identification of a crucial site for synoviolin expression

MOLECULAR AND CELLULAR BIOLOGY, Aug. 2005, p. 7344–7356 Vol. 25, No. 160270-7306/05/$08.00�0 doi:10.1128/MCB.25.16.7344–7356.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification of a Crucial Site for Synoviolin ExpressionKaneyuki Tsuchimochi,1,2 Naoko Yagishita,1 Satoshi Yamasaki,1 Tetsuya Amano,1

Yukihiro Kato,1 Ko-ichi Kawahara,3 Satoko Aratani,1 Hidetoshi Fujita,1Fengyun Ji,1 Akiko Sugiura,1 Toshihiko Izumi,1,2 Asako Sugamiya,1

Ikuro Maruyama,3 Akiyoshi Fukamizu,4 Setsuro Komiya,2Kusuki Nishioka,1 and Toshihiro Nakajima1*

Department of Genome Science, Institute of Medical Science, St. Marianna University School of Medicine,2-16-1 Sugao Miyamae-ku, Kawasaki, Kanagawa 216-8512,1 Departments of Orthopedic Surgery2 andLaboratory and Molecular Medicine,3 Kagoshima Graduate School of Medical and Dental Sciences,

Kagoshima 890-8520, and Institute of Applied Biochemistry and Center for TsukubaAdvanced Research Alliance, University of Tsukuba,

Tsukuba, Ibaraki 305-8577,4 Japan

Received 5 December 2004/Returned for modification 16 January 2005/Accepted 18 May 2005

Synoviolin is an E3 ubiquitin ligase localized in the endoplasmic reticulum (ER) and serving as ER-associated degradation system. Analysis of transgenic mice suggested that synoviolin gene dosage is implicatedin the pathogenesis of arthropathy. Complete deficiency of synoviolin is fatal embryonically. Thus, alternationof Synoviolin could cause breakdown of ER homeostasis and consequently lead to disturbance of cellularhomeostasis. Hence, the expression level of Synoviolin appears to be important for its biological role in cellularhomeostasis under physiological and pathological conditions. To examine the control of protein level, weperformed promoter analysis to determine transcriptional regulation. Here we characterize the role of syno-violin transcription in cellular homeostasis. The Ets binding site (EBS), termed EBS-1, from position �76 to�69 of the proximal promoter, is responsible for synoviolin expression in vivo and in vitro. Interestingly,transfer of EBS-1 decoy into NIH 3T3 cells conferred not only the repression of synoviolin gene expression butalso a decrease in cell number. Fluorescence-activated cell sorter analysis using annexin V staining confirmedthe induction of apoptosis by EBS-1 decoy and demonstrated recovery of apoptosis by overexpression ofSynoviolin. Our results suggest that transcriptional regulation of synoviolin via EBS-1 plays an important rolein cellular homeostasis. Our study provides novel insight into the transcriptional regulation for cellularhomeostasis.

Synoviolin is a molecule cloned from synoviocytes of pa-tients with rheumatoid arthritis (RA) and characterizes RAsynovial cells (RASCs) based on its high expression level inthese cells (4). Indeed, immunohistochemical analysis showedmarked expression of Synoviolin in synovial tissue of RA pa-tients relative to that of patients with osteoarthritis. Otherstudies indicated that Synoviolin is an endoplasmic reticulum(ER)-resident membrane protein and is the human homologueof the yeast E3-ubiquitin ligase (Hrd1p), which functions as anER-associated degradation (ERAD) system in yeast (7, 21,59). Furthermore, Synoviolin was found to have an E3 ligaseactivity and to function in the ERAD system, similar to Hrd1p(4, 28, 33, 37).

The biological role of Synoviolin was first investigated withtransgenic mice. Interestingly, Synoviolin caused arthropathywith synovial hypertrophy in over 30% of transgenic mice,which was associated with significant suppression of apoptosis(4). In contrast, destruction of the synoviolin gene heterozy-gote, i.e., 50% half gene dosage mice, was almost completelyprotective against collagen-induced arthritis (CIA) due to en-

hanced apoptosis of synovial cells (4). These results confirmthe involvement of Synoviolin in the onset of arthropathy andthat synoviolin gene dosage correlates significantly with theonset of arthropathy; i.e., increased expression of Synoviolinappears to be important for synovium overgrowth and trigger-ing of arthropathy (4).

In other studies, we also demonstrated that the synoviolingene is involved in the maintenance of embryonic life, sincehomozygote mice deficient in synoviolin died in utero at 13.5days postconception (dpc) because of aberrant apoptosis (60).Furthermore, in a culture system using small interfering RNA(siRNA), down-regulation of the synoviolin gene was vulnera-ble to various ER stress reagents such as tunicamycin, thapsi-gargin, and dithiothreitol, leading to apoptosis, whereas over-expression conversely rescued the apoptosis (4). These resultsindicate that alternation of the Synoviolin expression level canmodulate the resistance to apoptosis caused by disruption ofERAD function. Furthermore, reduction of constitutive ex-pression of the synoviolin gene could result in deterioration ofER homeostasis, consequently leading to a breakdown of cel-lular homeostasis and eventual apoptosis of the cell. Sincemost cells are exposed to a flux of newly synthesized proteinseven under physiological conditions and consequently some ofthese proteins accumulate as misfolded and unfolded proteinsin the ER, Synoviolin has to eliminate such proteins in order tomaintain ER homeostasis, namely, to protect against any dis-

* Corresponding author. Mailing address: Department of GenomicScience, Institute of Medical Science, St. Marianna University Schoolof Medicine, 2-16-1 Sugao Miyamae-ku, Kawasaki, Kanagawa 216-8512, Japan. Phone: 81-44-977-8111, ext. 4113. Fax: 81-44-977-9772.E-mail: [email protected].

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ruption of cellular homeostasis. Therefore, constitutive expres-sion of Synoviolin might be responsible for maintaining ERhomeostasis for cell survival in vivo. The aforementioned find-ings emphasize the importance of transcriptional regulation ofthe synoviolin expression level in cellular homeostasis. Thepresent study was designed to determine the mechanism(s)involved in the transcriptional regulation of synoviolin expres-sion in the cells.

MATERIALS AND METHODS

Cell culture. NIH 3T3 cells were cultured in Dulbecco’s modified Eagle’smedium (DMEM) (Life Technologies, Rockville, MD) supplemented with 10%heat-inactivated fetal calf serum. Stable cell lines were maintained in DMEMsupplemented with 10% fetal calf serum and containing 1% penicillin-strepto-mycin and 500 �g/ml G418.

Construction of plasmids. DNA constructs including various parts of the5�-flanking region of the synoviolin gene were generated as follows. The ampli-fication of the promoter sequences was performed by PCR using a subclonedplasmid of synoviolin, and the fragment (�2055 to �845) was subcloned intoPGV-B2 (PicaGene Basic vector2; TOYO INK, Japan) with the use of XhoI andNcoI to yield SyG�2055/�845 (full-length promoter). Next, deletion constructswere generated by excision of the 5�-end promoter region by restriction digestionwith AflII, SacII, BssHII, NruI, PvuII, and EcoRV, yielding SyG�1175/�845,SyG�1062/�845, SyG�320/�845, SyG�199/�84, SyG�410/�845, andSyG�800/�845, respectively. For further deletion constructions, a series of var-ious truncated 5�-end fragments were generated by PCR using SyG�2055/�845as the template with the following primers: SyG-106, 5�-GGCGGTACCTACGGTCCACTCCGCCGC; SyG-82, 5�-GGCGGTACCCGCCGCCGGAAGTGAGGTGT; SyG-71, 5�-GGCGGTACCGTGAGGTGTCTTACCCCCGA; SyG-63, 5�-GGCGGTACCTCTTACCCCCGAAGTTCC; SyG-37, 5�-GGCGGTACCGGGGGTGGGGAGTGTTGTTAA; and SyG-10, 5�-GGCGGTACCGCTGCCGCAGTCGCGGTG. The amplified PCR products were then gel purified, digested with theKpnI/PvuII enzyme, and cloned into the KpnI/PvuII sites upstream of SyG�199/�845. In this study the six constructs are designated SyG�106/�845, SyG�82/�845, SyG�71/�845, SyG�63/�845, SyG�37/�845, and SyG�10/�845. Thename of each plasmid construct included the borders of the inserted DNAfragment in base pairs relative to the transcription start site. Each mutation wasconstructed as follow. Plasmids containing the mutations (see Fig. 3A) wereconstructed using the plasmid SyG�199/�845 as the template by an overlapextension PCR protocol (43a). Briefly, two separate PCR products were gener-ated with either an antisense- or a sense-mutated oligonucleotide and one out-side primer. The two products were mixed, and a second PCR was then per-formed using the two outside primers. The product was digested with KpnI/PvuIIand ligated into KpnI/PvuII sites of SyG�199/�845. The inserts were sequencedto confirm the intended mutations. Plasmids for mammalian expression of Sy-noviolin-hemagglutinin (HA) were generated by insertion of the cDNA encodingsynoviolin, which had been described previously (4), into the site upstream ofHA-pcDNA3. PBK-CMV/His-muGABP� for expression of GA binding protein� (GABP�) and PBK-CMV/His-muGABP� for expression of GABP�1 werekindly provided by Barbara J. Graves and Nancy A. Speck (49). The dominantnegative (DN) construct of GABP�1 (DN-GABP�1) was generated by PCRusing primers 5�-GTAATACGACTCACTATAGGGC-3� (sense) and 5�-CTATCATTCTGCACATTCCACCC-3� (antisense) and cloning the PCR product intoPBK-CMV/His-muGABP�1 with BamHI/EcoRI to yield DN-GABP�1 (deletedfrom position 1121 to the end) (8). All constructions were confirmed by DNAsequencing (ABI PRISM3100 genetic analyzer; Applied Biosystems, Foster City,CA).

Transfection and reporter assays. For transient transfection into NIH 3T3cells, test plasmids (100 ng) and internal control DNA (cytomegalovirus [CMV]–�-galactosidase [�-gal] expression vector; 50 ng) were transfected withFUGENE6 (Roche, Mannheim, Germany) and added to 24-well plates as de-scribed by the manufacturer. After 30 h, the cultures were aspirated and cellswere added to 100 �l of Passive lysis buffer (Promega, Madison, WI). The celldebris were pelleted, and the supernatants were collected and immediately an-alyzed for luciferase and �-galactosidase activities. Luminescence was measuredin a MicoLumat Plus (Perkin-Elmer Cetus, Foster City, CA). �-Galactosidaseassays were performed by adding 7 �l of the cell extract to 100 �l of assay buffer(60 mM Na2HPO4, 40 mM NaH2PO4, 1 mM MgCl2, 50 mM �-mercaptoethanol,and 0.665 mg/ml o-nitrophenyl �-D-galactoside), followed by incubation at 37°Cfor 30 min. The reactions were terminated by the addition of 160 �l of 1.0 M

Na2CO3, and absorbance was measured at 420 nm. All luciferase measurementswere normalized for transfection efficiency to �-galactosidase expressed in theplasmid CMV–�-galactosidase, which was termed the relative luciferase activity.These values were normalized by setting the average relative luciferase activityfor SyG�199/�845.

EMSA. Double-stranded DNA oligonucleotides were annealed and labeledwith T4 kinase (Invitrogen, San Diego, CA) and [�-32P]ATP (Amersham Bio-sciences, Arlington Heights, IL), following the instructions provided by themanufacturer. The probes and competitor double-stranded oligodeoxynucle-otide (ODN) sequences were as follow: EBS-1 probe (�83 to �68) (16-mer),5�-GCGCCGCCGGAAGTGA-3�; mutated EBS-1 (G-74T) probe (�83 to �68)(16-mer), GCGCCGCCGTAAGTGA. The EBS-1 nucleotide sequence is de-rived from the mouse synoviolin promoter. Electrophoretic mobility shift assay(EMSA) was performed using 25 fmol of �-32P-labeled probe in 15 �l at roomtemperature in 20 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM dithio-threitol, 2 mM MgCl2, 5% glycerol, and 1 �g of poly(dI-dC). After 30 min ofincubation, the reaction mixtures were electrophoresed on 6% polyacrylamidegels in 0.25� Tris-borate-EDTA (22.5 mM Tris-borate, 0.5 M EDTA), followedby autoradiography (FLA-2000; FujiFILM, Tokyo). The band intensity wasquantified with Image Gauge version 3.0 (FujiFILM). Competition assays wereperformed by using a 100-fold molar excess of homologous unlabeled probes.The supershift assay was performed as follow. Polyclonal antiserum (2 or 4 �g)was added and the reaction mixture was incubated for another 1 h on ice priorto loading on gel. Antibodies used for supershift assay were GABP� (C-20),GABP� (N-20), Ets-1 (C-20), Ets-2 (C-20), Elk-1 (I-20), Erg-1/2 (C-20), andPea3 (16) (all purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Chromatin immunoprecipitation (CHIP) assay. To cross-link DNA and pro-tein, 1 � 108 NIH 3T3 cells were treated with 1% formaldehyde for 5 min atroom temperature. A chromatin solution was then prepared as described previ-ously (36). For immunoprecipitation, 4 �g of GABP� (C-20) (Santa Cruz Bio-technology) was incubated overnight with chromatin solution at 4°C on a rotationwheel. The immunocomplexes were collected with salmon sperm DNA–proteinA-Sepharose beads and washed sequentially with 200 �l of each of the followingbuffers containing a protease inhibitor mixture (Sigma Chemical Co., St. Louis,MO): twice with wash buffer I (0.1% sodium dodecyl sulfate [SDS], 1% TritonX-100, 20 mM Tris, pH 8.1, 2 mM EDTA, and 150 mM NaCl), twice with washbuffer II (0.1% SDS, 1% Triton X-100, 20 mM Tris, pH 8.1, 2 mM EDTA, and500 mM NaCl), twice with wash buffer III (10 mM Tris, pH 8.1, 1 mM EDTA0.25%, M LiCl, 1% NP-40, and 1% deoxycholate), and twice with TE buffer (10mM Tris, pH 8.1, and 1 mM EDTA). For elution, 100 �l of elution buffer I (1%SDS, 10 mM Tris, pH 8.1, and 1 mM EDTA) was added to the immunocom-plexes, followed by incubation at 65°C for 15 min. After centrifugation, thesupernatant was transferred to clean tubes. Pellets were added to 150 �l ofelution buffer II (0.67% SDS, 10 mM Tris, pH 8.1, and 1 mM EDTA), and theeluates were combined in the same tube. For reversing formaldehyde cross-linking, proteinase K (Wako Pure Chemicals, Osaka, Japan) was added to theeluate at a final concentration of 50 �g/ml prior to overnight incubation at 65°C.The mixture was then extracted with phenol-chloroform, the DNA was precip-itated with ethanol and resuspended in 20 �l of H2O, and the DNA solution wasthen used for PCR amplification of synoviolin promoter regions. The primers forthe synoviolin promoter were 5�-CGACCACACGTCACAGCTCT (bp �198 to�179 from the transcriptional start site) and 3�-AACAACACTCCCCACCCCCT (bp �38 to �19 from the transcriptional start site). The resulting product,which was 180 bp for Synoviolin, was separated by agarose gel electrophoresis.

Construction of synoviolin promoter transgene. DNA fragments free from theplasmid sequence were prepared from each construct by digestion with SacII andthen microinjected into the pronuclei of fertilized eggs from BDF1 mice (JapanSLC, Inc.). The generated embryos were sacrificed at 11.5 dpc and 13.5 dpc, andthe established transgenic mice were sacrificed at 7 to 10 weeks and subsequentlysubjected to expression analysis of the reporter gene. Transgenic embryos wereidentified by PCR analysis of genomic DNA extracted from the yolk sac asdescribed previously (4), and transgenic mice were ascertained by Southernblotting. All constructs were confirmed by DNA sequencing (ABI PRISM3100Genetic Analyzer; Applied Biosystems).

In vivo �-galactosidase staining. �-Gal activity was analyzed by staining withX-Gal (5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside) (Sigma). The em-bryos and some organs of adult mice were fixed in 4% paraformaldehyde for 20min and stained with X-Gal (0.5 mg/ml X-Gal, 44 mM HEPES buffer, pH 7.9, 3mM potassium ferricyanide, 15 mM NaCl, and 1.3 mM MgCl2) in phosphate-buffered saline (PBS) for 12 to 24 h at 37°C. The reaction was stopped by washingin PBS and postfixing in 4% paraformaldehyde.

Induction of arthritis. Arthritis was induced in mice by a combination ofmonoclonal antibodies (MAb cocktail) and lipopolysaccharide (LPS) (MAb-

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FIG. 1. Identification of the element responsible for synoviolin promoter activity. (A and B) Structure and sequence of the 5�-flanking regionof the mouse synoviolin gene and comparison of the human and mouse synoviolin promoters. (A) In the proximal conserved region, there are twotranscription factor binding sites for Sp1, two for the Ets family, and one for AML1, which were named Sp1 binding sites (SBS-1 and SBS-2), Etsfamily binding sites (EBS-1 and EBS-2), and AML1 binding site (ABS), respectively. As for EBS-1, the flanking sequences to EBS are identicalto the binding site of GABP. The consensus binding sites are shown in boxes. The arrowhead indicates the transcriptional start site. (B) The mapshows the structure of the synoviolin promoter. Two highly conserved regions exist in the synoviolin promoter. The percentages of conservation in

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LPS-induced arthritis) by using the method described by the supplier (Chondrex,LLX) and Amano et al. (4). Three mice from each group with arthritis (hetero-zygote, SyL 2.9k wt, and SyL 1.0k wt) and mice from the control group free ofarthritis (each line) were sacrificed at day 7 after injection. The periarticularsynovia of these mice were stripped and then mechanically homogenized, and thetissue extracts were analyzed for �-gal activity as described previously (17). The�-gal activity in the synovial tissue of each mouse was quantitated using the �-galassay.

Western blotting. Antisynoviolin monoclonal antibody was previously de-scribed (4). Western blot analysis was performed as described previously withminor modifications (4). Briefly, cell cultures were harvested and lysed in 1%NP-40, 25 mM Tris-HCl, pH 6.8, 0.25% SDS, 0.05% 2-mercaptoethanol, and0.1% glycerol. Aliquots of clear cell lysates were separated on SDS-polyacryl-amide gels, transferred to nitrocellulose membranes, and immunoblotted withantisynoviolin monoclonal antibody. Bound antibody was detected by peroxi-dase-conjugated sheep anti-mouse immunoglobulin G and the ECL detectionsystem (Amersham Pharmacia Biotech).

siRNAs and oligodeoxynucleotides. Phosphorothioate DNA with 20 nucleo-tides (Hokkaido System Science, Hokkaido, Japan) and RNA with 21 nucleo-tides (Japan Bio Service, Saitama, Japan) were chemically synthesized. DecoyODNs were prepared by annealing of sense and antisense phosphorothioateODNs. Twenty-four hours before transfection, cells in the exponential growthphase were trypsinized and then placed on 24-well plate (1 � 104 per well).Transfection was carried out for 84 h with 200 nmol/liter of decoy ODNs per wellwith the FUGENE6 (Roche) reagent or with 25 nmol/liter of siRNAs per wellwith Lipofectamine 2000 (Invitrogen) according to the instructions provided bythe supplier. The following siRNAs and phosphorothioate ODN sequences wereused in this study: mouse Synoviolin (m1374), AUGGUGACUGGUGCUAAGATT; green fluorescent protein (GFP), GGCUACGUCCAGGAGCGCATT;EBS-1 (�83 to �64), 5�-GCGCCGCCGGAAGTGAGGTG-3� and 3�-CACCRCACTTCCGGCGGCGC-3�; and Scramble, 5�-TTGCCGTACCCTACTTAGCC-3� and 3�-GGCTAAGTAGGGTACGGCAA-5�.

Establishment of Synoviolin-overexpressing stable cell lines. Synoviolin ex-pression vector Synoviolin-HA/pcDNA3, and HA/pcDNA3 vector were trans-fected into NIH 3T3 cells by using the Transfectamine 2000 reagent (Invitrogen).Transformants were selected at a final concentration of 1 mg/ml for G418.Independently isolated clonal lines were established. Clonal cell lines were main-tained in DMEM containing 10% fetal bovine serum and 500 �g/ml of G418.

Annexin V staining and fluorescence-activated cell sorter analysis. NIH 3T3cells were trypsinized; washed twice with ice-cold PBS, pH 7.4; resuspended in1� annexin-binding buffer (Vybrant apoptosis assay kit; Invitrogen); and thendiluted in 1� annexin-binding buffer to 1 � 106 cells/ml, preparing a sufficientvolume for 100 �l per assay. In the next step, 5 �g of fluorescein isothiocyanate(FITC)-annexin V was added to 100 �l of cell suspension. The cells wereincubated at room temperature for 15 min. After the incubation period, 400 �lof 1� annexin-binding buffer was added and mixed gently, and the samples werekept on ice. The stained cells were then analyzed with a FACSCalibur (BectonDickinson, Mountain View, CA).

Statistical analysis and ethical considerations. Data were expressed as mean standard error of the mean (SEM) or standard deviation. Differences betweengroups were examined for statistical significance by using Student’s t test. A Pvalue of less than 0.05 indicated the presence of a statistically significant differ-ence. All experimental protocols described in this study were approved by theEthics Review Committee of St. Marianna University School of Medicine.

RESULTS

Core region of synoviolin promoter. We first isolated a mousesynoviolin genomic clone by plaque hybridization using cDNAof human synoviolin (4) as a probe from the EMBL3SP6/T7library (Clontech Laboratories, Palo Alto, CA). The clonecontained a 7.5-kb insert consisting of 2.2 kb of the 5�-flankingregion. The 5�-flanking region and the first exon and intron

were confirmed by sequencing to be similar to a sequenceregistered in GenBank (accession no. AK004688). The tran-scription initiation site of the mouse synoviolin gene was de-termined by 5� rapid amplification of cDNA ends (data notshown). The portion containing the transcriptional start site is

FIG. 2. Binding of GABP to the endogenous synoviolin pro-moter.(A) GABP� binds EBS-1. EMSA was performed using a mix-ture containing approximately 3 � 104 cpm of the wild-type probe, 10�g of NIH 3T3 cell nuclear extract, and the indicated amounts ofunlabeled competitor corresponding to sequences between bp �83and �68 of the synoviolin promoter. The competitors either repre-sented the wild-type promoter sequence (WT) or contained mutations(MT) in the indicated EBS-1. “Supershift” assays were performed withthe 32P-labeled EBS-1 probe (�83/�68) and NIH 3T3 cell nuclearextracts in the absence or presence of antibodies to GABP� andGABP�. The positions of the supershift are indicated on the left.(B) Chromatin immunoprecipitation assays. CHIP from NIH 3T3 cellswas performed with antibodies to GABP� and Fli-1. Input corre-sponds to PCR mixtures containing 0.5% of the total amount of pro-teins used in immunoprecipitation reactions. IgG, immunoglobulin G.

the distal conserved region (�684 to �515) and proximal conserved region (�1 to �94) are 81.9% and 97.8%, respectively. (C and D) Analysisof promoter activity of the 5�-flanking region of the mouse synoviolin gene. Promoter activities were measured with a luciferase reporter plasmidcontaining one of the indicated fragments of the synoviolin promoter (left panel) and with a �-galactosidase gene plasmid driven by the CMVpromoter, as described in Materials and Methods. Values are averages standard deviations for duplicate transfections, with similar resultsobtained in two independent experiments.



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shown in Fig. 1A. Although a putative TATA box was notobserved in the promoter region, a GC-rich region was notednear the initiation site, in addition to two Ets binding sites(EBS-1 and EBS-2), one AML1 binding site, and two SP1binding sites (SBS-1 and SBS-2) (Fig. 1A). These features wereconsistent with those observed in promoters of certain house-keeping genes such as thymidylate synthase (43). To clarify theimportance of the promoter region of synoviolin in evolution,we compared the mouse and human synoviolin promoters andfound two highly conserved regions, which were termed thedistal and proximal conserved regions (81.9% and 97.8% iden-tity, respectively) (Fig. 1B), suggesting that these two regionsare crucial elements in the transcriptional regulation of syno-violin.

Next, we determined the cis-acting element in the transcrip-tional regulation of synoviolin. For this purpose, we con-structed a reporter plasmid containing two conserved regions,approximately 2.9 kb from the translational start site. Theplasmid was deleted from the 5� end to give constructs termedthe SyG series. Using the SyG series, deletion assays wereperformed. SyG�199/�845 still exhibited the full promoteractivity, whereas SyG�410/�845 showed almost complete dis-ruption of the promoter activity. These results indicate thatsynoviolin promoter activity is within bp �199 to �845 (Fig.1C). We next examined more refined deletions within thisregion. The deletion from �82 to �71 resulted in a furtherdecrease of the promoter activity to 3.8% (Fig. 1D). Similarresults were obtained from experiments using several cell lines,such as ATDC5 cells, HeLa cells, and rheumatoid synovialcells, consistent with the ubiquitous expression of Synoviolin invivo and in vitro in humans and mice (data not shown). Con-sidered together, these results indicate that in this region, 11bp containing EBS-1, is important for the constitutive tran-scriptional activity of synoviolin.

Binding of GABP�/� complex to EBS-1 on the synoviolinpromoter. The cis-acting element contained an EBS-1. SinceEBS-1 is the binding site for the ets family, which is composed

FIG. 3. Involvement of GABP in EBS-1-dependent synoviolin pro-moter activity. (A) Effect of mutation on synoviolin promoter strength.The site-directed mutants shown in the middle panel were transientlyexpressed in NIH 3T3 cells as described in Materials and Methods.After normalization of luciferase activity to that of �-galactosidase, therelative luciferase activity was expressed as a percentage of that ofSyG�199/�845. Data represent the means SEMs from three inde-

pendent experiments. The top panel is a schematic representation ofthe proximal promoter region of synoviolin. The middle panel repre-sents three mutated sequences and each wild-type sequence (Sp1, Ets,and AML1 binding sites) used in these reporter assays. (B) EBS-1 isimportant for transcriptional regulation of synoviolin by GABP�/�complexes. Reporter assays were performed using the following pro-cedure. NIH 3T3 cells were prepared at 2 � 104/well in 24-well plates.After 24 h, the indicated plasmids, GABP� and GABP�, or emptyvector was transfected with 50 �g of either SyG�199/�845 orSyg�199/�845 (G-74T) and 25 ng of CMV–�-galactosidase expressionvector at a total amount of 125 ng into NIH 3T3 cells by using FU-GENE6 (Roche) reagents. Thirty-six hours after transfection, the cellswere harvested and promoter activities were measured. Promoter ac-tivities are expressed relative to that of a reporter without effector andare expressed as a percentage of relative activity (luciferase activity/�-galactosidase). Data are means SEMs from three independent ex-periments. (C) DN-GABP�1 represses synoviolin promoter activity viaEBS-1. As a reporter 50 ng of SyG�2055/�845 or SyG�2055/�845(G-74T) was used in reporter assays with the indicated amount ofDN-GABP�1 and 25 ng of CMV–�-galactosidase expression vector.Reporter assays were performed according to the same proceduredescribed for panel B. Promoter activities of Syg�2055/�845 andSyG�2055/�845 (G-74T) are shown as white and black bars, respec-tively. Data are means SEMs from at least three independent ex-periments.



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of many transcription factors (47), and the specificity of thebinding of members of the Ets family to the target promoter isdependent on the flanking sequences of the core element,GGAA/T, we searched the consensus motif of each ets familymember. Interestingly, the flanking sequences of EBS-1 con-sisting of “CGGAAGTG” were identical to the consensus mo-tif of GABP�, an ets family member (51) which was initiallyidentified as a rat liver transcription factor (34, 52, 55). Arecent study demonstrated that the two ets motifs are requiredfor GABP to function as an initiator interacting with SP1 (63)for basal transcription of housekeeping genes that contain theGC-rich and TATA-less promoter context. Since the synoviolinproximal promoter shared these features (Fig. 1A), we consid-ered that GABP was a candidate transcription factor involvedin the regulation of the synoviolin promoter.

GABP is composed of two distinct proteins. One is GABP�,which has an ets domain and not a transactivation domain, andthe other is GABP�1, which is a specific cofactor; togetherthey form complexes for transactivation (6, 12, 19, 41, 50).Thus, to ascertain whether GABP can bind EBS-1 of the sy-noviolin promoter in NIH 3T3 cells, we prepared an EBS-1probe (�83/�68) including EBS-1 (�76/�69) and then per-formed EMSA using this probe. Several complexes wereformed by the EBS-1 probe and nuclear extracts in NIH 3T3cells (Fig. 2A, lane 2). Furthermore, competition assays con-firmed that the nonradiolabeled EBS-1 probe (wild type) al-most inhibited the formation of the bands (Fig. 2A, lane 3),while nonradiolabeled mutant probe, with a mutated G to T atbp �74, did not affect the formation of the bands (Fig. 2A, lane4), indicating that these bands were specific to the formation ofcomplexes between the EBS-1 probe and NIH 3T3 nuclearextracts.

Supershift assays revealed shifts of three of these bandsfollowing the addition of antibody against GABP� (Fig. 2A,lanes 5 and 6), indicating that GABP� bound the EBS-1 probe.Furthermore, formation of complex I was inhibited by using anantibody against GABP�1/2, suggesting an inhibitory effect ofGABP�1/2 antibody on the formation of the heterotetramer,as shown in Fig. 2A, lanes 7 and 8 [(�/�)2]. On the other hand,antibodies against other Ets families did not affect these bands(data not shown), including Ets-1 (C-20), Ets-2 (C-20), Elk-1(I-20), Erg-1/2 (C-20), and Pea3 (16), indicating the specificityof GABP� binding to the EBS-1 probe.

To confirm the binding of GABP to the endogenous pro-moter of synoviolin in NIH 3T3 cells, we performed a CHIPassay. The results confirmed the binding of GABP� to theproximal promoter at �19 to �198 in vivo (Fig. 2B), but notthat of Fli-1, which is another Ets family member. These re-sults indicate that GABP� constitutively binds the synoviolinproximal promoter in vivo.

EBS-1-dependent transcriptional regulation of the synovio-lin promoter by GABP. Next, we examined the role of EBS-1 insynoviolin promoter activity. As a template for SyG�199/�845,we constructed a mutant reporter plasmid in which G wasreplaced with T at bp �74, and the point-mutated constructwas termed mEBS-1. Moreover, two other binding sites nearthe region were converted to mutants (mSBS-1 and mABS),and the activity of each promoter was studied. mEBS-1, but notmSBS-1 or mABS, caused disruption of the promoter activityof synoviolin to 12% (Fig. 3A). These results suggest that

EBS-1 is a crucial element for the basal transcriptional activityof the synoviolin promoter.

Next, we performed reporter assays to examine the tran-scriptional activation of GABP�/� complexes on the synoviolinpromoter. The GABP�/� complex activated the promoter, andsuch activation was EBS-1-dependent (Fig. 3B). To verify thatthe GABP�/� complex-dependent transcriptional regulationof synoviolin is mediated via EBS-1, we performed reporterassays using DN-GABP�1, which lacks a transactivation do-main (8). DN-GABP� repressed synoviolin promoter activityin a dose-dependent manner (Fig. 3C). Taken together, theseresults indicate that synoviolin is regulated via EBS-1 by theGABP�/� complex.

EBS-1 is crucial for Synoviolin expression in vivo. To ascer-tain the effect of EBS-1 on in vivo expression of Synoviolin, weproduced transgenic mice that overexpressed the synoviolinpromoter gene combined with the lacZ gene (Fig. 4A). In eachtransgenic mouse, the promoter region contained approxi-mately 2.9 kbp from the translational start site (SyL 2.9k wtand the EBS-1 mutant SyL 2.9k mt) or approximately 1 kbfrom the translational start site (SyL 1.0k wt and the EBS-1mutant SyL 1.0k mt). To determine the distribution of Syno-violin, X-Gal staining of whole-mount mouse embryos andsome organs of adult mice was performed (Fig. 4B and C). Weestablished lines positive for �-gal staining for each transgenicmouse. The proportions of transgenic mice that stained posi-tively for �-gal are shown in Table 1.

Since we previously targeted synoviolin by using a knockoutconstruct encoding LacZ, the �-gal staining pattern of theheterozygote animal can be used as a positive control for thepattern of gene expression observed using various promoterconstructs (4). Thus, the expression pattern of the heterozy-gote was compared to those of SyL 2.9k wt and SyL 1.0k wt.�-gal staining of the organs of these transgenic mice showedthat systemic expression of Synoviolin was evident and wasespecially high in the granular and Purkinje layers of the cer-ebellum and the renal pelvis of the kidney (Fig. 4B). Further-more, in these transgenic embryos, extensive expression ofSynoviolin, such as in mesenchymal condensations and theneural tube, was observed (Fig. 4C). These results indicatedthat the expression pattern of transgenic mice containing 1 kbupstream from the translation start site (SyL 1.0k wt) is iden-tical to that of the heterozygote (Fig. 4B,C), suggesting that 1kb from the translation start site contained the endogenousactive promoter region.

The distribution patterns of each line in two transgenic mice(SyL 2.9k wt and SyL 1.0k wt) were identical (Fig. 4B and C).Surprisingly, those of mutations SyL 2.9k mt and SyL 1.0k mtexhibited random staining patterns (Fig. 4C). A number ofstudies (27, 38, 56, 57) explained that this could reflect con-stituents around the plasmid insertion site, the so-called posi-tional effect, and that these effects are probably exerted moststrongly on transgenes that do not contain strong promoters,enhancers, or other modulating sequences (3). Thus, it is con-ceivable that the disruption of the core promoter rendered theexpression of �-gal random in the promoter transgenic mice.Thus, these results indicate that the EBS-1 on synoviolin iscrucial for basal transcription of Synoviolin in vivo; namely, itsexpression in vivo requires EBS-1.



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FIG. 4. EBS-1 is crucial for expression of Synoviolin in mouse embryos under physiological condition and CIA. (A) Construction of transgenesand schematic diagram of the transgenes. (B) Presence of several enhancers of expression in various tissues within 1 kb from the translational startsite in adult mice. The patterns of �-gal staining are identical among heterozygote (b, f, and j), SyL-2.9k wt (c, g, and k), and SyL-1.0k wt (d, h,and l). Various tissues, including the granular (Gr) and Purkinje (Pu) layers of the cerebellum (f, g, and h) and renal pelvis (RP) (j, k, and l), exhibit



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Induced synoviolin expression in arthritis is due to sequencewithin 1.0 kbp of the synoviolin promoter. Our previous datashowed the pivotal role of Synoviolin overexpression in arthri-tis (4). Furthermore, we demonstrated that EBS-1 is a crucialelement for Synoviolin expression in vivo. To investigate therole of EBS-1 in arthritis, we used MAb- and LPS-inducedarthritis as experimental models for arthritis and evaluated�-gal activities in the synovia of three transgenic mice (hetero-zygote, SyL 2.9k wt, and SyL 1.0k wt) with induced arthritis, bycomparing the activities with those in mice free of arthritis. Asexpected, �-gal activity in heterozygote mice with induced ar-thritis was significantly higher than that in mice free of arthritis(Fig. 5A and B). We also found that the �-gal expression levelwas higher in SyL 2.9k wt and SyL 1.0k wt mice with MAb- andLPS-induced arthritis than control mice without arthritis (Fig.5B) (induction ratios of heterozygote, SyL 2.9k wt, and SyL1.0k wt mice compared to the control mice were, 2.49, 2.38,and 1.86, respectively). These results indicate that Synoviolinoverexpression in arthritic mice is due to the transcriptionalregulation of synoviolin promoter, especially within 1.0 kbp.Given that EBS-1 is the core element for synoviolin transcrip-tion, transcriptional regulation via EBS-1 could be one candi-date responsible for the increased expression of Synoviolin inarthritis.

Enhanced apoptosis by EBS-1 decoy induced-disruption ofSynoviolin expression. To confirm the functional effect ofEBS-1 on Synoviolin expression, we generated a decoy ODN(�83/�64) that targets EBS-1 (�76/�69), termed EBS-1 de-coy, and verified its effect on the regulation of synoviolin bytransfer of EBS-1 decoy into NIH 3T3 cells. Before the assays,immunocytochemical analysis using FITC confirmed that theefficacy of EBS-1 decoy transfer into NIH 3T3 cells was over80% (data not shown). Luciferase assays using cell extractstreated with the EBS-1 decoy showed repression of its tran-scription (Fig. 6A). In addition, Western blotting showed thatEBS-1 decoy transfer significantly reduced the expression ofSynoviolin (Fig. 6B). These results strongly indicate that theEBS-1 decoy is responsible for synoviolin promoter activity.Interestingly, transfection of EBS-1 decoy into NIH 3T3 cellsyielded only a few cells (Fig. 6B). Thus, Synoviolin might havean antiapoptotic role in cellular homeostasis, based on the

following evidences (4). In the synovia of heterozygote micewith CIA, enhanced apoptosis was observed compared withwild-type mice (4), and aberrant apoptosis was detected inembryos of homozygotes (60). Moreover, in culture systems,synoviolin-specific down-regulation by RNA interferencecaused cell apoptosis under normal condition in RASCs (4).Taken together, the results suggest that signaling via EBS-1targets a set of genes including synoviolin to prevent apoptosis

FIG. 5. Induction of synoviolin promoter activity in arthritis.(A) Macroscopic appearance of cut surfaces of joints with (a to d) andwithout (e to h) arthritis. The rate of induction of arthritis by a com-bination of a MAb cocktail and LPS was 100%. Induced synoviolinpromoter activity was observed in heterozygotes (a and b) and bothsynoviolin promoter-overexpressing transgenic mice, SyL 2.9k wt (c)and SyL 1.0k wt (d), with arthritis but not in the respective control mice(e, f, g, and h). These photographs were taken from heterozygotes withexperimentally induced arthritis (n 3), SyL2.9k wt (n 3), SyL 1.0kwt (n 3), and respective control mice (n 3 each). Asterisks markthe synovium. Magnifications: a and e, �1; b, c, d, f, g, and h, �3.(B) Correlation between induction of arthritis and synoviolin promoteractivity. Periarticular synovia of these mice were homogenized andincubated with di-�-D-galactopyranoside and analyzed by �-gal assay.The synoviolin promoter activity of each sample is expressed relative tothat of the control.

TABLE 1. Expression patterns in LacZ transgenic mice

Transgenic constructNo. of

transgenicembryos

No. oftransgenicembryos

expressing �-galactosidase

X-Gal-stainedtissue

SyL 1.0k wt (�199/�845) 8 3 UbiquitousSyL 1.0k mt (�199/�845) 5 3 RandomSyL 2.9k wt (�2055/�845) 6 3 UbiquitousSyL 2.9k mt (�2055/�845) 6 3 Random

identical patterns of expression. These results indicate that several enhancers of synoviolin expression are systemically distributed in tissues of adultmice. (C) One-point mutation disrupts the expression of LacZ derived from the synoviolin promoter in mouse embryos. �-Galactosidase expressionin transgenic embryos bearing synoviolin-lacZ constructs at 11.5 dpc (a to n) and 13.5 dpc (o to u) is shown. LacZ expression is seen ubiquitouslyin heterozygote embryos (a, b, and o), SyL 2.9k wt (�2055/�845) (i, j, and s), and SyL 1.0k wt (�199/�845) (c, d and p) transgenic mice. Incontrast, LacZ expression in mice containing a mutant promoter showed disruption of the expression pattern (e, f, g, h, k, l, m, n, q, r, t, and u).



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under normal conditions. Therefore, EBS-1 might play an im-portant role in cell survival.

To confirm the results on synoviolin shown in Fig. 6B, firstwe investigated the effect of synoviolin knockdown in NIH 3T3cells. siRNA for Synoviolin was applied to NIH 3T3 cells, andthe results showed a decrease in cell number (Fig. 6C). Theseresults suggest that a low transcript level of synoviolin pro-moted deterioration of cellular homeostasis and consequentlyresulted in fewer cells.

Next, we verified the relationship between transcriptionalregulation of synoviolin via EBS-1 and apoptosis. Since GABPtargets several genes that are considered important in apopto-tic signaling (35, 42, 53), to determine the role of transcrip-tional regulation of synoviolin in the induction of the observedapoptosis, we carried out the following experiments. Using twotypes of stable cell lines, Western blotting with an antibodyagainst Synoviolin confirmed that Synoviolin-HA driven by aCMV promoter overexpressed in cell lines, named syno, is notaffected by EBS-1 decoy, because the CMV overexpressionsystem is independent of EBS-1-mediated transcriptional reg-ulation. On the other hand, pcDNA3-overexpressing cell linesshowed that mock, endogenous Synoviolin was repressed byEBS-1 decoy (Fig. 7A). Furthermore, the rate of apoptosisinduced by EBS-1 decoy transfer was comparable, and thesecomparisons indicated a significant inhibition of apoptosis ofSynoviolin-overexpressing stable cells compared with mockcells (proportion in annexin V-positive Synoviolin-overexpress-ing stable cell line, 29.8%; that in pcDNA3 stable cell line,51.1%) (Fig. 7B). Taken together, these results indicate thatthe transcriptional regulation of synoviolin via EBS-1 is a cru-cial factor in the control of resistance to apoptosis and hencepromotion of cell survival.

DISCUSSION

Most cells secrete various products, such as hormones, ex-tracellular matrix proteins, and a variety of growth factors. Thenewly produced proteins enter the ER, where they are modi-fied for protein folding and subjected to posttranslational mod-ification. However, although chaperones help in the refoldingof these proteins, many protein molecules (more than 80% forsome proteins) translocated into the ER fail to achieve properfolding (1). Such proteins are exported from the ER lumenthrough translocon into the cytosol, where they are ubiquiti-nated and degraded. These processes are called ERAD (58,64), in which Synoviolin functions as an E3 ligase to degradesuch misfolded and unfolded proteins. Our previous study re-vealed that mice deficient in the synoviolin gene die beforebirth (60), and synoviolin-specific knockdown by RNA inter-ference approaches results in deterioration of cell survival incell cultures, suggesting that Synoviolin is involved in theERAD system and implicated in cell survival.

Several studies examined quality control in the ER (10, 23,61). When the ER is exposed to stress, such as increasedprotein synthesis, the cells have specific signal responses called

FIG. 6. Induction of apoptosis by repression of synoviolin.(A) EBS-1 decoy represses the promoter activity of synoviolin. NIH3T3 cells were prepared at 2 � 104/well in 24-well plates. Twenty-fourhours after the preparation, decoy ODNs were transfected at 200 nMinto the cells by using FUGENE6 (Roche) reagents. Twenty-fourhours after transfection, SyG �199/�845 as a reporter plasmid andCMV–�-galactosidase as an internal control were transfected. Thirty-six hours later, the cells were harvested and lysed with passive lysisbuffer and the whole extracts were subjected to luciferase assay. Dataare means standard deviations. (B) Twenty-four hours after prepa-ration of NIH 3T3 cells, decoys for either EBS-1 or Scramble at 200nM were transfected using FUGENE6 (Roche) reagents and incu-bated for 84 h. Western blotting using decoy for either EBS-1 orScramble is shown in the upper panel. Photographs taken 84 h aftertransfection are shown in the lower panels at a magnification of �100.Mouse polyclonal antibody to Synoviolin (4) and mouse monoclonalantibody to �-actin (clone AC-15; Sigma) were used in these experi-ments. (C) As in panel B, siRNAs for either GFP or synoviolin at 25nM were transfected using Lipofectamine 2000 (Invitrogen) reagents

and incubated for 84 h. Western blotting is shown in the upper panel,and photographs taken 84 h after transfection are shown in the lowerpanels at a magnification of �100.



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the unfolded protein response to deal with such perturbationin the ER (30, 31, 40, 64), which induces a set of genes such asBip for refolding and synoviolin for degradation (29, 54, 62).However, under normal conditions, the unfolded protein re-sponse is not activated, and thus the constitutively expressedgenes (which are known to cope with misfolded and unfoldedproteins produced under nonstress conditions) are necessary inorder to control ER quality. It is conceivable that maintenanceof ER homeostasis requires constitutive expression of synovio-lin. In the present study, we identified a cis-acting element,CGGAAGTG, termed EBS-1, which was found to be essentialfor maintenance of cellular homeostasis. Furthermore, down-

regulation of synoviolin targeted by EBS-1 decoy induced apo-ptosis, and the induction of apoptosis was significantly rescuedby overexpression of Synoviolin (Fig. 7). That is, transcrip-tional regulation for constitutive expression of synoviolin viaEBS-1 might be required continuously for ER homeostasis byeliminating unfolded and misfolded proteins produced in theER. Therefore, down-regulation of Synoviolin leads to disrup-tion of ER homeostasis and consequently leads to apoptosis.

Our findings point to a fundamental mechanism underlyingthe transcriptional regulation of synoviolin via EBS-1 for theSynoviolin expression pattern. Using transgenic mice carryingthe promoter of the synoviolin gene, our analysis showed ubiq-

FIG. 7. Synoviolin expression protects against apoptosis induced by EBS-1 decoy. (A) Establishment of a stable cell line for Synoviolinoverexpression and empty expression vector (pcDNA3) overexpression. For stable cell lines, either Synoviolin-HA/pcDNA3 or HA/pcDNA3 emptyvector was transfected into NIH 3T3 cells by Lipofectamine 2000 (Invitrogen) reagents. After addition of selective medium (containing 0.5 �g/mlG418) on the following day, the media were changed every 3 days and the concentration of G418 increased gradually up to 1.0 �g/ml. After colonyformation, a limited dilution was performed in order to prepare a clone for stable expression of Synoviolin-HA/pcDNA3 expression vector orHA/pcDNA3 empty vector. One of the established clones was used in the following experiments. To ascertain the expression from plasmids in celllines, Western blotting was performed using antibody against HA tag. Furthermore, Western blotting using Synoviolin antibody was performed toensure the extent of repression for Synoviolin. �-Actin antibody was used as a control. (B) Synoviolin overexpression protects the cells againstapoptosis induced by EBS-1 decoy. The percentage of apoptotic cells was determined after treatment with EBS-1 decoy. Eighty-four hours afterEBS-1 transfection by FUGENE6 (Roche) reagents, the cells were harvested and collected in microtubes and then labeled with annexin V-FITC.Magnification, �100. The distribution patterns of live and apoptotic cells were determined by fluorescence-activated cell sorter analysis. Percentdata represent the percentages of apoptotic cells.



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uitous distribution of synoviolin expression, although a strongerexpression was observed in some tissues, such as the granularand Purkinje layers of the cerebellum and the renal pelvis (Fig.4B). In embryos, for example, synoviolin was highly expressedin mesenchymal cells such as somites (data not shown), mes-enchymal condensation of limb buds, and neural tube (Fig.4C). The promoter region located 1.0 kb from the translationstart site of the synoviolin gene was sufficient for its constitutiveexpression (Fig. 4B and C, SyL 1.0k wt). Furthermore, tran-scriptional regulation of synoviolin for constitutive expressionin various tissues and the abundant expression in mesenchymalcells are dependent on the promoter region within 1 kbp fromthe translation start site (Fig. 4B and C, SyL 1.0k wt). Ourstudies using mutation of EBS-1 in transgenic mice revealedthat EBS-1 is crucial for the regulation of Synoviolin expres-sion in vivo, suggesting that EBS-1 is the core element fortranscription of the synoviolin gene in vivo. These results indi-cate that EBS-1 is responsible for the Synoviolin expressionpattern, namely, for constitutive expression as well as abundantexpression.

The Ets binding site (EBS), GGAA/T, is a binding site forEts family members that regulate a number of viral and cellu-lar genes (47). In the present study, we demonstrated thatEBS-1 is bound by GABP�, one of the Ets family members,and that the GABP�/� complex regulates the synoviolin pro-moter via EBS-1. GABP� is expressed ubiquitously and targetshousekeeping genes for constitutive expression (11, 13, 39, 43).On the other hand, GABP regulates lineage-restricted genesfor differentiation in specific cells such as myeloid cells (9, 41)and neuromuscular-specific cells (8, 14, 15, 18, 32, 45), whereGABP is targeted by phosphorylation events that lie down-stream of specific signal transduction pathways such as c-Jun–N-terminal kinase (JNK) (24) and extracellular signal-regu-lated kinase (ERK) (5, 24) or by the physiological andfunctional interaction of GABP� and GABP� with each otherand with other transcription factors and cofactors (9, 15, 41).Since GABP receives signaling for various biological settingsand then regulates a set of genes for adaptive responses, suchas mitochondrial function (11, 46), protein synthesis (16), andcell cycle events (25, 26, 43, 44, 48), it is known as an integratorof intracellular signaling pathway (42). Therefore, GABPmight work as an integrator to regulate synoviolin via EBS-1for constitutive expression and specific expression under cer-tain conditions, such as development and maintenance of celllife. In addition, aberration of this GABP-dependent regula-tion of synoviolin, that is, the activated regulation or associa-tion with another transcriptional factor by which Synoviolinexpression was increased, might lead certain cells to a patho-logical state such as proliferation of synovial cells.

Since EBS-1 is responsible for transcriptional regulation im-plicated in synoviolin constitutive ubiquitous expression in tis-sues and stronger expression in some tissues, we first assessedthe implication of GABP in the constitutive expression of sy-noviolin. Taking the features of GABP for transcriptional reg-ulation of target genes into consideration, GABP is known toregulate housekeeping genes for basic cellular activities (39,42), which are expressed in virtually all cell types (2) and tendto be GC rich, TATA-less, and with no initiation element (11,43). These features of GABP target genes are consistent withthose of the synoviolin promoter structure (Fig. 1A). In addi-

tion, given that Synoviolin has an ubiquitous distribution (60),that deficiency of the synoviolin gene caused death in utero ofhomozygote mice (60), and that the RING finger domain ofsynoviolin is evolutionarily highly conserved from yeast to hu-man, synoviolin is likely to play an important role in maintain-ing cell life, similar to that of housekeeping genes. Takentogether, it is likely that GABP regulates constitutive expres-sion of synoviolin via EBS-1. During protein synthesis, theERAD system is required for the degradation of unfolded andmisfolded proteins because many new proteins are misfoldedand unfolded in the ER (1). Therefore, GABP probably tran-scriptionally regulates the constitutive expression of synoviolin,keeping the function of the ERAD system to cope with them.This is the first report to demonstrate that the signaling path-way, integrated by GABP, targets EBS-1 for constitutive ex-pression of synoviolin, which is implicated in the ERAD system(Fig. 3).

With regard to the transcriptional regulation of synoviolinfor more specific and stronger expression, Synoviolin is over-expressed in the synovia of mice with experimentally inducedarthritis (4). In the present study, the synovia of transgenicmice carrying SyL 1.0k wt (�2055/�845) showed significantinduction of synoviolin in response to the onset of arthritis (Fig.5A and B). This result suggests that a certain transcriptionalmechanism regulates this cell-specific expression of Synoviolinin response to arthritis induction. Our study indicated that theelement responsible for the increased expression of Synoviolinin arthritis is within 1.0 kbp of the synoviolin promoter, includ-ing the EBS-1 core promoter element. However, in the case ofarthritis, it remains unknown whether the increased expressionof Synoviolin in the synovium is due to increased binding ofGABP to EBS-1 (by modification or by association with othertranscriptional factors) or to the change of binding to EBS-1from GABP to other Ets family member transcription factorson the synoviolin promoter. Given that EBS-1 plays an impor-tant role in transcriptional regulation of synoviolin for cellularhomeostasis and that GABP regulates the constitutive expres-sion of synoviolin via EBS-1, it is possible that certain specificsignals such as JNK and ERK (since GABP is known to lie inthese signaling pathways) (5, 20, 23), are involved in the in-creased expression of Synoviolin via EBS-1. In particular, inthe case of rheumatoid arthritis, JNK is known to be highlyactivated in RASCs and synovium (22). Therefore, aberrantsignals of JNK could target certain transcriptional factors, e.g.,GABP, which subsequently activate the binding to EBS-1 ofthe synoviolin promoter, leading to increased synoviolin expres-sion in the synovium followed by high expression of synoviolin,and consequently leading to overgrowth of synovial cells. Thishypothesis needs to be confirmed by detailed analysis of thesignal transduction in arthritis.

Our results confirmed that EBS-1 is a crucial site for theregulation of Synoviolin expression, which is implicated in sy-novial outgrowth and onset of arthropathy. These results en-hance our understanding of the mechanism of augmented ex-pression of Synoviolin in the arthritic synovium. Furthermore,we demonstrated that the 1.0 kbp within the proximal pro-moter of synoviolin is responsible for the increased expressionof Synoviolin in arthritis (Fig. 5A and B). Taking into consid-eration that EBS-1 is the core element for Synoviolin expres-sion, the EBS-1 decoy could be potentially useful in the treat-



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ment of RA based on its effective suppression of Synoviolinexpression and induction of apoptosis. Studies that examinethe effect of EBS-1 decoy transfer in a mouse experimentalmodel of arthritis are under way in our laboratories.

In conclusion, we identified EBS-1 as a crucial site for theexpression of Synoviolin. We also demonstrated that synoviolinis a novel target for GABP and revealed its role in homeostasisand cell survival. Our results provide important informationregarding the transcriptional regulation of synoviolin in themaintenance of cellular homeostasis.

ACKNOWLEDGMENTS

We thank all members of T. Nakajima’s laboratory and E. Shono forhelpful discussions; F. Issa for reading and editing the manuscript; andF. Amano, W. Ohkawa, S. Shinkawa, K. Suzuki, Y. Suzuki, N. Takagi,Y. Nakagawa, M. Hayashi, M. Hinata, and M. Yui for technical assis-tance. We also thank Barbara J. Graves and Nancy A. Speck forproviding the murine GABP� and murine GABP�1 expression vec-tors.

This work was supported financially by LocomoGene Inc.; the Jap-anese Ministry of Education, Science, Culture and Sports; the Japa-nese Ministry of Health and Welfare; the Japan Science and Technol-ogy Corporation; the Human Health Science Foundation; theMemorial Yamanouchi Foundation; the Kato Memorial Trust forNanbyo Research; Kanagawa Academy of Science and Technologyresearch grants; the Japan Medical Association; the Nagao MemorialFund; the Kanae Foundation for Life and Socio-Medical Science; theJapan Research Foundation for Clinical Pharmacology; the KanagawaNanbyo Foundation; the Japan College of Rheumatology; the Naka-jima Foundation; the Mitsubishi Pharma Research Foundation; theNew Energy and Industrial Technology Development Organization;Mochida Pharmaceutical Co., Ltd.; the Pharmaceuticals and MedicalDevices Agency; the Kanagawa High-Technology Foundation; Kana-gawa Academy of Science and Technology research grants; the Min-istry of Education, Culture, Sports and Technology; the Japan Societyfor Promotion of Science; the Ministry of Health, Labor and Welfare;and the Kanto Bureau of Economy, Trade and Industry.

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identification of a crucial site for synoviolin expression

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