripheral zone of normal prostate show intense nuclear immunosignals. The stromal cells also show nuclear staining. B, The dysplastic epithelial cells in high-grade PIN lesions (micropapillary pattern) (*) show positive nuclear staining but with reduced intensity, compared with the normal epithelial cells. C, The neoplastic cells in adenocarcinoma lesion (Gleason grade 2) are heterogeneously stained in their nuclei. E–H, ERRß. E, The nuclei of the glandular epithelial cells in the same zone of normal prostate are intensely stained. F, The dysplastic epithelial cells in high-grade PIN lesion (*) are positively stained in their nuclei. G, The nuclei of neoplastic cells in well-differentiated adenocarcinoma lesion (Gleason grade 2) are variably stained. I–K, ERR{gamma}. I, The glandular epithelial cells are positively stained in their nuclei and cytoplasm. J, The dysplastic epithelial cells in high-grade PIN lesion (*) are weakly stained, whereas the basal epithelial cells show stronger immunosignals. K, The neoplastic cells in adenocarcinoma lesion are variably stained in their nuclei and cytoplasm. D and H, Control sections stained with ERR{alpha} and ERRß antisera preabsorbed with their respective ERR cognate peptides: epithelial cells show negative reaction. L, Immunostaining of high-molecular-weight (HMW) keratins in basal cells in PIN lesions. Magnifications in all figures are shown at x200 except C, G, and K (x400).

 
Subcellular localization of GFP-tagged ERRs in prostatic cells

To demonstrate the intracellular localization of transiently expressed ERR subtypes in cultured prostatic cells, full-length ERRs fused to the C terminus of GFP were expressed in PC-3 cells by transfection with pEGFP-C3-ERR expression vectors. The three expressed GFP-tagged ERR subtypes were exclusively localized to the nuclei of PC-3 cells, whereas their nuclear localization was not affected by the presence or absence of serum (Fig. 7Go, A–C and E–G). On the contrary, the untagged GFP was distributed evenly in the cytoplasm of PC-3 cells transfected with the empty vector pEGFP-C3 (Fig. 7Go, D and H). The localization of GFP-tagged ERRs in PC-3 cells was confirmed by immunofluorescence using ERR antisera. The results showed that the ERR antisera recognized and localized the expressed GFP-tagged ERRs in the nuclei of GFP-ERR transfected PC-3 cells, the same as in GFP fluorescent localization (Fig. 7Go, I–K). The results also confirmed the specificities of the ERR antisera.



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  		FIG. 7. Subcellular localization and immunofluorescence of GFP-tagged ERRs in transient transfected PC-3 cells under serum-present or -free conditions. A–C, In the absence of serum, the expressed GFP-tagged ERR{alpha} (A), ERRß (B), and ERR{gamma} (C) are exclusively localized to the nuclei of PC-3 cells transfected with pEGFP-C3-ERR{alpha}/ß/{gamma}. E–G, Similarly, in the presence of serum, the expressed GFP-tagged ERR{alpha} (E), ERRß (F), and ERR{gamma} (G) are also localized to the nuclei of the transfected cells. However, in cells transfected with the empty vector pEGFP-C3, the expressed untagged GFP is distributed throughout the cytoplasm (D and H). I–L, Immunofluorescence of GFP-tagged ERR{alpha} (I), ERRß (J), and ERR{gamma} (K) demonstrates identically a nuclear staining in the same PC-3 cells transfected with pEGFP-C3-ERR{alpha}/ß/{gamma} for fluorescent GFP localization as shown in E–G, respectively. However, the ERR antisera do not recognize the untagged GFP expressed in PC-3 cells. A representative result using the ERR{alpha} antiserum shows no immunofluorescent signal in cells transfected with the empty vector pEGFP-C3 (L). Bar, 10 µm; scale bar is applied to all figures.

 
Transactivation of ERE/SF-1-controlled reporters by ERRs in prostatic cells

Luciferase assay showed that the three expressed GFP-tagged ERRs were functional because they could transactivate both ERE- and SF-1-driven luciferase reporters in cotransfected PC-3 cells in a ligand-free manner (Fig. 8Go). The results also revealed a differential transactivation activity among the three expressed GFP-tagged ERRs on ERE3-Luc and SF-13-Luc reporters in PC-3 cells, with ERR{gamma} showing higher-induced luciferase activity (4- to 6-fold induction) than ERRß and ERR{alpha}. The reporter gene assay was repeated in serum-free condition to demonstrate whether serum could affect the ERR transactivation in PC-3 cells. The results showed that the expressed GFP-tagged ERRs could also transactivate the ERE3-Luc and SF-13-Luc reporters in the absence of serum. We also observed that the transcriptional activities of ERRß on ERE3-Luc or SF-13-Luc reporters were reduced under serum-free conditions. On the contrary, the SF-1 transactivation mediated by the transfected ERR{gamma} was significantly enhanced in the absence of serum.



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  		FIG. 8. Transactivation of ERE- and SF-1-driven luciferase reporter genes by transfecting GFP-tagged ERR subtypes in PC-3 cells under serum-present or -free conditions. A, ERE3-driven luciferase activation activity. ERR{alpha} shows a modest activation on ERE3-Luc reporter, whereas ERR{gamma} and ERRß show higher transactivation activities in both serum-present and serum-free conditions. B, SF-13-driven luciferase activation activity. Similar to ERE3-Luc activation activity, transfected ERRs show transactivation on SF-13-Luc reporter in an increasing order of ERR{alpha}, ERRß, and ERR{gamma}. It is also noted that the ERRß-mediated transactivation of ERE and SF-1 driven luciferase activity in the presence of serum is significantly higher than that of serum-free conditions. On the other hand, ERR{gamma}-mediated SF-1 transactivation is significantly increased under serum-free conditions. Each bar represents the mean ± SD of three independent experiments (*, P < 0.05 vs. pEGFP empty vector; #, P < 0.05 vs. serum-present conditions).

 
Effect of ectopic expression of ERRs on prostatic cell proliferation and ER expression

The transfection efficiency of PC-3 cells was determined to be 30–40% by X-gal staining. The results of transient transfection showed that the growth of PC-3 cells at 72 h post transfection was significantly inhibited by ectopic overexpression of all three ERR subtypes, compared with control cells transfected with the empty vector pSG5 (Fig. 9Go). However, no significant growth inhibition was seen in ERR-transfected cells at 48 h post transfection. Similarly, we also observed a growth inhibition in an immortalized cell line, PNT2, transfected with the three ERR subtypes (data not shown). In addition, we also examined the effect of transient expression of ERR{alpha} on the mRNA expression of ER{alpha}, ERß, and ERR{gamma} in PC-3 cells by semiquantitative RT-PCR to demonstrate whether a cross-talk between ERRs and ERs could be possible at the transcriptional level. The results showed that overexpression of ERR{alpha} could reduce the transcript levels of ER{alpha} and ERR{gamma} but not ERß in PC-3 cells (Fig. 10Go).



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  		FIG. 9. Effect of transient expression of ERR subtypes on cell growth of PC-3 cells. A, Representative RT-PCR results of ERRs in ERR-transfected PC-3 cells. Transcript levels of ERRs are significantly increased in cells after transfection with the respective pSG5-ERR{alpha}/ß/{gamma}. B, ERR{alpha}/ß/{gamma}-transfected PC-3 cells grow significantly slower than the empty vector pSG5-transfected cells at 72 h post transfection, whereas no significant growth inhibition is seen in ERR-transfected cells at 48 h. Each bar represents the mean ± SD of four independent experiments (*, P < 0.001 vs. pSG5 empty vector).

 


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  		FIG. 10. Semiquantitative RT-PCR of ERR{alpha}, ERR{gamma}, ER{alpha}, and ERß expression in ERR{alpha}-transfected PC-3 cells. Representative RT-PCR results are shown. Relative abundance of ERR{alpha}, ERR{gamma}, ER{alpha}, and ERß in transfected PC-3 cells is normalized to ß-actin. ERR{alpha} levels are significantly increased in pSG5-ERR{alpha}-transfected cells, compared with cells transfected with empty vector pSG5 (A). In the same ERR{alpha}-transfected cells, transcript levels of ERR{gamma} (B) and ER{alpha} (C) are significantly reduced, whereas no change in ERß levels is seen (D). Each bar is the mean ± SD of three independent experiments (*, P < 0.05 vs. pSG5).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrated for the first time the expression profiles of three ERR subtypes in normal PrECs, prostatic cell lines, prostate cancer xenografts, and prostatic tissues. Our RT-PCR results showed that PrECs, all prostatic cell lines, and CWR22 xenografts expressed differentially one to three ERR subtypes. Among the three ERR subtypes, ERR{alpha} and ERR{gamma} showed similar expression patterns that both transcripts were widely expressed in most cell lines and CWR22 xenografts, whereas ERRß was detected at high levels in PrECs and four immortalized cell lines derived from normal prostate but barely detectable or not expressed in cancer cell lines. Similar ERR expression patterns were also demonstrated in clinical hyperplastic and neoplastic prostate samples. Results of Western blottings and immunohistochemistry also confirmed similar expression patterns that ERR proteins were mainly expressed as nuclear proteins in glandular epithelial cells, whereas their expression levels became either reduced or undetected in neoplastic prostatic cells. The results of expression studies suggest that expressions of ERRs are down-regulated in the progression of prostate cancer development. Previous studies on the molecular cloning of ERR subtypes report positive expression of ERR{alpha} and ERR{gamma} but not ERRß in human prostate by Northern blotting or RT-PCR (12, 15, 50). ERR{gamma} protein is structurally more closely related to ERRß than ERR{alpha}, particularly in the N-terminal activation factor-1 and DBD domains (14, 19). ERRß and ERR{gamma} transcripts were variably expressed in clinical hyperplastic tissues and down-regulated in neoplastic tissues, although such expression patterns were not apparent in prostatic cell lines.

The specific roles of ERRs in prostatic functions and progression of prostate cancer are currently unknown. Because ERRs and ERs are positively coexpressed in prostate and prostate cancer cells, it is interesting to speculate that ERRs could transactivate sets of genes, which are also targeted by ERs, based on their similar characteristics of binding to ERE and ERE-related response elements in target genes and interactions with the same transcriptional coactivators. Some of these genes, including lactoferrin, osteopontin, and pS2, which are known to be expressed in normal prostate and prostate cancer, could be commonly regulated by both ERRs and ERs. The distinct expression patterns of the three ERR subtypes displayed in prostatic cell lines also suggest that ERRs may perform distinct roles in the prostate gland and prostate cancer. The almost ubiquitous expression pattern of ERR{alpha} in normal PrECs, prostatic cell lines, and tissues suggests that ERR{alpha} expression is essential to the prostatic cells. Its broad expression pattern in prostatic cells and tissues may be related to its recently revealed functions in regulation of cellular energy metabolism as demonstrated in brown fat and adipocytes. ERR{alpha} is a direct regulator of an enzyme (medium-chain acyl coenzyme A dehydrogenase) involved in mitochondrial fatty acid ß-oxidation (18, 31). A recent study in ERR{alpha}-knockout mouse further indicates that ERR{alpha} plays a physiological role in adipogenesis and energy metabolism via its regulation on enzymes involved broadly in lipid, prostaglandin, and steroid metabolisms in adipose tissue (51). Other studies also indicate that ERR{alpha} and its coactivator, peroxisome proliferator-activated receptor-{gamma} coactivator-1, are involved in the control of gene expression in mitochondrial biogenesis (43, 44, 45, 46, 52).

Based on the broad and similar expression patterns of ERR{gamma} and ERR{alpha} as displayed in prostatic cells and tissues, it is tempting to speculate that ERR{gamma} might perform similar functions or regulate similar cellular processes as ERR{alpha}. Currently genes regulated by ERR{gamma} as well as its physiological functions are poorly understood. It is shown that both ERR{alpha} and ERR{gamma} share a similar and board tissue expression pattern, with particularly high levels in certain organs and tissues with high metabolic rate, including fetal and adult brain, heart, kidney, and skeletal muscle (13, 15, 19, 53, 54). Interestingly, these organs and tissues also express high levels of peroxisome proliferator-activated receptor-{gamma} coactivator-1, suggesting that ERR{gamma} may perform similar functions as ERR{alpha} in energy metabolism. On the other hand, studies by others (39) as well as our present observations show that compared with ERR{alpha} and ERRß, ERR{gamma} displays higher transcriptional activities on reporters driven by some known response elements, suggesting that ERR{gamma} could regulate some gene expression program distinct from that of ERR{alpha} and -ß. It was recently shown that ERR{gamma} is an activator of an orphan nuclear receptor gene, small heterodimer partner (SHP), and its transactivation by ERR{gamma} is mediated through a distinct SF-1-related response element known as sft4 (39). SHP in turn can regulate the transcriptional activities of many nuclear receptors, including ER. Therefore, it would be interesting to speculate that ERR{gamma} could modulate the ER signal pathway via SHP in prostatic cells.

Compared with ERR{alpha} and ERR{gamma}, ERRß mRNA and protein were detected at high levels in normal PrECs and a few immortalized cell lines but barely detectable or negative in cancer cell lines and neoplastic tissues. This expression pattern suggests that ERRß expression is down-regulated in prostate cancer. Previous studies in mouse embryos show that ERRß expression is strictly confined to extraembryonic tissues and embryonic gonads, and a requisite function in development is implicated. In situ hybridization shows that ERRß mRNA is transiently expressed in trophoblast progenitor cells and primordial germ cells during early embryogenesis and becomes diminished at later stages of development (16, 55). Functional study by gene knockout shows that ERRß is important for placental development because ERRß-null mice die during gestation due to impaired placenta formation (56). Giguère et al. (12) previously reported that ERRß signal is not detected in human prostate by Northern blot analysis. However, our present results by RT-PCR and immunodetection show that ERRß is expressed in adult prostatic tissues.

The differential expression patterns of ERRs, particularly ERRß, shown in immortalized cell lines suggest that these cell lines may not represent the same state in terms of their ERR expressions, although they are all nontransformed, nontumorigenic (except RWPE-2), and derived from normal prostate epithelial cells, which are immortalized by either the large T antigen gene of Simian virus 40 or E6 and E7 genes of human papillomavirus-16 and -18. We speculate that genetic (aneuploidy) and epigenetic changes (DNA methylation and histone deacetylation) induced in these immortalized cell lines could be the factors contributing to their differential ERR expressions. However, further experiments are required to elucidate the mechanism of ERR gene regulation in these cell lines.

Our transient transfection studies confirmed that the three ERR subtypes, ectopically expressed in PC-3 prostate cancer cells, were nuclear proteins and transcriptional active in the absence of ß-estradiol, and their nuclear localization was not affected by serum. We also observed a differential transactivation of ERE- and SF-1-driven reporter elements mediated by the transfected ERRs in PC-3 cells, with ERR{gamma} displaying higher transcriptional activity than ERRß and ERR{alpha}. A similar pattern on transcriptional activities has been demonstrated in transactivation of ERE- and pS2 promoter-driven reporters by ERRs and fusion proteins of GAL4-ligand-binding domain of ERRs (15, 25, 57). We also observed that the transcriptional activity of ERRß on ERE and SF-1 response elements was enhanced by the presence of serum, whereas the SF-1 transactivation by ERR{gamma} was increased significantly under serum-free conditions, and also the transactivation on these elements by transfected ERRs were reduced when heat-treated serum was used (not shown), suggesting that an unknown serum factor could modulate the transcriptional activity of ERRs in prostatic cells. Vanacker et al. (34) have shown that an unknown regulatory serum factor, which can be removed by charcoal treatment, is necessary for the transcriptional activity of ERR{alpha}, and the authors argue that ERR{alpha} could be ligand regulated but not a true orphan receptor. However, other studies show that all ERR subtypes are constitutively active and can bind to coactivators in charcoal-filtered serum or serum-free conditions (19, 22, 23).

Our transient transfection study also provides evidence for the first time that ERRs could play a role in growth control of prostatic cells because overexpression of ERRs can inhibit the cell proliferation of an immortalized prostatic epithelial cell (PNT2) and a prostate cancer cell line (PC-3). In a preliminary study, we also confirmed this growth-inhibitory effect in ERR{gamma}-stable transfectants of DU 145 and PNT2 cells, and the inhibitory effect could be due to a decrease in growth rate and a disturbance in cell cycle distribution rather than induced apoptosis (not shown). So far, study on the role of ERRs in growth control in normal or cancer cells is absent. Our present observation appears to be similar to two recent studies of ERs in prostate cancer cells that overexpression of ER{alpha} and ERß by stable transfection or infection can inhibit the proliferation of two ER{alpha}-negative prostate cancer cell lines, ARCaP and DU 145, which is mediated by either a G1 cell cycle arrest or induction of apoptosis (58, 59).

Our transfection experiments showed a functional cross-talk between ERRs and ERs at the transcriptional level in prostatic cells in which both receptors are coexpressed because transient overexpression of ERR{alpha} repressed the transcript levels of ERR{gamma} and ER{alpha} but not ERß in PC-3 cells. The mechanism of this negative modulation on ER{alpha} and ERR{gamma} transcription by ERR{alpha} is unclear and remains to be elucidated. One possibility is that ERR{alpha} could be a direct transcriptional repressor of ER{alpha} and ERR{gamma} by binding to their promoters. Kraus et al. (26) identified a functional domain in the C terminal of ERR{alpha}, which can repress the estrogen-stimulated and ERE-dependent transcription. Alternatively, this repressing effect could be cell type specific because ERR{alpha} could function as an activator or a repressor of ERE-dependent transcription, depending on the relative amounts of ERR{alpha} and ligand-activated ER{alpha} in the cells. The authors (26) also show that overexpression of ERR{alpha} can stimulate the ERE-dependent transcription in ER-negative HeLa cells but repress the transcriptional activity in ER-positive MCF-7 cells. It was shown recently that transient expression of ER{alpha} in the presence of estrogen can stimulate ERR{alpha} expression in a human endometrial cancer cell line HEC-1B, and multiple steroid hormone response element half-sites present in the ERR{alpha} promoter are shown to be responsible for this estrogen-responsive transcriptional activation (60).

Some preclinical studies show that certain SERMs could be used as promising chemopreventive and therapeutic agents for prostate cancer. SERMs, such as tamoxifen, 4-hydroxytamoxifen, and raloxifene can induce apoptosis in cultured prostate cancer cells (61, 62, 63, 64), whereas toremifene and trioxifene can inhibit cancer development and metastasis in two animal models of prostate cancer (65, 66). These authors generally believe that the antiproliferative effects of SERMs on neoplastic prostatic cells are mediated via ERs due to their positive expression in prostate gland. However, two recent studies (67, 68) show that diethylstilbestrol, tamoxifen, and 4-hydroxytamoxifen can bind specifically to ERR{gamma} and ERRß and act as antagonists, resulting in abolishing their constitutive transcriptional activities. Recently we demonstrated that two organochlorine pesticides act as antagonists for ERR{alpha}-1, whereas some isoflavone and flavone phytoestrogens behave as agonists for ERRs (49, 69). Our data suggest that SERMs or xenoestrogens, which exhibit growth effects on prostatic cells, can also target ERRs, besides ERs.

Taken together, our present study shows for the first time that members of ERRs and ERs are coexpressed in prostatic cells and tissues. Based on these observations, we propose that besides ERs, additional estrogen signaling pathway mediated by the ligand-independent ERRs is also involved in the regulation of prostatic growth and function. However, it is unclear whether ERRs or ERs function independently or together in regulation of target genes during normal and abnormal growths of prostate. Our transfection studies also provide evidence showing that a functional relationship between ERRs and ERs is present in target cells as ERRs could modulate the ER-mediated signaling pathway via interference on transcription of ERs in prostatic cells, although the molecular basis of the cross-talk between ERRs and ERs at transcriptional level remains to be determined. The positive and differential expressions of the three ERR subtypes in prostatic cells and tissues suggests that each receptor may perform distinct roles in the prostate and prostate cancer cells. Because both ERRs and ERs can activate ERE-controlled genes, it will be of interest to elucidate the roles of ERRs, relative to ERs, in gene regulation in prostatic cells.


   Acknowledgments
 
The authors are grateful to Dr. Uwe Borgmeyer (Universität Hamburg, Hamburg, Germany) for the supply of plasmids and ERR{gamma} antibody and Ms. H. L. Choi for her excellent technical assistance.


   Footnotes
 
This work was supported by Direct Grants for Research Grant 2002.1.005 and 2004/2005 from the Research Grants Council of Hong Kong (to F.L.C.).

First Published Online December 14, 2004

Abbreviations: AR, Androgen receptor; DBD, DNA-binding domain; EGFP, enhanced GFP; ER, estrogen receptor(s); ERE, estrogen-responsive element; ERR, ER-related receptor; FBS, fetal bovine serum; GFP, green fluorescent protein; GR, glucocorticoid receptor; His, polyhistidine; KLH, keyhole limpet hemocyanin; PIN, prostatic intraepithelial neoplasia; PR, progesterone receptor; PrEC, prostate epithelial cell; PSA, prostate-specific antigen; SDS, sodium dodecyl sulfate; SERM, selective ER modulator; SF-1, steroidogenic factor-1; SHP, small heterodimer partner.

Received July 19, 2004.

Accepted December 7, 2004.


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 Introduction
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 Discussion
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