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Departments of Biochemistry (W.Z., J.-h.L., D.B.L.), Animal Sciences (Z.L., E.A., D.B.L.), and Child Health (D.B.L.), Dalton Cardiovascular Research Center and Department of Biomedical Sciences (J.W., S.M.H.), University of Missouri Center for Phytonutrient and Phytochemical Studies (W.Z., J.-h.L., D.B.L.), University of Missouri, Columbia, Missouri 65211
Address all correspondence and requests for reprints to: Dr. Dennis B. Lubahn, Room 110A ASRC, 920 East Campus Drive, University of Missouri, Columbia, Missouri 65211. E-mail: lubahnd{at}missouri.edu.
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Abstract |
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Objective: Two novel splicing isoforms of human (h) ERRß, hERRß2-
10 and short-form hERRß, were identified during the cloning of previously reported hERRß-hERRß2. We aim to investigate the functional differences of these three human ERRß-splicing isoforms.
Results and Conclusions: A genomic sequence comparison within and flanking the ERRß genes of eight species demonstrated that short-form hERRß lacks an F domain and is the matched homolog of mouse and rat ERRß proteins in humans. However, hERRß2-10 and the previously reported hERRß2 isoforms are primate specific. RT-PCR analysis showed that short-form hERRß has a wide distribution in the 24 of 27 human tissues and cell lines tested, whereas hERRß2 and hERRß2-10 were only expressed in testis and kidney. The three human ERRß-splicing isoforms have different transcriptional activities when measured on an estrogen response element-driven luciferase reporter in transfection assays. The localization of a nuclear localization signal of short-form hERRß was also determined. Interestingly, the F domain of hERRß2 alters the function of the nuclear localization signal. Therefore, the ERRß isoforms are likely to have diverse biological functions in vivo, and characterizing the three isoforms of ERRß will lead to an understanding of the multiple levels of gene regulation involved in steroid receptor-signaling pathways in humans and may provide novel therapeutic targets for human diseases.
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and ß (ER and ERß; NR3A1 and NR3A2). However, another subfamily, the estrogen-related receptors (ERR), exists in the nuclear receptor superfamily and shares sequence similarity, target genes, coregulatory proteins, some man-made ligands, and action sites with the ERs (reviewed in Ref.1). This subfamily contains three members: ERR, -ß and -
. Recent studies have shown that ERRs may play important roles in physiology and pathology.
ERRß was one of the first two orphan nuclear receptors reported by Giguere et al. in 1988 (2) using reduced stringency hybridization and is essential for embryo development. Targeted disruption of the ERRß gene in mice resulted in severely impaired placental formation, and the embryo died at 10.5 d post coitum (3). ERRß is expressed during mouse mammary gland development, and the expression of the estrogen-inducible pS2 gene, a human breast cancer prognostic marker, can be regulated by ERRß (4). It has also been shown that ERRß can repress the transcriptional activity of glucocorticoid receptor, but not progesterone receptor (PR) in a cell type-dependent manner (5). ERRß is also believed to be involved in estrogen-regulated pathways because it can bind the estrogen response element (ERE), activate transcription independent of exogenous ligands and share coactivators with ER and -ß (6, 7). It has also been shown that micromolar concentrations of tamoxifen and its active metabolite, 4-hydroxytamoxifen as well as diethylstilbestrol can suppress the transcription activity and coactivator interaction of ERRß (8, 9, 10). Additionally, three isoflavones (genistein, daidzein, and biochanin A) and one flavone (6,3',4'-trihydroxyflavone) were shown to act as agonists of ERR and ERRß (11).
Rat ERRß (NR3B2, GenBank accession no. X51417) was the first ERRß gene reported (2, 12). Mouse ERRß (no. X89594) was cloned in 1996 (13), encoding a polypeptide of 433 amino acids (aa), the same length as rat ERRß. In 1999, Chen et al. (12) identified human (h) ERRß2 (GenBank no. AF094517, reviewed as NM_004452 in GenBank) as the human homolog of ERRß, which encodes a polypeptide of 500 aa, with 67 extra aa at the C terminus compared with rat ERRß and mouse ERRß.
In this study we report the identification and tissue distribution of two novel mRNA alternative splicing isoforms of human ERRß, short-form hERRß and hERRß2-10 as well as the tissue distribution of the previously known isoform hERRß2. Short-form hERRß, which is identified for the first time, is the actual human ortholog of mouse ERRß (no. X89594) and rat ERRß (no. X51417). Transfection assays showed that these three splicing isoforms have different transcriptional activities on an ERE-driven luciferase reporter, but do not affect the ability of PR to activate progesterone response element (PRE)-driven luciferase activity. The nuclear localization signal (NLS) of short-form hERRß was also determined. Surprisingly, the F domain of hERRß2 inhibits the function of the NLS. Our results will allow researchers to study the appropriate tissue-specific isoform of human ERRß and better understand the regulation mechanisms of nuclear receptors by alternative splicing.
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Materials and Methods |
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Human testis total RNA was obtained from OriGene Technologies, Inc. (Rockville, MD). Human Total RNA Master Panel II, which includes 20 tissue RNAs: cerebellum, brain (whole), fetal brain, fetal liver, heart, kidney, lung, placenta, prostate, salivary gland, skeletal muscle, spleen, testis, thymus, trachea, uterus, colon with mucosal lining, small intestine, spinal cord and stomach, was obtained from BD Clontech (Palo Alto, CA). Human fetal heart total RNA, human adult ovary total RNA, human adult breast total RNA, human cervix total RNA, additional human kidney, uterus (postmenopause), skeletal muscle, and placenta total RNA were obtained from Stratagene (La Jolla, CA). A third placenta total RNA was purchased from BioChain Institute, Inc. (Hayward, CA). Human uterus (premenopause) total RNA was purchased from Chemicon International, Inc. (Temecula, CA). Total RNA of human breast cancer cell lines MCF-7 and C4-12-5 (14), human endometrial adenocarcinoma line Ishikawa, as well as the total RNA of mouse testis were prepared using Tri-Reagent (Sigma-Aldrich Corp., St. Louis, MO) following manufacturer’s protocols. All of the above anonymous human RNAs and human kidney tissue lysate described in the following Western blot section were properly handled according to the University of Missouri-Columbia institutional review board. 17ß-Estradiol (E2) and progesterone were obtained from Sigma-Aldrich Corp.
Materials and reagents used in Western blot and immunofluorescence confocal microscopy will be described in detail in later sections.
Cloning of human ERRß isoforms by RT-PCR
A two-step RT-PCR system was applied to clone the three isoforms of the hERRß gene. Briefly, the first RT step was carried out in a 10-µl volume containing 1 µg total RNA, a final concentration of 1 mM dNTPs (Invitrogen Life Technologies, Inc., Carlsbad, CA), 20 mA260 units random hexamer primer (Roche, Indianapolis, IN), 20 U avian myeloblastosis virus reverse transcriptase (Roche), 12.5 U ribonuclease inhibitor (Roche), and 2 µl 5x single-strength avian myeloblastosis virus reverse transcriptase buffer (Roche). The reaction was performed at 42 C for 60 min. The second PCR amplification step was carried out with the Expand High Fidelity PCR System (Roche) using primers specifically designed for different isoforms in a "touch-up-then-down" PCR program: 2 min at 94 C, then 20 cycles for 40 sec at 94 C, 40 sec at 65 C, with the temperature increasing by 0.5 C every cycle, 2 min at 68 C, followed by 25 cycles for 40 sec at 94 C, 45 sec at the final annealing temperature of 55 C, 2 min at 68 C, and a final extension step at 68 C for 7 min. The whole open reading frame (ORF) of short-form hERRß (GenBank accession no. AY451389) was cloned from human fetal heart total RNA (Stratagene) with primers hERRB2f261 (5'-act ttg agg cca gag gtg atc cag tga ttt-3') and hERR2r1690 (5'-cgg tct gtc cgt ttg tct gtc tgt agg t-3'). The whole ORFs of hERRß2 mRNA (GenBank no. NM_004452 or AF094517) and hERRß2-10 (GenBank accession no. AY451390) were cloned from human testis total RNA (OriGene Technologies, Inc.) with primer pair hERRB2f261 and hERRB2r2057 (5'-gcc aga tgt tac atg gtg agc cag aga t-3'). Negative controls were performed using RNA without the reverse transcriptase step. Amplified DNA products were ligated into pGEM-T vector (Promega Corp., Madison, WI), and clones corresponding to each isoform were selected by checking the insert fragment sizes. Finally, sequences of each isoform clones were verified using the 377 DNA Sequencer with BigDye version 3.1 chemistry (Applied Biosystems, Foster City, CA) at the DNA core of University of Missouri-Columbia.
Determination of tissue-specific mRNA isoform expression
To detect the distribution patterns of short-form hERRß, hERRß2, and hERRß-10 in different human tissues/cell lines, two-step RT-PCR was used again. Six pairs of primers were applied separately to each RT reaction product to investigate the distribution patterns of the three isoforms: hERRB2f963 (5'-acc aag att gtc tca tac cta ctg gt-3') and hERRB2r1265 (5'-ctc ctc atc cat gat gta gtc ctc-3') were used to detect the existence of hERRß (any isoform) in targeting tissue/cell line, hERRB2f1334 (5'-gct caa ggt gga gaa gga gga gtt tgt g-3') and hERRB2r1868 (5'-ctt gac att ctt tca tcc ttg gga gat cct-3') were used to detect the existence of both hERRß2 and hERRß2-10; hERR2f1328 (5'-caa gaa gct caa ggt gga gaa gga gga g-3') and hERR2r1690 were used to specifically amplify short-form hERRß; hERRB2f1607 (5'-ctt cct gga gat gct gga ggc caa ggt t-3'), which covers the boundary of exons 9 and 11, and hERRB2r2151 (5'-tct gct aga ggg gct ctg aag tga ggt c-3') were used to detect hERRß2-10 specifically; hERRB2f1565 (5'-cta tag cgt caa act gca ggg caa agt g-3') and hERRB2r1833 (5'-ctg ctc ttg gcc aac ctg ccc tct-3'), which covers the boundary of exons 10 and 11, were used to specifically amplify hERRß2; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward (5'-acc cac tcc tcc acc ttt g-3') and GAPDH reverse (5'-ctc ttg tgc tct tgc tgg g-3') were used as a positive control to amplify the housekeeping gene GAPDH. For total RNAs, including testis, kidney, placenta, uterus, and skeletal muscle from different sources, two pairs of primers designed for amplifying the N-terminal part of hERRß were also used in addition of above six pairs of primers: hERRBf261 (5'-act ttg agg cca gag gtg atc cag tga ttt-3') and hERRBr753 (5'-ggc agc tgt act caa tgt tcc ctt gga t-3'); hERRB2f315 (5'-ctc aga ggg ctg ctg aac agg atg tc-3') and hERRBr753 (5'-ggc agc tgt act caa tgt tcc ctt gga t-3'). The RT reaction was produced as described above. During the amplification step, each reaction tube contained 1 µl RT reaction product (equal to 0.1 µg total RNA), a final concentration of 0.2 mM deoxy-NTPs (Invitrogen Life Technologies, Inc.), 0.5 U Expand High Fidelity Enzyme Mix (Roche), and single-strength PCR buffer in a final volume of 10 µl. A touch-down PCR protocol was used: 2 min at 94 C, then 10 cycles for 20 sec at 94 C, 30 sec at 65 or 60 C, with the temperature decreasing by 0.5 C every cycle, 45 sec at 72 C, followed by 25 cycles for 20 sec at 94 C, 30 sec at the final annealing temperature (60 C or 55 C), 45 sec at 72 C, and a final extension step at 72 C for 7 min. Negative controls were performed using RNA without the reverse transcriptase step. The amplified DNA was fractionated electrophoretically on a 2% agarose gel, stained with ethidium bromide, and visualized under UV light.
In silico bioinformatics approach
Genome Browser screenshots and genome sequences were taken from http://genome.ucsc.edu (15, 16).
Cell culture
ER-negative MCF-7 cells, C4-12-5, were maintained in complete medium consisting of phenol red-free Eagle’s MEM (Sigma-Aldrich Corp.) supplemented with insulin (6 ng/ml), HEPES (10 mM), and 5% charcoal-stripped calf serum (Invitrogen Life Technologies, Inc., Gaithersburg, MD). The wild-type PR-positive parental T47D breast cancer cell line was maintained in phenol red-free DMEM:Ham’s F-12 (Invitrogen Life Technologies, Inc., Carlsbad, CA), supplemented with 10% fetal calf serum (JRH Bioscience, Lenexa, KS). COS-1 cells were maintained in DMEM supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Inc.). Ishikawa cells were maintained in complete medium consisting of phenol red-free Eagle’s MEM (Sigma-Aldrich Corp.) with insulin (6 ng/ml) and HEPES (10 mM), supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Inc.).
Expression and reporter vector constructs
pcDNA3.1+zeo short-form hERRß (AY451389), pcDNA3.1+zeo hERRß2 (NM_004452), and pcDNA3.1+zeo hERRß2-10 (AY451390) were constructed by subcloning the related ORFs (described above in the RT-PCR section) from pGEM-T vector (Promega Corp.) into pcDNA3.1+zeo expression vector (Invitrogen Life Technologies, Inc.) with NotI and ApaI restriction sites. 5'-Myc (c-myc epitope: Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu)-tagged short-form hERRß, hERRß2, hERRß2-10, and short-form hERRß deletion constructs (Fig. 8
; 101–433, 169–433, 210–433, 1–210, and 1–169 aa) were constructed by standard molecular biology methods in pcDNA3.1+ vector (Invitrogen Life Technologies, Inc.).
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hERR (NM_001438) ORF was amplified from human kidney multiple choice cDNA (Origene Technologies, Inc.) using forward primer (5'-gcg cgc tag cgc aca tgg att cgg tag aac ttt gc-3') and reverse primer (5'-gcg cgg atc cgt cag acc ttg gcc tcc aac att tc-3'), then cloned into pcDNA3.1+zeo expression vector (Invitrogen Life Technologies, Inc.) by NheI and BamHI restriction sites.
pCMX-hERR (2) and vERE-thymidine kinase (TK)-luciferase (luc), which contains one copy of ERE (AGG TCA CAG TGA CCT; underlined nucleotides are core sequences of vERE), were provided by Dr. Vincent Giguere.
The full length ER expression vector has been described previously (17).
The PvuII-SmaI fragment of pPRE/GRE.E1b.CAT was excised and inserted into the SmaI site of pGL3Basic from Promega Corp. pPRE/GRE.E1b has two copies of the consensus PRE linked to the TATA element from E1b (provided by Dr. Zafar Nawaz, Creighton University, Omaha, NE).
Renilla luciferase vector pRL-simian virus 40 (SV40)luc vector and pRL-cytomegalovirus (CMV)luc vector were obtained from Promega Corp.
Transient transfection and luciferase assay
Two days before transfection, C4-12-5 cells were seeded in 24-well plates in phenol red-free medium, then transfected with different vectors as indicated using Plus and Lipofectamine reagents (Invitrogen Life Technologies, Inc.). Fifty nanograms of ER and ERR expression vectors, 0.5 µg vERE-tk-luciferase reporter vector, and 20 ng Renilla luciferase control pRL-SV40luc vector (Promega Corp.) were used. After 12–16 h, transfected cells were then treated with ethanol vehicle (EtOH) and 100 nM E2, respectively. After 24-h incubation, cells were rinsed with PBS twice and lysed to measure the luciferase level using the dual luciferase assay kit (Promega Corp.).
T47-D cells were transfected as follows. Cells were grown in DMEM supplemented with 10% fetal bovine serum and plated at 3 x 105 cells/well in Falcon six-well dishes in 5% dextran-coated charcoal-stripped serum 24 h before transfection with the indicated plasmids using Superfect reagent (QIAGEN, Valencia, CA) according to the manufacturer’s guidelines. Cells were washed with PBS and incubated in DMEM:Ham’s F-12 and 5% serum in the presence of hormones as indicated. Cells were lysed after 20 h, and luciferase activity was measured using the dual luciferase reporter assay system (Promega Corp.). Experiments were performed in triplicate and repeated at least twice. Data were normalized to Renilla luciferase (pRL-CMV plasmid or pRL-SV40 plasmid, Promega Corp.) activity.
Western blot
Anti-ERRß (rat ERRß aa 4–100, GenBank accession no. X51417) mouse monoclonal antibody was purchased from R&D Systems, Inc. (Minneapolis, MN). Anti-actin (pan) polyclonal antibody was obtained from Cytoskeleton, Inc. (Denver, CO). Normal human tissue kidney lysate was obtained from ProSci, Inc. (San Diego, CA).
C4-12-5 and Ishikawa cells cultured in six-well plates were transfected respectively with 1 µg empty vector pcDNA3.1+zeo, short-form hERRß, hERRß2, or hERRß2-10 using Plus and Lipofectamine reagents (Invitrogen Life Technologies, Inc.). Total cell lysates were collected 48 h later. Protein concentrations were determined by bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL). Total cell lysate protein were then loaded on 10% SDS-PAGE gel and subjected to electrophoresis, membrane transfer, and Western blot. Western blot images were collected using a FujiFilm LAS-3000 imaging system (Tokyo, Japan).
Immunofluorescence assay
COS-1 or Ishikawa cells were grown as a single layer on glass coverslips in six-well plates. Cells were transfected with 1 µg (each) of short-form hERRß, hERRß2, hERRß2-10, 5'-Myc-tagged short-form hERRß, 5'-Myc-tagged hERRß2, 5'-Myc-tagged hERRß2-10, and 5'-Myc-tagged short-form hERRß deletion construct expression plasmids using Plus and Lipofectamine reagents (Invitrogen Life Technologies, Inc.). Cells were fixed in 100% methanol for 10 min at –20 C. Fixed cells were washed twice with 1x PBS (Mediatech, Inc., Herndon, VA) and once with 1x PBS containing 0.1% Triton X-100 (Sigma-Aldrich Corp.), 10 min each wash. Then cells were incubated with blocking buffer [1x PBS containing 3% BSA, 3% goat serum, 0.1% Micr-O-Protect (Roche)] for 1 h at 37 C. After three 10-min washes in 1x PBS containing 0.1% Triton X-100, cells were incubated with primary antibody, anti-ERRß monoclonal antibody (R&D Systems, Inc.) for short-form hERRß, hERRß2, and hERRß2-10 transfection; or anti-Myc polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 5'-Myc-tagged short-form hERRß, hERRß2, hERRß2-10, or 5'-Myc-tagged short-form hERRß deletion construct transfection at a dilution of 1:100 in 1x PBS containing 10% blocking buffer at 37 C for 1 h. Cells were washed three times with 1x PBS (0.1% Triton-100) for 10 min each wash. Secondary antibody, AlexaFluor 488 goat antimouse IgG(H+L) (Molecular Probes, Sunnyvale, CA; Invitrogen Life Technologies, Inc.) for short-form hERRß, hERRß2, and hERRß2-10 transfection, AlexaFluor 568 goat antirabbit IgG(H+L) (Molecular Probes; Invitrogen Life Technologies, Inc.) for 5'-Myc-tagged construct transfection, was diluted at 1:50 in 1x PBS containing 10% blocking buffer. Nuclear dye TO-PRO-3 (Molecular Probes; Invitrogen Life Technologies, Inc.) was also diluted at a final concentration of 10 µM with second antibody. Cells were incubated with second antibody and nuclear dye for 1 h at 37 C. Coverslips were washed three times in 1x PBS (0.1% Triton X-100) and mounted on glass slides in Prolong Gold Antifade Reagent (Molecular Probes; Invitrogen Life Technologies, Inc.) and sealed with clear nail polish. At least 250 positive cells were scored for subcellular localization of short-form hERRß, hERRß2, and hERRß2-10. Images were obtained with a Bio-Rad Radiance 2000 confocal system (Bio-Rad Laboratories, Inc., Hercules, CA) coupled to an Olympus IX70 inverted microscope (Olympus, New Hyde Park, NY) at Molecular Cytology Core, University of Missouri-Columbia.
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10
During our RT-PCR amplification of the full length of hERRß2 (GenBank accession no. NM_004452) ORF, we obtained two DNA bands (Fig. 1A). Sequence analysis of these two bands showed that although the larger band had the expected sequence of hERRß2, the shorter band revealed a splicing jump from the ninth exon to the 11th exon (Fig. 1B). This variant splicing resulted in a frame shift and moved the stop codon to the 12th exon. This new isoform, which we call hERRß2-10, encodes a putative polypeptide of 508 aa (Fig. 1C). Its predicted last 76 aa, which start at the variant splicing site, are totally different from the last 68 aa of hERRß2 because of a frame shift (Fig. 1D).
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The new isoform, hERRß2-10, has an altered F domain that might potentially affect the ligand binding and AF-2 domains. Based upon this hypothesis, we began to look for its homolog in other species.
The examination of human, chimpanzee, dog, rat, mouse, chicken, fugu, and zebrafish genome sequences within and flanking the ERRß gene revealed another unexpected result: exons 10, 11, and 12 in hERRß2 do not have a comparable homologous region in the rat, mouse, chicken, fugu, or zebrafish genome (Fig. 2, A and B). This indicates that no homolog isoform of similar aa content exists in rat, mouse, chicken, or fish. Even if a similar alternative splicing event should happen, it would introduce a stop codon just 24 bp downstream (Fig. 2C, putative stop codons, TAG, TGA, and TAG, shown as bold characters in mouse, rat, and chicken genome sequences comparable to the 10th exon of hERRß2). Even though dog genome has comparable sequence (64% identity in nucleotide) to the 10th exon of hERRß2, the putative aa encoded by this region only have 23% identity to that of the 10th exon of hERRß2 encoded. Furthermore, dog genome does not have comparable sequences to the 11th and 12th exons of hERRß2 (data not shown). This surprising result indicates that the F domains of hERRß2 and hERRß2-10 are primate specific. However, to investigate whether such splicing events occur in other species, RT-PCR experiments were performed with mouse testis and kidney total RNA with mouse-specific primers, but there is no evidence supporting a similar alternative splicing event in mice (data not shown).
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). The first six nucleotides of the intron between hERRß2 exons 9 and 10, gtgtga (or gtctga and gtttga in dog, chicken, and Fugu genomes), encode for the last aa, valine, of mERRß/rERRß and the stop codon (shown as underlined characters in Fig. 2C). This led us to hypothesize that a shorter human ERRß isoform may exist that does not splice to exon 10 or 11, but extends the length of exon 9, like the last exons in mouse and rat. This shorter hERRß would encode a protein of 433 aa, the same length as mERRß and rERRß. We hypothesized that hERRß2 and hERRß2-10 are tissue-specific variant splicing isoforms of hERRß.
We designed a forward primer in exon 8, hERR2f1328, and two reverse primers in the intron between exons 9 and 10, hERR2r1680 and hERR2r1690 (Fig. 1B), to determine whether this putative shorter form exists. RT-PCR was conducted using human testis, fetal heart, and breast cancer cell lines MCF-7 and C4-12-5 (14) total RNA. DNA bands of the expected sizes appeared in all the RNA that we tested. Sequencing analysis of the cDNA fragment amplified from human fetal heart verified our hypothesis (data not shown). Later, the whole ORF of this isoform, named short-form hERRß, was amplified from human fetal heart total RNA and verified by sequencing.
Tissue distribution expression patterns of the three mRNA isoforms of hERRß by RT-PCR
In total, five pairs of primers were designed to distinguish the tissue distribution expression patterns of the three hERRß isoforms (Fig. 3 and Materials and Methods). Among them, primer hERRB2f1607 (5'-ctt cct gga gat gct gga ggc caa ggt t-3'), is designed to specifically amplify hERRß2-10. This primer covers the boundary of exons 9 and 11. The first 25 nucleotides of forward primer hERRB2f1607 belong to the 3' end of exon 9, and the last three nucleotides belong to the 5' end of exon 11. This design ensures that only the cDNA of hERRß2-10, but not hERRß2 or short-form hERRß, can be amplified (Fig. 3B). For the same strategy, hERRB2r1833 (5'-ctg ctc ttg gcc aac ctg ccc tct-3'), which covers the boundary of exons 10 and 11, was used to specifically amplify the cDNA of hERRß2.
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RT-PCR results showed that although short-form hERRß mRNA is widely expressed in the 24 of 27 tissues/cell lines tested, hERRß2 and hERRß2-10 mRNA are only detectable in testis and kidney (Fig. 3B and Table 1). There were no splicing isoforms of human ERRß detected in uterus and Ishikawa cells under our RT-PCR conditions. Three different sources of placenta total RNA showed different results: two samples showed no existence of any isoform of human ERRß (Fig. 3B), and one sample has short-form hERRß expression (Table 1 and data not shown).
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When transiently expressed in C4-12-5 (MCF-7-derived, ER-negative) (14) cells, the three splicing isoforms of hERRß showed statistically different transcriptional activation on a reporter gene controlled under the basal TK promoter and single copy of vERE (Fig. 4). Short-form hERRß gives the strongest activation among three isoforms, hERRß2-10 gives an intermediate activation, and hERRß2 gives no activation, which is not different from the empty control vector, pcDNA3.1+zeo. Mouse ERRß, the homolog of short-form hERRß, showed similar activation on vERE-TK-Luc reporter as short-form hERRß. All ERR activation on vERE-TK-Luc reporter did not change when treated with 100 nM E2.
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Using pRL-CMV Renilla luciferase reporter as an internal transfection efficiency control, transient transfection and luciferase assay in T47D cells revealed that neither short-form hERRß nor hERRß2-10 isoform inhibited endogenous PR transcriptional activity in response to progesterone (Fig. 5A). However, the Renilla luciferase activity of the internal control plasmid pRL-CMV was about 2- to 3-fold higher when transfected with hERRß2 than when transfected with short-form hERRß, hERRß2-10, or empty vector pcDNA3.1+zeo (data not shown). For this reason, we switched to the pRL-SV40 Renilla luciferase reporter as an internal control when performing hERRß2 transfection analysis in T47D cells (Fig. 5B). The hERRß2 isoform did not influence PR activity when pRL-SV40 Renilla luciferase reporter was used as an internal control (Fig. 5B).
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Western blot revealed three major bands representing, respectively, short-form hERRß, hERRß, and hERRß2-10 in C4-12-5 and Ishikawa cells transiently transfected with coordinated expression plasmids (Fig. 6A). In agreement with RT-PCR results (Table 1), C4-12-5 cells expressed endogenous short-form hERRß (Fig. 6, mock lanes), whereas Ishikawa cells did not express ERRß protein (Fig. 6A, mock lane). A shadow band seen in hERRß2-10 transfection in Ishikawa cells was sometimes observed.
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): a faster migrating band the size of short-form hERRß, and slower migrating band, which may represent longer isoforms, hERRß2 and/or hERRß2-10, that with only an eight-aa difference in length are too close in size to distinguish. This Western result is in agreement with our RT-PCR results (Table 1) where all three isoforms are expressed. Different subcellular localizations of three splicing isoforms of human ERRß
To investigate the subcellular localizations of the three human ERRß isoforms, immunofluorescence confocal microscopy was used with anti-ERRß antibody. As expected, short-form hERRß and hERRß2-10 were primarily found located in the nucleus (96.5% and 97%, respectively; Fig. 7 and Table 2), similar to ER (18, 19). However, we were surprised to find that hERRß2, with an F domain different from that of hERRß2-10 (Fig. 1D), lost much of its ability to exclusively localize to the nucleus. More than 50% of stained COS-1 cells showed hERRß2 localized mostly in cytoplasm (Table 2 and Fig. 7). Similar results were obtained in Ishikawa cells transiently transfected with these three constructs expressing the three human ERRß isoforms (data not shown). In addition, COS-1 cells transfected with 5'-Myc-tagged short-form hERRß, hERRß2, and hERRß2-10 constructs revealed the same localization patterns of the three human ERRß splicing isoforms when detected with anti-Myc antibody (data not shown).
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To determine where the NLS of short-form hERRß is located, 5'-Myc-tagged short-form hERRß deletion mutants were constructed: 101–433 aa (C, D, and E domains), 169–433 aa (D and E domains), 210–433 aa (E domain), 1–210 aa (A/B, C, and D domains), and 1–169 aa (A/B and C domains). From these data, the NLS is found in the D domain of short-form hERRß. The constructs containing D domain (101–433, 169–433, and 1–210 aa) all locate within the nucleus, whereas the constructs lacking D domain (210–433 and 1–169 aa) are not in the nucleus (Fig. 8A).
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10. Tissue distribution studies, transfection assays, and subcellular localization studies of these two novel isoforms of human ERRß as well as a previously known isoform, hERRß2, suggest that these ERRß splicing isoforms have diverse biological functions. Alternative RNA splicing is an important molecular mechanism for the creation of protein diversity derived from a single gene in eukaryotes. The completion of the draft of the human genome revealed that the human genome appears to have only 30,000–40,000 protein-coding genes (20, 21), which is only 2- to 3-fold more than the number of genes of Caenorhabditis elegans and Drosophila melanogaster. However, it is currently estimated that at least 35–59% of human genes are alternatively spliced (20, 22, 23, 24). This indicates that instead of increasing gene numbers to achieve greater complexity, gene alternative splicing may be used to regulate complex functions in advanced organisms.
Many nuclear receptor genes undergo alternative RNA splicing (reviewed in Ref.25). Multiple RNA splicing variants for both ER and ERß genes have been reported (26, 27, 28). N-terminal mRNA splicing variants have also been reported for ERR and ERR (29, 30, 31), but no C-terminal splicing variants have been reported for ERR and ERR to date. For ERRß, no RNA variant splicing isoform has been reported before this manuscript, except for the website
www.ncbi.nih.gov/IEB/Research/Acembly/av.cgi?db=human&c=Gene&l=ESRRB
based upon 26 sequences from 19 cDNA clones and products, predicted the existence of six different human ERRß protein isoforms produced by alternative splicing: a, b, c, d, e, and f, encoding 500, 245, 186, 128, 99, and 102 aa, respectively. The ERRß form a is actually the hERRß2 reported by Chen et al. in 1999 (12). The remaining five isoforms are severely truncated proteins that only contain the N-terminal part of human ERRß, and none of them is the alternatively spliced products demonstrated in this paper. These five isoforms are unlikely to have characteristics similar to the full-length ERRß protein.
This report describes three human ERRß RNA splicing variants at the C terminus. Additionally, the human ERRß gene is able to use human-specific genome sequence to produce RNA alternative splicing variants not found in other species. Therefore, humans/primates may potentially achieve more finely tuned regulation of ERRß function than other species. Although human, rat, and mouse all express the common ERRß mRNA form coding 433 aa, humans/primates specifically express the hERRß2 and hERRß2-10 mRNA isoforms, coding for 500 and 508 aa, respectively.
Our results on the tissue distribution of human ERRß isoforms showed that although hERRß2 and hERRß2-10 expression is limited to testis and kidney, short-form hERRß is widely expressed in 24 of 27 tissue/cell lines we tested, including MCF-7 cell (Fig. 3B and Table 1). Three different sources of placenta total RNA gave different results: two samples revealed no expression of any ERRß isoform, and one sample expressed short-form hERRß mRNA. Only uterus and Ishikawa cells were free of ERRß detection under our RT-PCR conditions (Materials and Methods).
Previous literature reported that ERRß expression is limited to only a few tissues. Giguere et al. (2) reported that by Northern blot, a 4.8-kb mRNA band was found in rat kidney, heart, testis, hypothalamus, hippocampus, cerebellum, and prostate, but not in human placenta and prostate. Using the same method, Pettersson et al. (13) detected a 4.3-kb mRNA band in undifferentiated F9 embryonic carcinoma cells, undifferentiated embryonic stem cells, and a few adult tissues, including kidney and heart. Chen et al. (12) cloned hERRß2 cDNA from a human testis cDNA library, and their Northern blot showed that an approximately 5.5-kb transcript was expressed at a low level in heart, kidney, liver, skeletal muscle, and stomach. Interestingly, testis from which the cDNA was derived had no detectable signal with Northern analysis. Lu et al. (4) reported that ERRß is expressed during all stages of mammary gland development in the mouse. However, no ERRß was detected in the human breast cancer cell lines (including the MCF-7 cell line) and normal epithelial cell lines that they tested by Northern blot (4).
Our observations differ from previous reports of ERRß’s tissue distribution. However, we believe that this discrepancy is mainly due to the sensitivity of the detection method: Northern blot vs. RT-PCR. Generally, a standard Northern procedure is less sensitive than RT-PCR. This is consistent with the observation that testis from which hERRß2 was cloned had no detectable hERRß2 signal by Northern analysis (12). Using real-time quantitative PCR, Ariazi et al. (32) found that ERRß mRNA levels were quite low in human primary breast cancers (
1.2 x 103 copies/ng cDNA in tumor samples; n = 38) compared to ERR (1.768 x 106 copies/ng cDNA in tumor samples; n = 38). This may explain why ERR, but not ERRß, can be detected in human breast cancer cell lines using Northern blot (4). There also remains the possibility that different individuals have different mRNA expression patterns, such as the three different placenta total RNA samples with contradictory RT-PCR results (Fig. 3B and Table 1).
Several studies have shown that the F domain of ER can influence the receptor’s transcriptional activity and ligand-binding affinity (33, 34, 35). Not all nuclear receptors have the F domain, and the function of the F domain is still not clearly elucidated. Except for hERRß2 and hERRß2-10, no other members of the ERR family have an F domain. Because hERRß2 and hERRß2-10 have specific tissue distribution patterns, we speculate that hERRß2 and hERRß2-10 isoforms have different transcriptional activities or binding affinities with unknown ligands and/or transcriptional factors than the short-form hERRß isoform. The result of transient transfection in C4-12-5 (MCF-7-derived, ER-negative) (14) cells supports this hypothesis: the three splicing isoforms of human ERRß exhibit differential activation of the firefly luciferase reporter when driven by single copy of ERE, with short-form hERRß the strongest and hERRß2 the weakest (Fig. 4).
Furthermore, transfection of human ERRß isoforms into T47D cells did not provide evidence that the transcriptional activity of PR through PRE in response to progesterone is blocked by any hERRß isoforms, in agreement with a previous report showing lack of inhibition of PR activity by rat ERRß in CV-1 and SK-N-MC cell lines (5). However, unexpectedly, the Renilla luciferase activity of the internal control plasmid pRL-CMV is about 2- to 3-fold higher when transfected with hERRß2 than when transfected with short-form hERRß, hERRß2-10, or empty vector pcDNA3.1+zeo (data not shown). For this reason, we switched to the pRL-SV40 Renilla luciferase reporter as an internal control when performing hERRß2 transfection analyses in T47D cells (Fig. 5B). Clearly, this differential interaction of hERRß2 on the CMV promoter strengths the view that the ERRß isoforms could have differential biological activities.
Subcellular localization studies of the three human ERRß splicing isoforms by immunofluorescence confocal microscopy unveiled additional surprising information about the F domain function, in addition to its contribution to the differential biological activities of the three isoforms. Although short-form hERRß and hERRß2-10 proteins localize mainly in the nucleus (>95%), hERRß2 loses its ability to localize exclusively within the nucleus; more than 50% of stained cells show that hERRß2 preferentially localizes in the cytoplasm and not the nucleus (Fig. 7 and Table 2). Deletion constructs of short-form hERRß revealed that the NLS of short-form hERRß is located within the D domain (Fig. 8). hERRß2’s F domain, which is totally different in aa sequences from hERRß2-10’s F domain, inhibits the function of the NLS of short-form hERRß. This is the first example to our knowledge that the F domain can interfere with a nuclear receptor’s subcellular localization. It is possible that nuclear receptors may use this alternative splicing strategy to finely regulate their biological activity by changing their subcellular localization.
We also compared the D domain (170–210 aa) of short-form hERRß with the known NLS sequence (256–303 aa) of human ER (19). The first two basic stretches of ER NLS, aa 256–260 and 266–271, have been shown to be conserved through human, mouse, rat, and chicken ER (19). Interestingly, there are two similar basic stretches, 174–178 and 184–188 aa, in the D domain of short-form hERRß (Fig. 8B). ER and short-form hERRß even share the same gap lengths (5 aa) between these two basic stretches and three of the gap aa are identical. Considering the overall low similarity (27%) of the D domains between ER and short-form hERRß, it is very likely that the sequences responsible for short-form hERRß nuclear localization are aa 174–188.
ERRß was one of the first two orphan nuclear receptors discovered more than 15 yr ago. However, unraveling its function is still in its infancy. With this paper we finally know the correct human isoform to study. Additionally, the lack of a known natural ligand has prevented full understanding of how ERRß is regulated in vivo and how ERRß regulates other genes. Additional characterization of the new appropriate ERRß isoforms will probably provide novel gene targets for future treatment of human diseases.
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, and Leilani Castleman for her help in cloning mouse ERRß. This work was supported by National Institute of Environmental and Health Sciences Grant P01-ES-10535 and Army Concept Proposal Award DAMD17-03-1-0561 (to D.B.L.) and in part by National Institutes of Health Grant CA-86916 (to S.M.H.).
First Published Online December 6, 2005
Abbreviations: aa, Amino acid; CMV, cytomegalovirus; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; ERRß, estrogen-related receptor ß; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; h, human; luc, luciferase; NLS, nuclear localization signal; ORF, open reading frame; PR, progesterone receptor; PRE, progesterone response element; SV40, simian virus 40; TK, thymidine kinase.
Received October 11, 2004.
Accepted November 23, 2005.
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, but not by ERß. EMBO J 18:4270–4279[CrossRef][Medline]. Endocrinology 142:4572–4575. Proc Natl Acad Sci USA 98:8880–8884 in estrogen receptor -negative MCF-7 cells restores both estrogen and insulin-like growth factor-mediated signaling and growth. Cancer Res 61:5771–5777 gene are generated by alternative splicing and promoter usage. Mol Endocrinol 12:1939–19541 functionally binds as a monomer to extended half-site sequences including ones contained within estrogen-response elements. Mol Endocrinol 11:342–352, a third member of the estrogen receptor-related receptor (ERR) subfamily of orphan nuclear receptors: tissue-specific isoforms are expressed during development and in the adult. Mol Endocrinol 14:382–392 and estrogen-related receptor associate with unfavorable and favorable biomarkers, respectively, in human breast cancer. Cancer Res 62:6510–6518 alter its response to estradiol and 4-hydroxytamoxifen. J Biol Chem 277:13202–13209This article has been cited by other articles:
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) or 10–8 M progesterone (P; 
). B, T47D cells were transfected with empty vector pcDNA3.1+zeo, or hERRß2 (1 or 2 µg), 2 µg 2xPRE-luc reporter, and 0.2 µg pRL-SV40-luc internal control reporter. Transfected cells were treated with vehicle ethanol (C; 
