A novel estrogen receptor-related protein γ splice variant lacking a DNA binding domain exon modulates transcriptional activity of a moderate range of nuclear receptors

Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 181–192

A novel estrogen receptor-related protein ␥ splice variant lacking a DNA binding domain exon modulates transcriptional activity of a moderate range of nuclear receptors Hitoshi Kojo a,∗ , Kaoru Tajima a , Masao Fukagawa a , Takao Isogai b , Shintaro Nishimura a a

Advanced Technology Platform Research Laboratory, Fujisawa Pharmaceutical Co. Ltd., Japan b Helix Research Institute, 5-2-3 Tokodai, Tsukuba, Ibaraki 300-2698, Japan Received 6 July 2005; accepted 14 October 2005

Abstract A novel estrogen receptor-related protein (ERR) ␥ splice variant cDNA (ERR␥3) was found in human full-length cDNA libraries. ERR␥3 cDNA consists of 3362 base pairs and has an open reading frame of 1188 bp. The predicted peptide sequence of ERR␥3 differs from both ERR␥1 and ERR␥2 in missing 39 amino acid residues corresponding to the second zinc finger motif of the DNA binding domain (DBD). ERR␥3 gene consists of 8 exons including three unique 5 -terminal exons and lacks the exon encoding the second zinc finger motif. The expression of ERR␥3 was confined to adipocytes and prostate while that of ERR␥2 was fairly widespread. The ERR␥3 product was shown by transactivation assay to have no ability to activate ERE-controlled transcription. However, ERR␥3 has an ability to modulate the transcriptional activity of other nuclear hormone receptors. ERR␥3 augmented the ligand-dependent transcriptional activities of ER (estrogen receptor) ␣, ER␤, and thyroid receptor (TR) ␣ by 1.3-, 4-, and 2.1-fold whereas it inhibited fully the activity of glucocorticoid receptor (GR). However, ERR␥3 had no effect on Vitamin D3 receptor, retinoic acid receptor ␣ or peroxisome proliferator activated receptor ␣, ␤, and ␥. These findings will help to elucidate the physiological role of the ERR␥ subfamily. © 2006 Elsevier Ltd. All rights reserved. Keywords: Estrogen receptor-related protein ␥ (ERR␥); Splice variant; DNA binding domain (DBD); Modulation; Nuclear receptor; Steroid

1. Introduction Estrogen receptor-related protein ␥ (ERR␥ or ERR3) is part of a nuclear receptor subfamily comprising ERRs, which are orphan receptors closely related to the estrogen receptor [1–4]. All members of the ERR subfamily share an almost identical DNA binding domain (DBD), which has 69% amino acid homology with that of estrogen receptor (ER) ␣ [4]. All the ERRs bind not only to the estrogen response element (ERE) but also to the extended half-site of ERE and activate genes controlled by both response elements in the absence of any ligands [4–6]. No natural lig∗ Corresponding author. Present address: Biomarker Science Co. Ltd., 2-8 Honmachibashi, Chuo-ku, Osaka 540-0029, Japan. Tel.: +81 29 824 2435; fax: +81 29 824 2435. E-mail address: [email protected] (H. Kojo).

0960-0760/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2005.10.004

ands for ERRs are known, however, diethylstilbestrol (DES), tamoxifen and 4-hydroxytamoxifen were recently identified to have high affinity for ERRs [6–9]. DES was shown to release a coactivator from ERRs and consequently inhibit the constitutive transcriptional activity of ERRs [7]. Similarly, 4hydroxytamoxifen was verified to disrupt ERR␥-coactivator interaction and inhibit the constitutive transcriptional activity of ERR␥ [8]. Although the function of ERRs remains largely unknown, several lines of evidence imply a role for ERRs in regulating the function of other nuclear receptors [6,10,11]. ERRs were shown to modulate transcription of ER target genes such as the pS2 breast cancer marker gene and lactoferrin gene through their overlapping control of ERE [6,10]. ERR␤ was also reported to repress transcriptional activity of the glucocorticoid receptor [11]. Recent rapid progress in studies on genomic and cDNA sequences has accelerated the identification of splice vari-

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ants of genes and led us to recognize their importance in securing genetic polymorphism. The number of splice variants of genes of the nuclear receptor superfamily has also been increasing and analyses of function of the splice variants have contributed to elucidating the physiological regulations and roles of respective nuclear receptors [12–18]. ERR␥ has been reported to have two splice variants, one of which, named ERR␥2, differs from the other variant ERR1 by having an additional 23 N-terminal amino acid residues [1,3,4,19]. Although the expression patterns of the two splice variants in fetal and adult tissues were confirmed to differ from each other, differences in their function remain unknown [4]. To uncover the physiological roles of ERRs, we have searched intensively for novel splice variants of ERRs among human full-length cDNA-enriched libraries prepared by the oligocap method [17]. Finally we discovered a novel splice variant of ERR␥, named ERR␥3, which lacks an exon encoding the second zinc finger motif of DBD. Splice variants generated by alternative splicing in the DBD of steroid receptors have been identified from studies on ER␣ [20–23], ER␤ [24–26], androgen receptor (AR) [27], retinoic acid receptor (RAR) ␣ [28] and NER (identical to liver X receptor (LXR) ␤) [29]. The splicing mode of these variants differs. The splice variants of AR and ER␤ lack exon3 encoding the second zinc finger within the DBD [25,27] and the NER variant lacks exon5 encoding the entire DBD [29]. The splice variants of ER␣ are generated by either a combination of exons 2 and 3 which span the whole DNA binding domain or each alone [22] while those of RAR␣ are generated by either a combination of exons 4 and 5 which span the whole DBD or exon 4 alone [28]. All of these splice variants were identified in neoplastic tissues such as breast cancer cell lines, lymphocytes and primary cancers. The association of these splice variants with oncogenesis implies possible roles in the development and/or progression of cancers. However, most of these variant products were predicted based on the disruption of the DBD to be unable, or to have reduced ability, to bind to their response element and to activate transcription. However, the functions of ER␤ [25] and RAR␣1 [28] variants were analyzed in detail as follows. The ER␣ variant was shown to lose the ability to enhance transcription from promoters containing ERE but retain the ability to enhance transcription from AP1 containing promoters in the presence of an agonist such as estradiol [25] while the RAR␣ variant was revealed not to bind nor to transactivate RARE by itself but to retain the ability to interact with RXR␣ and to transactivate DR5 RARE (RARE consisting of direct repeats of the consensus sequence separated by 5 nucleotides) in the presence of all trans-retinoic acid as an RXR␣/RAR variant heterodimer [28]. In this paper, we characterized a novel human ERR␥ splice variant ERR␥3 and investigated whether this variant has the ability to interact with other nuclear receptors and to modulate their transactivation from their own response elements to elucidate the function of this variant.

2. Materials and methods 2.1. Materials 4-Hydroxytamoxifen was purchased from Nacalai Tesque (Kyoto, Japan) and ␤-estradiol was from Sigma (St. Louis, MO, USA). Restriction enzymes were purchased from Toyobo (Osaka, Japan) and Takara Shuzo (Otsu, Japan) and DNA primers were from Sawady (Tokyo, Japan). All other chemicals and enzymes used in this study were of reagent grade. 2.2. Preparation of human full-length enriched cDNA libraries Human full-length cDNA libraries were constructed by the oligo-capping method as described previously [30,31]. Briefly, the cap structure of the mRNA was replaced with a synthetic 5 -oligo-ribonucleotide in three consecutive enzymatic reactions, namely (1) hydrolysis of the phosphate of truncated mRNA by bacterial alkaline phosphatase, (2) removal of the cap structure by tobacco acid pyrophosphatase with the phosphate at the 5 -end left and (3) ligation of the oligo-ribonucleotide to the 5 -end of mRNA originally having the cap structure by RNA ligase. First-strand cDNA was synthesized with SuperScriptII (Gibco BRL, Gaithersburg, MD, USA) using a dT adapter primer. After removal of the template RNA by alkaline hydrolysis, the first-strand cDNA was amplified using an XL PCR kit (Perkin-Elmer, Boston, MA, USA). PCR fragments were digested with SfiI and sizefractionated by agarose gel electrophoresis. Products longer than 2 kb were cloned into DraIII-digested pME18S-FL3 in an orientation-defined manner. Plasmid DNAs of the clones of the libraries were isolated with a PI-200 robot (KURABO, Osaka, Japan) and the sequences were determined with an ABI 377XL auto-sequencer (ABI, Foster City, CA, USA). 2.3. In silico analysis Annotation of the DNA sequences of the clones from fulllength cDNA libraries was performed with bioSCOUT/SRS (LION Bioscience, Heidelberg, Germany). In silico genomic mapping was performed by making a BLASTN search of cDNA sequences in a draft human genome sequence database (Sanger Institute) [32]. 2.4. Analysis of gene expression by PCR amplification The tissue distribution of human ERR␥2 and ERR␥3 cDNAs was determined by PCR amplification. Commercially available human cDNA from heart, kidney, brain (cDNA Panels, CLONTECH, Palo Alto, CA, USA), liver, spleen, skeletal muscle, adipocyte and prostate (Gene Pool cDNA, ResGen, Carlsbad, CA, USA) were used as templates. The ERR␥ isoform-specific PCR primers used were 5 -CTTTTTCCCTGCACTACGA-3 /5 -GACCTCCACGTACTCTGTC-3 for ERR␥2 and 5 -CCACTGAG-

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AAAGGGAATAAGGCT-3 /5 -TGTTATATGGCTTTTTGGCTGGCT-3 for ERR␥3. Reaction mixtures consisting of 1 ␮g of cDNA, 100 pmoles each of the forward and reverse primers, 4 nmoles of each dNTP, 5 units of Ex Taq DNA poolymerase (Takara Shuzo), 2 ␮l of 10 × Ex Taq buffer and 1 ␮l of Perfect Match PCR Enhancer (STRATAGENE, La Jolla, CA, USA) in 20 ␮l were subjected to 35 cycles of amplification (1 min at 94 ◦ C, 2 min at 55 ◦ C and 3 min at 72 ◦ C). PCR was simultaneously performed using primers for human glyceraldehyde-3-phosphate dehydrogenase (Takara Shuzo) as a control. PCR products from the same cDNA were separated by electrophoresis on the same 1% agarose gel. The predicted sizes of PCR products amplified with ERR␥2- and ERR␥3-specific primers are 588 and 532 bp, respectively.

2.5. Construction of reporter and expression plasmids The ER- and ERR␥-activated luciferase reporter plasmid pGVP2ERE was constructed as follows. Synthetic oligonucleotides corresponding to the sense and antisense ER response element (ERE), 5 -GATCTAGGTCACAGTGACCTA-3 /5 -GATCTA GGTCACTGTGACCTA-3 were phosphorylated with T4 DNA polynucleotide kinase (Takara Shuzo), mixed and annealed gradually after heating at 65 ◦ C for 15 min. The resulting double-stranded oligonucleotide was cloned into the BglII site of the reporter vector PGVP2 (Promega, Madison, WI, USA) and the plasmid clone containing four sense-oriented tandem copies of ERE was selected for use by sequencing of the resulting recombinant DNA clones and named pGVP2-ERE. ERR␥ isoform expression plasmids were constructed by inserting a coding sequence of each ERR␥ isoform into the expression vector pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA). The coding sequence of ERR␥2 was amplified by PCR with the primers 5 -TTTGGATCCTCGCACATGGATTCGGTA-3 /5 -GGGCTCGAGAAGAAAGAGGAAAGAAGA-3 using human skeletal muscle cDNA as template. The coding sequence of ERR␥3 was amplified with 5 -CGCGGATCCTCTGCAGAATGTCAAAC-3 /5 CGCCTCGAGGAAAGAAACTCATCAGG-3 using pME 18SFL3-C-BRAWH2017250 as template. The amplified products were separated by agarose gel electrophoresis after digestion with BamHI and XhoI. The resulting fragments were cloned respectively into the expression vector pcDNA3.1(+) cleaved with BamHI and XhoI. Sequences of expresson plasmid DNA from the transformants were confirmed using an ABI Prism 7700 auto-sequencer (ABI). Human estrogen receptor (ER) ␣, ER␤, thyroid hormone receptor (TR), retinoic acid receptor (RAR) ␣, glucocorticoid receptor (GR), and rat Vitamin D3 receptor (VDR) coding sequences were cloned by PCR amplification using commercially available cDNAs as templates and after nucleotide sequences of the amplified fragment were determined, PCR-derived mutations of the sequences were

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removed by swapping subfragments cleaved by appropriate restriction enzymes. Expression plasmids for each nuclear hormone receptor were constructed by inserting cloned coding sequence into the expression vector pcDNA3.1. Reporter plasmids for the nuclear receptors were constructed by inserting a tandem repeat of nuclear hormone receptor responsive elements into the multiple cloning site of the pGVP2 reporter vector (Promega). The nuclear hormone receptor responsive elements were prepared by ligation of renatured complementary synthetic oligonucleotides whose two termini were designed to produce cohesive ends and whose 5 -termini were previously phosphorylated. 2.6. Transient transfection assay CV-1 cells (ATCC, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (Gibco-BRL) supplemented with penicillin, streptomycin and 10% heat inactivated fetal calf serum (Hyclone, Logan, UT, USA). Cells were seeded at 2 × 105 per well in 6-well culture dishes and after overnight culture transiently transfected with 1 ␮g each of pGVP2-ERE luciferase reporter plasmid and ERR␥ expresson plasmids together with the control Renilla luciferase expression plasmid RL-TK (Promega) using Lipofectamine 2000 (Gibco-BRL). Cells were seeded at 2 × 105 per well in 6-well culture dishes and after overnight culture transiently transfected with 1 ␮g each of pGVP2-ERE luciferase reporter plasmid, ERR␥3 expresson plasmid, and other nuclear receptor expression plasmids together with control Renilla luciferase expression plasmid RL-TK (Promega) by using Lipofectamine 2000 (Gibco-BRL). Cells were harvested 4 h after transfection and plated again at 1.6 × 104 per well in 96-well plates. The drugs, ␤-estradiol and/or 4-hydroxytamoxifen dissolved in dimethyl sulfoxide were added to the culture and the cells were incubated at 37 ◦ C for 24 h. After being washed with PBS(−), cells were lysed with passive lysis buffer (PLB) (Promega) and the lysates were used for the reporter assay. The expression of the reporter was evaluated by measuring the activity of firefly luciferase using the dual luciferase reporter assay system (Promega) and ARVO HTS 1420 multilabel counter (Wallac, Boston, MA, USA) as a luminometer according to the instructions of the manufacturer. Firefly luciferase activity was corrected for tranfection efficiency based on the activity of internal control Renilla luciferase.

3. Results 3.1. Identification of a novel ERRγ splice variant To identify novel human cDNA species, full-length cDNA libraries were constructed from various human tissues by the oligo-capping method and after the novelty of the sequences had been checked by 5 and 3 one pass sequencing of the cDNA clones, entire sequences of full-length cDNAs were

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Fig. 1. Nucleotide sequence and deduced amino acid sequence of ERR␥3 (C-BRAWH2017250) cDNA. Nucleotides are numbered on the upper side. The coding region was deduced using GENETYX-WIN software and the deduced amino acid sequence of ERR␥3 is shown in the single-letter code below the nucleotide sequence.

determined and accumulated as a sequence database. We found a novel cDNA clone named C-BRAWH2017250 (GenBank Accession No. AK132293) showing high sequence homology with the human ERR␥ on a blast search of the sequence database of the full-length cDNAs. This cDNA has a nucleotide sequence of 3362 bp and an open reading frame of 1188 bp (684–1871) (Fig. 1). The nucleotide sequence of the cDNA was compared with the sequence of two known splice

variants of ERR␥, namely ERR␥1, a short form and ERR␥2, a long form. The predicted N-terminal peptide sequence of C-BRAWH2017250 cDNA is identical with that of ERR␥1 but lacks the N-terminal 23 amino acid residues of ERR␥2. Furthermore, the predicted peptide sequence of the cDNA differs from both ERR␥1 and ␥2 in missing 39 amino acid residues corresponding to the second zinc finger motif of the DNA binding domain (Fig. 2). The cDNA sequence con-

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Fig. 2. ERR␥3 lacks the second zinc finger of DBD. The amino acid residues indicated in thin letters are missing in ERR␥3.

tains a stop codon in-frame with the coding sequence 96 bp upstream of the start codon and within the 96 bp, 4 stop codons in the other frames (Fig. 1). The sequence around the predicted start codon is quite different from the Kozak consensus sequence [33] but A at position −3 which plays a key role is identical with that of the consensus sequence. Furthermore, the predicted start codon is the same one used in the splice variant ERR␥1. These findings strongly support that C-BRAWH2017250 cDNA is a splice variant of the human ERR␥ gene and so it was named ERR␥3. To clarify the mode of splicing of ERR␥3 cDNA as well as to confirm that the cDNA is not an artificial product, we performed in silico genomic mapping of the cDNAs for ERR␥ by making a blast search for the cDNA sequences among draft human genome sequences (combined human chromosome databases of the Wellcome Trust Sanger Institute). Five contiguous BAC clones containing sequences identical to the

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ERR␥3 cDNA were identified on chromosome 1 at q41. By overlapping these clones, ERR␥3 was revealed to consist of 8 exons (Fig. 3). Distances between exons were calculated from sequence data but those between exons A and B and between exons B and C were not able to be determined because the sequence of exon B is in the opposite orientation to the sequences of the other exons probably due to an erroneous assembling of genomic sequences. ERR␥3 has all 5 of the 3 -terminal exons of ERR␥2 except exon F. Exon F, 117 bp in length, encodes the sequence corresponding to the second zinc finger motif of the DNA binding domain. Instead of the 5 -terminal exons (exon D and unidentified exon(s)) used by ERR␥2, ERR␥3 uses three unique 5 -terminal exons (A–C). All intron-exon boundaries between ERR␥3-specific exons A and B, between ERR␥3-specific exons B and C and between ERR␥3-specific exon C and common exon E comply with the GT/AG rule. These results support that ERR␥3 is a genuine ERR␥ splice variant. 3.2. Expression profile of ERRγ splice variant The expression of ERR␥3 in eight human tissues (brain, heart, kidney, liver, spleen, skeletal muscle, adipocytes and prostate) was analyzed by PCR amplification using commercially available cDNA from human tissues as templates. To discern between the two ERR␥ isoforms, ERR␥2 and ERR␥3,

Fig. 3. Comparison of genomic organization between ERR␥2 and ERR␥3. Schematic representation of the structure of the gene for ERR␥2 and ERR␥3 is shown in the middle diagram. Exons are indicated by boxes and named in alphabetical order (A–J). The sizes of each exon are shown in the table on the right, while the sizes of each intron (expressed in kb) are shown above the horizontal line. The white boxes indicate exons commonly used by both variants and the two kinds of gray boxes indicate exons used specifically by each variant. The structures of the ERR␥2 and ERR␥3 cDNAs are schematically represented below and above the diagram of gene structure, respectively. ERR␥2 cDNA includes the 5 - and 3 -terminal portions (26 and 31 bp, respectively), corresponding exons of which have not yet been identified. Moreover, exon J of ERR␥2 is 23 bp shorter than that of ERR␥3 and hence named J . The location of start and stop codons, and regions corresponding to the DNA binding domain and ligand binding domain are schematically indicated above or below the cDNA diagrams. The regions amplified by PCR with ERR␥2 and ERR␥3-specific primers were indicated by double-headed arrows.

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Fig. 4. Expression of ERR␥3 in human tissues. Expression of ERR␥3 in human prostate (A), adipose tissue (B), spleen (C) and heart (D) was determined by PCR amplification as described in the text. Lane 1 represents a 100 bp DNA ladder size marker (New England Biolabs). Lanes 2, 3 and 4 represent amplified products obtained using GAPDH, ERR␥2 and ERR␥3-specific primers, respectively.

we used isoform-specific sequences as PCR primers. While ERR␥2 was shown to be expressed in a fairly broad range of tissues such as heart, kidney, spleen, skeletal muscle, placenta and prostate, the expression of ERR␥3 was confined to adipocytes and prostate (Fig. 4). 3.3. ERRγ3 has no ability to activate ERE-directed transcription The most striking feature of the ERR␥3 product is that it lacks the second zinc finger of the DBD (Fig. 2). This characteristic led us to examine by dual luciferase assay whether the product is able to bind the response element of ERR␥ (ERE) and to transactivate a reporter gene placed downstream of ERE. The fibroblast cell line CV-1 was cotransfected with luciferase reporter plasmids and the ERR␥2 or ERR␥3 expression plasmid. As expected, the ERR␥3 product had no ability to activate ERE-controlled transcription of a luciferase gene while the ERR␥2 product markedly activated the transcription (Fig. 5).

scriptional activity of ER␣ or ER␤ together with ERR␥3. 4-Hydroxytamoxifen is an antagonist of estrogen receptor [44] and recently was also recognized as an inhibitor of ERR␥ [8,9]. This complicates the interpretation of the results, however, ␤-estradiol-dependent activation of ER␣ and ER␤ appears indispensable for the augmentation by ERR␥3 of the transcriptional activities of ER␣ and ER␤, because the augmentation was abolished in the presence of relatively low concentrations of 4-hydroxytamoxifen when ␤-estradiol was not added to the medium (Figs. 6A and 7A). The augmentation by ERR␥3 observed at low concentrations of 4hydroxytamoxifen could be derived from the presence of estrogen-like substances in the medium. In contrast, in the

3.4. ERRγ3 activates ligand-dependent transcriptional activities of ERα, ERβ, TR Next, we examined if the product has the ability to modulate the transcriptional activity of other nuclear hormone receptors. We were led to examine this by the resemblance of ERR␥3 to SHP, an atypical orphan nuclear hormone receptor which lacks a DBD and inhibits or augments the transcriptional activities of other nuclear hormone receptors [34–42]. Since ERR␥ is related most closely to estrogen receptors [1–4,43], ER␣ and ER␤ were selected as candidate partners for examining the influence of ERR␥3 on their transcriptional activity. Surprisingly, the ERR␥3 product enhanced the transcriptional activities of both ER␣ and ER␤ by 1.3- and 4-fold, respectively (Figs. 6 and 7). This is the first report that a splice variant of a nuclear receptor lacking a part of the DNA binding domain has the ability to modulate transcriptional activity of other nuclear hormone receptors. We investigated simultaneously the effect of 4-hydroxytamoxifen on the tran-

Fig. 5. ERR␥3 product alone has no transactivation ability. CV-1 cells were transiently transfected with a luciferase reporter gene controlled by ERE alone or together with the ERR␥2 or ERR␥3 expression plasmid as described in the text. After transfection, cells were incubated for 24 h in the absence or presence of indicated concentrations of 4-hydroxytamoxifen. For all transfections, the activation of luciferase was corrected for transfection efficiency with Renilla luciferase activity levels. The graph represents means from a representative experiment conducted three times. Error bar indicates S.E.M.

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Fig. 6. ERR␥3 enhances ␤-estradiol induced transactivation by ER␣. CV-1 cells were transiently transfected with a luciferase reporter gene controlled by ERE alone or together with ER␣, ERR␥3 or ER␣ plus ERR␥3 expression plasmid as described in the text. After transfection, cells were incubated for 24 h in the absence (A) or presence of 10−7 M ␤-estradiol (B) together with no or the indicated concentrations of 4-hydroxytamoxifen. The graph represents means of triplicate data of a representative experiment. Error bar indicates S.E.M.

presence of 10−7 M ␤-estradiol, the augmentation by ERR␥3 of the transcriptional activities of ER␣ and ER␤ was maintained in spite of the presence of 4-hydroxytamoxifen until 10−6 M and 10−5 M, respectively (Figs. 6B and 7B). The inhibition of enhancement at these concentrations is ascribed to the antagonistic activity of 4-hydroxytamoxifen against ER, because a similar inhibition curve for ER was observed. To examine the range of nuclear hormone receptors whose transcriptional activities are modulated by ERR␥3, we investigated a series of receptors for the modulation of their activities by ERR␥3. Consequently, we found that ERR␥3 also has the ability to augment the thyroxine-dependent transcriptional activity of TR by 2.1-fold (Fig. 8). No aug-

mentation was observed when thyroxine was not added to the medium. This augmentation was reduced slightly (20%) by the addition of 10−5 M 4-hydroxytamoxifen while 4hydroxytamoxifen did not affect the transcriptional activity of TR alone. 3.5. ERRγ3 inhibits the transcriptional activity of GR Although the effect of ERR␥3 was mostly augmentation, ERR␥3 was found to inhibit dexamethazone-dependent transcriptional activity of GR (Fig. 9). Almost full inhibition was exhibited by ERR␥3 and this inhibition was markedly decreased by the addition of 10−5 M 4-hydroxytamoxifen.

Fig. 7. ERR␥3 enhances ␤-estradiol-induced transactivation by ER␤. CV-1 cells were transiently transfected with a luciferase reporter gene controlled by ERE alone or together with ER␤, ERR␥3 or ER␤ plus ERR␥3 expression plasmid as described in the text. After transfection, cells were incubated for 24 h in the absence (A) or presence of 10−7 M ␤-estradiol (B) together with no or the indicated concentrations of 4-hydroxytamoxifen. The graph represents means of triplicate data of a representative experiment. Error bar indicates S.E.M.

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Fig. 8. ERR␥3 enhances thyroxine-induced transactivation by TR. CV-1 cells were transiently transfected with a luciferase reporter gene controlled by, TRE alone or together with TR, ERR␥3 or TR plus ERR␥3 expression plasmid as described in the text. After transfection, cells were incubated for 24 h in the absence (A) or presence of 10−7 M thyroxine (B) together with no or the indicated concentrations of 4-hydroxytamoxifen. The graph represents means of triplicate data of a representative experiment. Error bar indicates S.E.M.

3.6. ERRγ3 has no effect on the transcriptional activity of VDR, RARα, and PPARα, β and γ We further explored whether ERR␥3 has the ability to modulate the transcriptional activity of typical nuclear hormone receptors: VDR, RAR␣, PPAR␣, PPAR␤ and PPAR␥. ERR␥3 did not show any effect either on the all trans-retinoic acid-dependent transcriptional activity of RAR␣ (Fig. 10A) or on the calcitriol-dependent transcriptional activity of VDR (Fig. 10B). Furthermore, ERR␥3 did not affect the transcriptional activities of PPAR␣, ␤ and ␥ in the absence or presence of their respective ligands (Fig. 11).

4. Discussion We have identified a novel ERR␥ splice variant named ERR␥3 from full-length cDNA-enriched libraries. The use

of full-length cDNA-enriched libraries in the search for new splice variants provides an advantage over the use of conventional cDNA libraries or 5 - or 3 -RACE-PCR, because the combinatorial use of exons by splice variants can be fully elucidated only when the cDNA sequence is scanned along its entire length. Thus, we could clarify that ERR␥3 differs from the existing variant ERR␥2 in both the use of three 5 terminal exons and the skipping of exon F. In this paper, we revealed that a novel ERR␥ splice variant named ERR␥3 has an ability to modulate the transactivation by a moderate range of members of the steroid receptor superfamily despite missing the second zinc finger motif of DBD. A few papers have reported on the functions of splice variants which lack an exon encoding DBD [25,28]. Namely, both ER␤1␦3 and RAR␣1BC were shown to lose the ability to activate their response element-directed transcription but ER␤1␦3 retains the ability to enhance transcription from AP-1 containing promoters in the presence of ␤-estradiol [25]

Fig. 9. ERR␥3 attenuates dexamethasone-dependent transactivation by GR. CV-1 cells were transiently transfected with a luciferase reporter gene controlled by GRE alone or together with GR, ERR␥3 or GR plus ERR␥3 expression plasmid as described in the text. After transfection, cells were incubated for 24 h in the absence (A) or presence of 10−6 M dexamethazone (B) together with no or the indicated concentrations of 4-hydroxytamoxifen. The graph represents means of triplicate data of a representative experiment. Error bar indicates S.E.M.

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Fig. 10. ERR␥3 does not modulate transactivation by RAR and VDR. CV-1 cells were transiently transfected with a luciferase reporter gene controlled by RARE alone or together with RAR, ERR␥3 or RAR plus ERR␥3 expression plasmid (A) or by VDRE alone or together with VDR, ERR␥3 or VDR plus ERR␥3 expression plasmid (B) as described in the text. After transfection, cells were incubated for 24 h in the presence of 10−6 M all trans-retinoic acid (A) or in the presence of 10−7 M calcitriol (B) together with no or the indicated concentrations of 4-hydroxytamoxifen. The graph represents means of triplicate data of a representative experiment. Error bar indicates S.E.M.

and RAR␣1BC has an ability to activate RARE-directed transcription in the presence of all trans-retinoic acid by heterodimerization with RXR␣ [28]. However, this is the first report that a splice variant which lacks an exon encoding DBD has the ability to modulate transactivation by other members of the steroid receptor superfamily. This ability of ERR␥3 reminds us of short heterodimer partner (SHP), one of the members of the nuclear receptor superfamily lacking a DBD, which modulates the activities of a wide range of nuclear hormone receptors [34]. SHP has been shown to inhibit ligand-dependent transcriptional activity of RARs, thyroid hormone receptor (TR) [34], ERs [35], androgen receptor (AR) [39], glucocorticoid receptor (GR) [42], liver receptor homolog 1 (LRH-1) [37], liver X receptor (LXR) [41], hepatocyte nuclear factor-4 (HNF4) and RXR [36], or augment the transcriptional activity of PPAR␥ [38,40]. SHP interacts with the same nuclear hormone receptor site recognized by nuclear receptor coactivators and competes with the coactivators to bind to nuclear hormone receptors and also directly represses transcription by its C-terminal repressor domain [36]. Similarly, SHP augments the transcriptional activity of PPAR␥ by directly binding to PPAR␥ and competing with a nuclear receptor corepressor for binding to PPAR␥ [40]. ERR␥3 appears to be different from SHP in that the range of nuclear hormone receptors with which ERR␥3 interacts is comparatively narrower and that activation of the transcriptional activity of nuclear hormone receptors is the predominant interaction profile of ERR␥3 while inhibition of the transcriptional activity is that of SHP. Nonetheless, as ERR␥3 shares many common features with SHP, mechanisms of transactivation modulation by ERR␥3 are presumed to be similar to those by SHP. This presumption should be examined further. Splice variants missing the DBD exon were thought to be products of abnormal splicing in neoplastic tissues

[20,22,27,29], but evidence has been presented that this type of variant such as RAR␣1BC [28] and ER␤1␦3 [25] is also expressed in normal tissues. We confirmed that ERR␥3 is also expressed in human normal tissues such as adipocytes and prostate. This result contradicts the possibility that ERR␥3 is an artificial product. In this study we identified ER␣, ER␤, TR and GR as nuclear hormone receptors whose transcriptional activities are modulated by ERR␥3. The roles of these nuclear receptors in physiological events such as regulation of the energy balance and lipid metabolism are closely related to each other. These findings imply strongly that ERR␥3 plays an important physiological role, however, further studies such as expression profiling in animal disease models are needed to elucidate the physiological and pathological roles. ERR␥ was recently found to be another target of 4hydroxytamoxifen, one of the selective estrogen receptor modifiers (SERMs) [8]. Our study confirmed that the transcriptional activity of ERR␥2 is inhibited by 4hydroxytamoxifen but we could not conclude whether ERR␥3 is also a target of 4-hydroxytamoxifen as the antagonistic activity of 4-hydroxytamoxifen against ERs obscured the contribution of inhibition by 4-hydroxytamoxifen against the ability of ERR␥3 to enhance the transcriptional activity of ERs. Meanwhile, 4-hydroxytamoxifen explicitly inhibited the activation by ERR␥3 of the transcriptional activity of TR and inhibition by ERR␥3 of the transcriptional activity of GR. These results indicated that ERR␥3 should also be taken into consideration as one of the possible targets when the action mechanism of SERM is investigated. In conclusion, our study revealed that as a splice variant of the steroid receptor superfamily, ERR␥3 has a novel function to modulate transactivation by other members of the steroid receptor superfamily. This finding will help to elucidate the physiological role of the ERR␥ subfamily and may also urge

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Fig. 11. ERR␥3 does not modulate transactivation by PPAR␣, ␤ and ␥. CV-1 cells were transiently transfected with a luciferase reporter gene controlled by PPRE alone or together with PPAR␣, ERR␥3 or PPAR␣ plus ERR␥3 expression plasmid (A and B), with PPAR␤, ERR␥3 or PPAR␤ plus ERR␥3 expression plasmid (C and D) or with PPAR␥, ERR␥3 or PPAR␥ plus ERR␥3 expression plasmid (E and F) as described in the text. After transfection, cells were incubated for 24 h in the absence (A) or presence of 10−5 M bezafibrate (B), in the absence (C) or presence of 10−5 M carbacyclin (D) or in the absence (E) or presence of 10−6 M rosiglitazone (F) together with no or the indicated concentrations of 4-hydroxytamoxifen. The graph represents means of triplicate data of a representative experiment. Error bar indicates S.E.M.

us to reconsider the function of DBD-lacking splice variants of steroid receptors other than ERR␥. References [1] J.D. Eudy, S. Yao, M.D. Weston, M. Ma-Edmonds, C.B. Talmadge, J.J. Cheng, W.J. Kimberling, J. Sumegi, Isolation of a gene encoding

a novel member of the nuclear receptor superfamily from the critical region of Usher syndrome type IIa at 1q41, Genomics 50 (1998) 382–384. [2] T. Nagase, K. Ishikawa, M. Suyama, R. Kikuno, M. Hirosawa, N. Miyajima, A. Tanaka, H. Kotani, N. Nomura, O. Ohara, Prediction of the coding sequences of unidentified human genes. XII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro, DNA Res. 5 (1998) 355–364.

H. Kojo et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 181–192 [3] F. Chen, Q. Zhang, T. McDonald, M.J. Davidoff, W. Bailey, C. Bai, Q. Liu, C.T. Caskey, Identification of two hERR2-related novel nuclear receptors utilizing bioinformatics and inverse PCR, Gene 228 (1999) 101–109. [4] D.J. Heard, P.L. Norby, J. Holloway, H. Vissing, Human ERR␥, 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 (2000) 382–392. [5] H. Hong, L. Yang, M.R. Stallcup, Hormone-independent transcriptional activation and coactivator binding by novel orphan nuclear receptor ERR3, J. Biol. Chem. 274 (1999) 22618–22626. [6] D. Lu, Y. Kiriyama, K.Y. Lee, V. Giguere, Transcriptional regulation of the estrogen-inducible pS2 breast cancer marker gene by the ERR family of orphan nuclear receptors, Cancer Res. 61 (2001) 6755–6761. [7] G.B. Tremblay, T. Kunath, D. Bergeron, L. Lapointe, C. Champigny, J.-A. Bader, J. Rossant, V. Giguere, Diethylstilbestrol regulates trophoblast stem cell differentiation as a ligand of orphan nuclear receptor ERR ␤, Genes Dev. 15 (2001) 833–838. [8] P. Coward, D. Lee, M.V. Hull, J.M. Lehmann, 4-Hydroxytamoxifen binds to and deactivates the estrogen-related receptor ␥, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 8880–8884. [9] H. Greschik, J.-M. Wurtz, S. Sanglier, W. Bourguet, A. van Dorsselaer, D. Moras, J.-P. Renaud, Structural and functional evidence for ligand-independent transcriptional activation by the estrogen-related receptor 3, Mol. Cell. 9 (2002) 303–313. [10] N. Yang, H. Shigeta, H. Shi, C.T. Teng, Estrogen-related receptor, hERR1, modulates estrogen receptor-mediated response of human lactoferrin gene promoter, J. Biol. Chem. 271 (1996) 5795–5804. [11] T. Trapp, F. Holsboer, Nuclear orphan receptor as a repressor of glucocorticoid receptor transcriptional activity, J. Biol. Chem. 271 (1996) 9879–9882. [12] R. Mukherjee, L. Jow, G.E. Croston, J.R. Paterniti Jr., Identification, characterization, and tissue distribution of human peroxisome proliferator-activated receptor (PPAR) isoforms PPAR␥2 versus PPAR␥1 and activation with retinoid X receptor agonists and antagonists, J. Biol. Chem. 272 (1997) 8071–8076. [13] T. Tagami, P. Kopp, W. Johnson, O.K. Arseven, J.L. Jameson, The thyroid hormone receptor variant ␣2 is a weak antagonist because it is deficient in interactions with nuclear receptor corepressors, Endocrinology 139 (1998) 2535–2544. [14] P. Gervois, I.P. Torra, G. Chinetti, T. Grotzinger, G. Dubois, J.C. Fruchart, J. Fruchart-Najib, E. Leitersdorf, B. Staels, A truncated human peroxisome proliferator-activated receptor ␣ splice variant with dominant negative activity, Mol. Endocrinol. 13 (1999) 1535–1549. [15] R.H. Oakley, C.M. Jewell, M.R. Yudt, D.M. Bofetiado, J.A. Cidlowski, The dominant negative activity of the human glucocorticoid receptor ␤ isoform. Specificity and mechanisms of action, J. Biol. Chem. 274 (1999) 27857–27866. [16] B. Lu, E. Leygue, H. Dotzlaw, L.J. Murphy, L.C. Murphy, Functional characteristics of a novel murine estrogen receptor-␤ isoform, estrogen receptor-␤ 2, J. Mol. Endocrinol. 25 (2000) 229–242. [17] M.C. Zennaro, A. Souque, S. Viengchareun, E. Poisson, M. Lombes, A new human MR splice variant is a ligand-independent transactivator modulating corticosteroid action, Mol. Endocrinol. 15 (2001) 1586–1598. [18] D. Ren, T.N. Colingwood, E.J. Rebar, A.P. Wolffe, H.S. Camp, PPAR␥ knockdown by engineered transcription factors: exogenous PPAR␥2 but not PPAR␥1 reactivates adipogenesis, Genes Dev. 16 (2002) 27–32. [19] U. Susens, I. Hermans-Borgmeyer, U. Borgmeyer, Alternative splicing and expression of the mouse estrogen receptor-related receptor ␥, Biochem. Biophys. Res. Commun. 267 (2000) 532–535. [20] M.W. Madsen, B.E. Reiter, A.E. Lykkesfeldt, Differential expression of estrogen receptor mRNA splice variants in the tamoxifen

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32] [33]

[34]

[35]

[36]

[37]

[38]

191

resistant human breast cancer cell line, MCF-7/TAMR-1 compared to the parental MCF-7 cell line, Mol. Cell. Endocrinol. 109 (1995) 197–207. K.E. Friend, E.M. Resnick, L.W. Ang, M.A. Shupnik, Specific modulation of estrogen receptor mRNA isoforms in rat pituitary throughout the estrous cycle and in response to steroid hormones, Mol. Cell. Endocrinol. 131 (1997) 147–155. I. Poola, S. Koduri, S. Chatra, R. Clarke, Identification of twenty alternatively spliced estrogen receptor alpha mRNAs in breast cancer cell lines and tumors using splice targeted primer approach, J. Steroid. Biochem. Mol. Biol. 72 (2000) 249–258. J. Yang, A. Liu, R. Guzman, S. Nandi, Estrogen receptor variants in epithelial compartment of normal human breast, Endocrine 12 (2000) 243–247. R.H. Price Jr., N. Lorenzon, R.J. Handa, Differential expression of estrogen receptor beta splice variants in rat brain: identification and characterization of a novel variant missing exon 4, Brain Res. Mol. Brain Res. 80 (2000) 260–268. R.H. Price Jr., C.A. Butler, P. Webb, R. Uht, P. Kushner, R.J. Handa, A splice variant of estrogen receptor ␤ missing exon 3 displays altered subnuclear localization and capacity for transcriptional activation, Endocrinology 142 (2001) 2039–2049. I. Poola, J. Abraham, K. Baldwin, Identification of ten exon deleted ER␤ mRNAs in human ovary, breast, uterus and bone tissues: alternate splicing pattern of estrogen receptor ␤ mRNA is distinct from that of estrogen receptor ␣, FEBS Lett. 516 (2002) 133–138. X. Zhu, A.A. Daffada, C.M. Chan, M. Dowsett, Identification of an exon 3 deletion splice variant androgen receptor mRNA in human breast cancer, Int. J. Cancer 72 (1997) 574–580. A. Parrado, G. Despouy, R. Kraiba, C. Le Pogam, S. Dupas, M. Choquette, M. Robledo, J. Larghero, H. Bui, I. Le Gall, C. RochetteEgly, C. Chomienne, R.A. Padua, Retinoic acid receptor ␣1 variants, RAR␣1B and RAR␣1BC, define a new class of nuclear receptor isoforms, Nucleic Acids Res. 29 (2001) 4901–4908. H. Saito, S. Nakatsuru, J. Inazawa, T. Nishihira, J.-G. Park, Y. Nakamura, Frequent association of alternative splicing of NER, a nuclear hormone receptor gene in cancer tissues, Oncogene 14 (1997) 617–621. K. Maruyama, S. Sugano, Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides, Gene 138 (1994) 171–174. T. Ota, T. Nishikawa, Y. Suzuki, K. Maruyama, S. Sugano, T. Isogai, Full-length cDNA project toward a high throughput functional analysis, Microb. Comp. Genomics 2 (1997) 204. S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basic local alignment search tool, J. Mol. Biol. 215 (1990) 403–410. M. Kozak, Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes, Cell 44 (1986) 283–292. W. Seol, H.S. Choi, D.D. Moore, An orphan nuclear hormone receptor that lacks a DNA binding domain and heterodimerizes with other receptors, Science 272 (1996) 1336–1339. W. Seol, B. Hanstein, M. Brown, D.D. Moore, Inhibition of estrogen receptor action by the orphan receptor SHP (short heterodimer partner), Mol. Endocrinol. 12 (1998) 1551–1557. Y.K. Lee, H. Dell, D.H. Dowhan, M. Hadzopoulou-Cladaras, D.D. Moore, The orphan nuclear receptor SHP inhibits hepatocyte nuclear factor 4 and retinoid X receptor transactivation: two mechanisms for repression, Mol. Cell. Biol. 20 (2000) 187–195. B. Goodwin, S.A. Jones, R.R. Price, M.A. Watson, D.D. McKee, L.B. Moore, C. Galardi, J.G. Wilson, M.C. Lewis, M.E. Roth, P.R. Maloney, T.M. Willson, S.A. Kliewer, A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis, Mol. Cell. 6 (2000) 517–526. A. Kassam, J.P. Capone, R.A. Rachubinski, The short heterodimer partner receptor differentially modulates peroxisome proliferatoractivated receptor ␣-mediated transcription from the peroxisome

192

H. Kojo et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 181–192

proliferator-response elements of the genes encoding the peroxisomal ␤-oxidation enzymes acyl-CoA oxidase and hydratasedehydrogenase, Mol. Cell. Endocrinol. 176 (2001) 49–56. [39] J. Gobinet, G. Auzou, J.C. Nicolas, C. Sultan, S. Jalaguier, Characterization of the interaction between androgen receptor and a new transcriptional inhibitor, SHP, Biochemistry 40 (2001) 15369–15377. [40] H. Nishizawa, K. Yamagata, I. Shimomura, M. Takahashi, H. Kuriyama, K. Kishida, K. Hotta, H. Nagaretani, N. Maeda, M. Matsuda, S. Kihara, T. Nakamura, H. Nishigori, H. Tomura, D.D. Moore, J. Takeda, T. Funahashi, Y. Matsuzawa, Small heterodimer partner, an orphan nuclear receptor, augments peroxisome proliferatoractivated receptor gamma transactivation, J. Biol. Chem. 277 (2002) 1586–1592.

[41] C. Brendel, K. Schoonjans, O.A. Botrugno, E. Treuter, J. Auwerx, The small heterodimer partner interacts with the liver X receptor ␣ and represses its transcriptional activity, Mol. Endocrinol. 16 (2002) 2065–2076. [42] L.J. Borgius, K.R. Steffensen, J.A. Gustafsson, E. Treuter, Glucocorticoid signaling is perturbed by the atypical orphan receptor and corepressor SHP, J. Biol. Chem. 277 (2002) 49761– 49766. [43] V. Giguere, N. Yang, P. Segui, R.M. Evans, Identification of a new class of steroid hormone receptors, Nature 331 (1988) 91– 94. [44] V.C. Jordan, K.E. Allen, C.J. Dix, Pharmacology of tamoxifen in laboratory animals, Cancer Treat Rep. 64 (1980) 745–759.