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Molecular and Cellular Biology, August 2005, p. 7344-7356, Vol. 25, No. 16
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.16.7344-7356.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification of a Crucial Site for Synoviolin Expression

Kaneyuki Tsuchimochi,1,2 Naoko Yagishita,1 Satoshi Yamasaki,1 Tetsuya Amano,1 Yukihiro Kato,1 Ko-ichi Kawahara,3 Satoko Aratani,1 Hidetoshi Fujita,1 Fengyun Ji,1 Akiko Sugiura,1 Toshihiko Izumi,1,2 Asako Sugamiya,1 Ikuro Maruyama,3 Akiyoshi Fukamizu,4 Setsuro Komiya,2 Kusuki 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 Surgery,2 Laboratory and Molecular Medicine, Kagoshima Graduate School of Medical and Dental Sciences, Kagoshima 890-8520,3 Institute of Applied Biochemistry and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan4

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


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ABSTRACT
 
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 implicated in the pathogenesis of arthropathy. Complete deficiency of synoviolin is fatal embryonically. Thus, alternation of Synoviolin could cause breakdown of ER homeostasis and consequently lead to disturbance of cellular homeostasis. Hence, the expression level of Synoviolin appears to be important for its biological role in cellular homeostasis under physiological and pathological conditions. To examine the control of protein level, we performed promoter analysis to determine transcriptional regulation. Here we characterize the role of synoviolin 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 but also a decrease in cell number. Fluorescence-activated cell sorter analysis using annexin V staining confirmed the induction of apoptosis by EBS-1 decoy and demonstrated recovery of apoptosis by overexpression of Synoviolin. Our results suggest that transcriptional regulation of synoviolin via EBS-1 plays an important role in cellular homeostasis. Our study provides novel insight into the transcriptional regulation for cellular homeostasis.


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

The biological role of Synoviolin was first investigated with transgenic mice. Interestingly, Synoviolin caused arthropathy with synovial hypertrophy in over 30% of transgenic mice, which was associated with significant suppression of apoptosis (4). In contrast, destruction of the synoviolin gene heterozygote, i.e., 50% half gene dosage mice, was almost completely protective against collagen-induced arthritis (CIA) due to enhanced apoptosis of synovial cells (4). These results confirm the involvement of Synoviolin in the onset of arthropathy and that synoviolin gene dosage correlates significantly with the onset of arthropathy; i.e., increased expression of Synoviolin appears to be important for synovium overgrowth and triggering of arthropathy (4).

In other studies, we also demonstrated that the synoviolin gene is involved in the maintenance of embryonic life, since homozygote mice deficient in synoviolin died in utero at 13.5 days postconception (dpc) because of aberrant apoptosis (60). Furthermore, in a culture system using small interfering RNA (siRNA), down-regulation of the synoviolin gene was vulnerable to various ER stress reagents such as tunicamycin, thapsigargin, and dithiothreitol, leading to apoptosis, whereas overexpression conversely rescued the apoptosis (4). These results indicate that alternation of the Synoviolin expression level can modulate the resistance to apoptosis caused by disruption of ERAD function. Furthermore, reduction of constitutive expression of the synoviolin gene could result in deterioration of ER homeostasis, consequently leading to a breakdown of cellular homeostasis and eventual apoptosis of the cell. Since most cells are exposed to a flux of newly synthesized proteins even under physiological conditions and consequently some of these proteins accumulate as misfolded and unfolded proteins in the ER, Synoviolin has to eliminate such proteins in order to maintain ER homeostasis, namely, to protect against any disruption of cellular homeostasis. Therefore, constitutive expression of Synoviolin might be responsible for maintaining ER homeostasis for cell survival in vivo. The aforementioned findings emphasize the importance of transcriptional regulation of the synoviolin expression level in cellular homeostasis. The present study was designed to determine the mechanism(s) involved in the transcriptional regulation of synoviolin expression in the cells.


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MATERIALS AND METHODS
 
Cell culture. NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Rockville, MD) supplemented with 10% heat-inactivated fetal calf serum. Stable cell lines were maintained in DMEM supplemented with 10% fetal calf serum and containing 1% penicillin-streptomycin and 500 µg/ml G418.

Construction of plasmids. DNA constructs including various parts of the 5'-flanking region of the synoviolin gene were generated as follows. The amplification of the promoter sequences was performed by PCR using a subcloned plasmid of synoviolin, and the fragment (–2055 to +845) was subcloned into PGV-B2 (PicaGene Basic vector2; TOYO INK, Japan) with the use of XhoI and NcoI to yield SyG–2055/+845 (full-length promoter). Next, deletion constructs were generated by excision of the 5'-end promoter region by restriction digestion with AflII, SacII, BssHII, NruI, PvuII, and EcoRV, yielding SyG–1175/+845, SyG–1062/+845, SyG–320/+845, SyG–199/+84, SyG+410/+845, and SyG+800/+845, respectively. For further deletion constructions, a series of various truncated 5'-end fragments were generated by PCR using SyG–2055/+845 as 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 the KpnI/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. The name of each plasmid construct included the borders of the inserted DNA fragment in base pairs relative to the transcription start site. Each mutation was constructed as follow. Plasmids containing the mutations (see Fig. 3A) were constructed using the plasmid SyG–199/+845 as the template by an overlap extension PCR protocol (43a). Briefly, two separate PCR products were generated with either an antisense- or a sense-mutated oligonucleotide and one outside primer. The two products were mixed, and a second PCR was then performed using the two outside primers. The product was digested with KpnI/PvuII and ligated into KpnI/PvuII sites of SyG–199/+845. The inserts were sequenced to confirm the intended mutations. Plasmids for mammalian expression of Synoviolin-hemagglutinin (HA) were generated by insertion of the cDNA encoding synoviolin, which had been described previously (4), into the site upstream of HA-pcDNA3. PBK-CMV/His-muGABP{alpha} for expression of GA binding protein {alpha} (GABP{alpha}) and PBK-CMV/His-muGABPß for expression of GABPß1 were kindly provided by Barbara J. Graves and Nancy A. Speck (49). The dominant negative (DN) construct of GABPß1 (DN-GABPß1) was generated by PCR using primers 5'-GTAATACGACTCACTATAGGGC-3' (sense) and 5'-CTATCATTCTGCACATTCCACCC-3' (antisense) and cloning the PCR product into PBK-CMV/His-muGABPß1 with BamHI/EcoRI to yield DN-GABPß1 (deleted from position 1121 to the end) (8). All constructions were confirmed by DNA sequencing (ABI PRISM3100 genetic analyzer; Applied Biosystems, Foster City, CA).



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  		FIG. 3. Involvement of GABP in EBS-1-dependent synoviolin promoter activity. (A) Effect of mutation on synoviolin promoter strength. The site-directed mutants shown in the middle panel were transiently expressed in NIH 3T3 cells as described in Materials and Methods. After normalization of luciferase activity to that of ß-galactosidase, the relative luciferase activity was expressed as a percentage of that of SyG–199/+845. Data represent the means ± SEMs from three inde-pendent experiments. The top panel is a schematic representation of the proximal promoter region of synoviolin. The middle panel represents three mutated sequences and each wild-type sequence (Sp1, Ets, and AML1 binding sites) used in these reporter assays. (B) EBS-1 is important for transcriptional regulation of synoviolin by GABP{alpha}/ß complexes. Reporter assays were performed using the following procedure. NIH 3T3 cells were prepared at 2 x 104/well in 24-well plates. After 24 h, the indicated plasmids, GABP{alpha} and GABPß, or empty vector was transfected with 50 µg of either SyG–199/+845 or Syg–199/+845 (G-74T) and 25 ng of CMV-ß-galactosidase expression vector at a total amount of 125 ng into NIH 3T3 cells by using FUGENE6 (Roche) reagents. Thirty-six hours after transfection, the cells were harvested and promoter activities were measured. Promoter activities are expressed relative to that of a reporter without effector and are expressed as a percentage of relative activity (luciferase activity/ß-galactosidase). Data are means ± SEMs from three independent experiments. (C) DN-GABPß1 represses synoviolin promoter activity via EBS-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 of DN-GABPß1 and 25 ng of CMV-ß-galactosidase expression vector. Reporter assays were performed according to the same procedure described for panel B. Promoter activities of Syg–2055/+845 and SyG–2055/+845 (G-74T) are shown as white and black bars, respectively. Data are means ± SEMs from at least three independent experiments.

Transfection and reporter assays. For transient transfection into NIH 3T3 cells, test plasmids (100 ng) and internal control DNA (cytomegalovirus [CMV]-ß-galactosidase [ß-gal] expression vector; 50 ng) were transfected with FUGENE6 (Roche, Mannheim, Germany) and added to 24-well plates as described by the manufacturer. After 30 h, the cultures were aspirated and cells were added to 100 µl of Passive lysis buffer (Promega, Madison, WI). The cell debris were pelleted, and the supernatants were collected and immediately analyzed for luciferase and ß-galactosidase activities. Luminescence was measured in a MicoLumat Plus (Perkin-Elmer Cetus, Foster City, CA). ß-Galactosidase assays were performed by adding 7 µl of the cell extract to 100 µl of assay buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 1 mM MgCl2, 50 mM ß-mercaptoethanol, and 0.665 mg/ml o-nitrophenyl ß-D-galactoside), followed by incubation at 37°C for 30 min. The reactions were terminated by the addition of 160 µl of 1.0 M Na2CO3, and absorbance was measured at 420 nm. All luciferase measurements were normalized for transfection efficiency to ß-galactosidase expressed in the plasmid CMV-ß-galactosidase, which was termed the relative luciferase activity. These values were normalized by setting the average relative luciferase activity for SyG–199/+845.

EMSA. Double-stranded DNA oligonucleotides were annealed and labeled with T4 kinase (Invitrogen, San Diego, CA) and [{gamma}-32P]ATP (Amersham Biosciences, Arlington Heights, IL), following the instructions provided by the manufacturer. The probes and competitor double-stranded oligodeoxynucleotide (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 derived from the mouse synoviolin promoter. Electrophoretic mobility shift assay (EMSA) was performed using 25 fmol of {gamma}-32P-labeled probe in 15 µl at room temperature in 20 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 2 mM MgCl2, 5% glycerol, and 1 µg of poly(dI-dC). After 30 min of incubation, the reaction mixtures were electrophoresed on 6% polyacrylamide gels in 0.25x Tris-borate-EDTA (22.5 mM Tris-borate, 0.5 M EDTA), followed by autoradiography (FLA-2000; FujiFILM, Tokyo). The band intensity was quantified with Image Gauge version 3.0 (FujiFILM). Competition assays were performed by using a 100-fold molar excess of homologous unlabeled probes. The supershift assay was performed as follow. Polyclonal antiserum (2 or 4 µg) was added and the reaction mixture was incubated for another 1 h on ice prior to loading on gel. Antibodies used for supershift assay were GABP{alpha} (C-20), GABPß (N-20), Ets-1 (C-20), Ets-2 (C-20), Elk-1 (I-20), Erg-1/2 (C-20), and Pea3 (16) (all purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Chromatin immunoprecipitation (CHIP) assay. To cross-link DNA and protein, 1 x 108 NIH 3T3 cells were treated with 1% formaldehyde for 5 min at room temperature. A chromatin solution was then prepared as described previously (36). For immunoprecipitation, 4 µg of GABP{alpha} (C-20) (Santa Cruz Biotechnology) was incubated overnight with chromatin solution at 4°C on a rotation wheel. The immunocomplexes were collected with salmon sperm DNA-protein A-Sepharose beads and washed sequentially with 200 µl of each of the following buffers containing a protease inhibitor mixture (Sigma Chemical Co., St. Louis, MO): twice with wash buffer I (0.1% sodium dodecyl sulfate [SDS], 1% Triton X-100, 20 mM Tris, pH 8.1, 2 mM EDTA, and 150 mM NaCl), twice with wash buffer II (0.1% SDS, 1% Triton X-100, 20 mM Tris, pH 8.1, 2 mM EDTA, and 500 mM NaCl), twice with wash buffer III (10 mM Tris, pH 8.1, 1 mM EDTA 0.25%, M LiCl, 1% NP-40, and 1% deoxycholate), and twice with TE buffer (10 mM Tris, pH 8.1, and 1 mM EDTA). For elution, 100 µl of elution buffer I (1% SDS, 10 mM Tris, pH 8.1, and 1 mM EDTA) was added to the immunocomplexes, followed by incubation at 65°C for 15 min. After centrifugation, the supernatant was transferred to clean tubes. Pellets were added to 150 µl of elution buffer II (0.67% SDS, 10 mM Tris, pH 8.1, and 1 mM EDTA), and the eluates were combined in the same tube. For reversing formaldehyde cross-linking, proteinase K (Wako Pure Chemicals, Osaka, Japan) was added to the eluate at a final concentration of 50 µg/ml prior to overnight incubation at 65°C. The mixture was then extracted with phenol-chloroform, the DNA was precipitated with ethanol and resuspended in 20 µl of H2O, and the DNA solution was then used for PCR amplification of synoviolin promoter regions. The primers for the 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 the plasmid sequence were prepared from each construct by digestion with SacII and then microinjected into the pronuclei of fertilized eggs from BDF1 mice (Japan SLC, Inc.). The generated embryos were sacrificed at 11.5 dpc and 13.5 dpc, and the established transgenic mice were sacrificed at 7 to 10 weeks and subsequently subjected to expression analysis of the reporter gene. Transgenic embryos were identified by PCR analysis of genomic DNA extracted from the yolk sac as described previously (4), and transgenic mice were ascertained by Southern blotting. All constructs were confirmed by DNA sequencing (ABI PRISM3100 Genetic Analyzer; Applied Biosystems).

In vivo ß-galactosidase staining. ß-Gal activity was analyzed by staining with X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) (Sigma). The embryos and some organs of adult mice were fixed in 4% paraformaldehyde for 20 min and stained with X-Gal (0.5 mg/ml X-Gal, 44 mM HEPES buffer, pH 7.9, 3 mM 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 washing in PBS and postfixing in 4% paraformaldehyde.

Induction of arthritis. Arthritis was induced in mice by a combination of monoclonal antibodies (MAb cocktail) and lipopolysaccharide (LPS) (MAb-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 (heterozygote, SyL 2.9k wt, and SyL 1.0k wt) and mice from the control group free of arthritis (each line) were sacrificed at day 7 after injection. The periarticular synovia of these mice were stripped and then mechanically homogenized, and the tissue extracts were analyzed for ß-gal activity as described previously (17). The ß-gal activity in the synovial tissue of each mouse was quantitated using the ß-gal assay.

Western blotting. Antisynoviolin monoclonal antibody was previously described (4). Western blot analysis was performed as described previously with minor 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, and 0.1% glycerol. Aliquots of clear cell lysates were separated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with antisynoviolin monoclonal antibody. Bound antibody was detected by peroxidase-conjugated sheep anti-mouse immunoglobulin G and the ECL detection system (Amersham Pharmacia Biotech).

siRNAs and oligodeoxynucleotides. Phosphorothioate DNA with 20 nucleotides (Hokkaido System Science, Hokkaido, Japan) and RNA with 21 nucleotides (Japan Bio Service, Saitama, Japan) were chemically synthesized. Decoy ODNs were prepared by annealing of sense and antisense phosphorothioate ODNs. Twenty-four hours before transfection, cells in the exponential growth phase were trypsinized and then placed on 24-well plate (1 x 104 per well). Transfection was carried out for 84 h with 200 nmol/liter of decoy ODNs per well with the FUGENE6 (Roche) reagent or with 25 nmol/liter of siRNAs per well with Lipofectamine 2000 (Invitrogen) according to the instructions provided by the supplier. The following siRNAs and phosphorothioate ODN sequences were used 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 expression vector Synoviolin-HA/pcDNA3, and HA/pcDNA3 vector were transfected 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 maintained in DMEM containing 10% fetal bovine serum and 500 µg/ml of G418.

Annexin V staining and fluorescence-activated cell sorter analysis. NIH 3T3 cells were trypsinized; washed twice with ice-cold PBS, pH 7.4; resuspended in 1x annexin-binding buffer (Vybrant apoptosis assay kit; Invitrogen); and then diluted in 1x annexin-binding buffer to 1 x 106 cells/ml, preparing a sufficient volume for 100 µl per assay. In the next step, 5 µg of fluorescein isothiocyanate (FITC)-annexin V was added to 100 µl of cell suspension. The cells were incubated at room temperature for 15 min. After the incubation period, 400 µl of 1x annexin-binding buffer was added and mixed gently, and the samples were kept on ice. The stained cells were then analyzed with a FACSCalibur (Becton Dickinson, Mountain View, CA).

Statistical analysis and ethical considerations. Data were expressed as mean ± standard error of the mean (SEM) or standard deviation. Differences between groups were examined for statistical significance by using Student's t test. A P value of less than 0.05 indicated the presence of a statistically significant difference. All experimental protocols described in this study were approved by the Ethics Review Committee of St. Marianna University School of Medicine.


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RESULTS
 
Core region of synoviolin promoter. We first isolated a mouse synoviolin genomic clone by plaque hybridization using cDNA of human synoviolin (4) as a probe from the EMBL3SP6/T7 library (Clontech Laboratories, Palo Alto, CA). The clone contained a 7.5-kb insert consisting of 2.2 kb of the 5'-flanking region. The 5'-flanking region and the first exon and intron were confirmed by sequencing to be similar to a sequence registered in GenBank (accession no. AK004688). The transcription initiation site of the mouse synoviolin gene was determined by 5' rapid amplification of cDNA ends (data not shown). The portion containing the transcriptional start site is shown in Fig. 1A. Although a putative TATA box was not observed in the promoter region, a GC-rich region was noted near the initiation site, in addition to two Ets binding sites (EBS-1 and EBS-2), one AML1 binding site, and two SP1 binding sites (SBS-1 and SBS-2) (Fig. 1A). These features were consistent with those observed in promoters of certain housekeeping genes such as thymidylate synthase (43). To clarify the importance of the promoter region of synoviolin in evolution, we compared the mouse and human synoviolin promoters and found two highly conserved regions, which were termed the distal and proximal conserved regions (81.9% and 97.8% identity, respectively) (Fig. 1B), suggesting that these two regions are crucial elements in the transcriptional regulation of synoviolin.



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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 region of the mouse synoviolin gene and comparison of the human and mouse synoviolin promoters. (A) In the proximal conserved region, there are two transcription 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), Ets family binding sites (EBS-1 and EBS-2), and AML1 binding site (ABS), respectively. As for EBS-1, the flanking sequences to EBS are identical to the binding site of GABP. The consensus binding sites are shown in boxes. The arrowhead indicates the transcriptional start site. (B) The map shows the structure of the synoviolin promoter. Two highly conserved regions exist in the synoviolin promoter. The percentages of conservation inthe distal conserved region (–684 to –515) and proximal conserved region (–1 to –94) are 81.9% and 97.8%, respectively. (C and D) Analysis of promoter activity of the 5'-flanking region of the mouse synoviolin gene. Promoter activities were measured with a luciferase reporter plasmid containing one of the indicated fragments of the synoviolin promoter (left panel) and with a ß-galactosidase gene plasmid driven by the CMV promoter, as described in Materials and Methods. Values are averages ± standard deviations for duplicate transfections, with similar results obtained in two independent experiments.

Next, we determined the cis-acting element in the transcriptional regulation of synoviolin. For this purpose, we constructed a reporter plasmid containing two conserved regions, approximately 2.9 kb from the translational start site. The plasmid was deleted from the 5' end to give constructs termed the SyG series. Using the SyG series, deletion assays were performed. SyG–199/+845 still exhibited the full promoter activity, whereas SyG+410/+845 showed almost complete disruption of the promoter activity. These results indicate that synoviolin promoter activity is within bp –199 to +845 (Fig. 1C). We next examined more refined deletions within this region. The deletion from –82 to –71 resulted in a further decrease of the promoter activity to 3.8% (Fig. 1D). Similar results were obtained from experiments using several cell lines, such as ATDC5 cells, HeLa cells, and rheumatoid synovial cells, consistent with the ubiquitous expression of Synoviolin in vivo and in vitro in humans and mice (data not shown). Considered together, these results indicate that in this region, 11 bp containing EBS-1, is important for the constitutive transcriptional activity of synoviolin.

Binding of GABP{alpha}/ß complex to EBS-1 on the synoviolin promoter. The cis-acting element contained an EBS-1. Since EBS-1 is the binding site for the ets family, which is composed of many transcription factors (47), and the specificity of the binding of members of the Ets family to the target promoter is dependent on the flanking sequences of the core element, GGAA/T, we searched the consensus motif of each ets family member. Interestingly, the flanking sequences of EBS-1 consisting of "CGGAAGTG" were identical to the consensus motif of GABP{alpha}, an ets family member (51) which was initially identified as a rat liver transcription factor (34, 52, 55). A recent study demonstrated that the two ets motifs are required for GABP to function as an initiator interacting with SP1 (63) for basal transcription of housekeeping genes that contain the GC-rich and TATA-less promoter context. Since the synoviolin proximal promoter shared these features (Fig. 1A), we considered that GABP was a candidate transcription factor involved in the regulation of the synoviolin promoter.

GABP is composed of two distinct proteins. One is GABP{alpha}, which has an ets domain and not a transactivation domain, and the other is GABPß1, which is a specific cofactor; together they form complexes for transactivation (6, 12, 19, 41, 50). Thus, to ascertain whether GABP can bind EBS-1 of the synoviolin promoter in NIH 3T3 cells, we prepared an EBS-1 probe (–83/–68) including EBS-1 (–76/–69) and then performed EMSA using this probe. Several complexes were formed by the EBS-1 probe and nuclear extracts in NIH 3T3 cells (Fig. 2A, lane 2). Furthermore, competition assays confirmed that the nonradiolabeled EBS-1 probe (wild type) almost inhibited the formation of the bands (Fig. 2A, lane 3), while nonradiolabeled mutant probe, with a mutated G to T at bp –74, did not affect the formation of the bands (Fig. 2A, lane 4), indicating that these bands were specific to the formation of complexes between the EBS-1 probe and NIH 3T3 nuclear extracts.



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  		FIG. 2. Binding of GABP to the endogenous synoviolin promoter.(A) GABP{alpha} binds EBS-1. EMSA was performed using a mixture containing approximately 3 x 104 cpm of the wild-type probe, 10 µg of NIH 3T3 cell nuclear extract, and the indicated amounts of unlabeled competitor corresponding to sequences between bp –83 and –68 of the synoviolin promoter. The competitors either represented the wild-type promoter sequence (WT) or contained mutations (MT) in the indicated EBS-1. "Supershift" assays were performed with the 32P-labeled EBS-1 probe (–83/–68) and NIH 3T3 cell nuclear extracts in the absence or presence of antibodies to GABP{alpha} and GABPß. The positions of the supershift are indicated on the left. (B) Chromatin immunoprecipitation assays. CHIP from NIH 3T3 cells was performed with antibodies to GABP{alpha} and Fli-1. Input corresponds to PCR mixtures containing 0.5% of the total amount of proteins used in immunoprecipitation reactions. IgG, immunoglobulin G.

Supershift assays revealed shifts of three of these bands following the addition of antibody against GABP{alpha} (Fig. 2A, lanes 5 and 6), indicating that GABP{alpha} bound the EBS-1 probe. Furthermore, formation of complex I was inhibited by using an antibody against GABPß1/2, suggesting an inhibitory effect of GABPß1/2 antibody on the formation of the heterotetramer, as shown in Fig. 2A, lanes 7 and 8 [({alpha}/ß)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 specificity of GABP{alpha} binding to the EBS-1 probe.

To confirm the binding of GABP to the endogenous promoter of synoviolin in NIH 3T3 cells, we performed a CHIP assay. The results confirmed the binding of GABP{alpha} to the proximal promoter at –19 to –198 in vivo (Fig. 2B), but not that of Fli-1, which is another Ets family member. These results indicate that GABP{alpha} constitutively binds the synoviolin proximal promoter in vivo.

EBS-1-dependent transcriptional regulation of the synoviolin promoter by GABP. Next, we examined the role of EBS-1 in synoviolin promoter activity. As a template for SyG–199/+845, we constructed a mutant reporter plasmid in which G was replaced with T at bp –74, and the point-mutated construct was termed mEBS-1. Moreover, two other binding sites near the region were converted to mutants (mSBS-1 and mABS), and the activity of each promoter was studied. mEBS-1, but not mSBS-1 or mABS, caused disruption of the promoter activity of synoviolin to 12% (Fig. 3A). These results suggest that EBS-1 is a crucial element for the basal transcriptional activity of the synoviolin promoter.

Next, we performed reporter assays to examine the transcriptional activation of GABP{alpha}/ß complexes on the synoviolin promoter. The GABP{alpha}/ß complex activated the promoter, and such activation was EBS-1-dependent (Fig. 3B). To verify that the GABP{alpha}/ß complex-dependent transcriptional regulation of synoviolin is mediated via EBS-1, we performed reporter assays using DN-GABPß1, which lacks a transactivation domain (8). DN-GABPß repressed synoviolin promoter activity in a dose-dependent manner (Fig. 3C). Taken together, these results indicate that synoviolin is regulated via EBS-1 by the GABP{alpha}/ß complex.

EBS-1 is crucial for Synoviolin expression in vivo. To ascertain the effect of EBS-1 on in vivo expression of Synoviolin, we produced transgenic mice that overexpressed the synoviolin promoter gene combined with the lacZ gene (Fig. 4A). In each transgenic mouse, the promoter region contained approximately 2.9 kbp from the translational start site (SyL 2.9k wt and the EBS-1 mutant SyL 2.9k mt) or approximately 1 kb from the translational start site (SyL 1.0k wt and the EBS-1 mutant SyL 1.0k mt). To determine the distribution of Synoviolin, X-Gal staining of whole-mount mouse embryos and some organs of adult mice was performed (Fig. 4B and C). We established lines positive for ß-gal staining for each transgenic mouse. The proportions of transgenic mice that stained positively for ß-gal are shown in Table 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 transgenes and schematic diagram of the transgenes. (B) Presence of several enhancers of expression in various tissues within 1 kb from the translational start site in adult mice. The patterns of ß-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), exhibitidentical patterns of expression. These results indicate that several enhancers of synoviolin expression are systemically distributed in tissues of adult mice. (C) One-point mutation disrupts the expression of LacZ derived from the synoviolin promoter in mouse embryos. ß-Galactosidase expression in 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 ubiquitously in 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. In contrast, 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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  		TABLE 1. Expression patterns in LacZ transgenic mice

Since we previously targeted synoviolin by using a knockout construct encoding LacZ, the ß-gal staining pattern of the heterozygote animal can be used as a positive control for the pattern of gene expression observed using various promoter constructs (4). Thus, the expression pattern of the heterozygote was compared to those of SyL 2.9k wt and SyL 1.0k wt. ß-gal staining of the organs of these transgenic mice showed that systemic expression of Synoviolin was evident and was especially high in the granular and Purkinje layers of the cerebellum and the renal pelvis of the kidney (Fig. 4B). Furthermore, in these transgenic embryos, extensive expression of Synoviolin, such as in mesenchymal condensations and the neural tube, was observed (Fig. 4C). These results indicated that the expression pattern of transgenic mice containing 1 kb upstream from the translation start site (SyL 1.0k wt) is identical to that of the heterozygote (Fig. 4B,C), suggesting that 1 kb from the translation start site contained the endogenous active 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 mt exhibited random staining patterns (Fig. 4C). A number of studies (27, 38, 56, 57) explained that this could reflect constituents around the plasmid insertion site, the so-called positional effect, and that these effects are probably exerted most strongly on transgenes that do not contain strong promoters, enhancers, or other modulating sequences (3). Thus, it is conceivable that the disruption of the core promoter rendered the expression of ß-gal random in the promoter transgenic mice. Thus, these results indicate that the EBS-1 on synoviolin is crucial for basal transcription of Synoviolin in vivo; namely, its expression in vivo requires EBS-1.

Induced synoviolin expression in arthritis is due to sequence within 1.0 kbp of the synoviolin promoter. Our previous data showed the pivotal role of Synoviolin overexpression in arthritis (4). Furthermore, we demonstrated that EBS-1 is a crucial element for Synoviolin expression in vivo. To investigate the role of EBS-1 in arthritis, we used MAb- and LPS-induced arthritis as experimental models for arthritis and evaluated ß-gal activities in the synovia of three transgenic mice (heterozygote, SyL 2.9k wt, and SyL 1.0k wt) with induced arthritis, by comparing the activities with those in mice free of arthritis. As expected, ß-gal activity in heterozygote mice with induced arthritis was significantly higher than that in mice free of arthritis (Fig. 5A and B). We also found that the ß-gal expression level was higher in SyL 2.9k wt and SyL 1.0k wt mice with MAb- and LPS-induced arthritis than control mice without arthritis (Fig. 5B) (induction ratios of heterozygote, SyL 2.9k wt, and SyL 1.0k wt mice compared to the control mice were, 2.49, 2.38, and 1.86, respectively). These results indicate that Synoviolin overexpression in arthritic mice is due to the transcriptional regulation of synoviolin promoter, especially within 1.0 kbp. Given that EBS-1 is the core element for synoviolin transcription, transcriptional regulation via EBS-1 could be one candidate responsible for the increased expression of Synoviolin in arthritis.



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  		FIG. 5. Induction of synoviolin promoter activity in arthritis. (A) Macroscopic appearance of cut surfaces of joints with (a to d) and without (e to h) arthritis. The rate of induction of arthritis by a combination of a MAb cocktail and LPS was 100%. Induced synoviolin promoter activity was observed in heterozygotes (a and b) and both synoviolin 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 with experimentally induced arthritis (n = 3), SyL2.9k wt (n = 3), SyL 1.0k wt (n = 3), and respective control mice (n = 3 each). Asterisks mark the synovium. Magnifications: a and e, x1; b, c, d, f, g, and h, x3. (B) Correlation between induction of arthritis and synoviolin promoter activity. Periarticular synovia of these mice were homogenized and incubated with di-ß-D-galactopyranoside and analyzed by ß-gal assay. The synoviolin promoter activity of each sample is expressed relative to that of the control.

Enhanced apoptosis by EBS-1 decoy induced-disruption of Synoviolin expression. To confirm the functional effect of EBS-1 on Synoviolin expression, we generated a decoy ODN (–83/–64) that targets EBS-1 (–76/–69), termed EBS-1 decoy, and verified its effect on the regulation of synoviolin by transfer of EBS-1 decoy into NIH 3T3 cells. Before the assays, immunocytochemical analysis using FITC confirmed that the efficacy of EBS-1 decoy transfer into NIH 3T3 cells was over 80% (data not shown). Luciferase assays using cell extracts treated with the EBS-1 decoy showed repression of its transcription (Fig. 6A). In addition, Western blotting showed that EBS-1 decoy transfer significantly reduced the expression of Synoviolin (Fig. 6B). These results strongly indicate that the EBS-1 decoy is responsible for synoviolin promoter activity. Interestingly, transfection of EBS-1 decoy into NIH 3T3 cells yielded only a few cells (Fig. 6B). Thus, Synoviolin might have an antiapoptotic role in cellular homeostasis, based on the following evidences (4). In the synovia of heterozygote mice with CIA, enhanced apoptosis was observed compared with wild-type mice (4), and aberrant apoptosis was detected in embryos of homozygotes (60). Moreover, in culture systems, synoviolin-specific down-regulation by RNA interference caused cell apoptosis under normal condition in RASCs (4). Taken together, the results suggest that signaling via EBS-1 targets a set of genes including synoviolin to prevent apoptosis under normal conditions. Therefore, EBS-1 might play an important role in cell survival.



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  		FIG. 6. Induction of apoptosis by repression of synoviolin. (A) EBS-1 decoy represses the promoter activity of synoviolin. NIH 3T3 cells were prepared at 2 x 104/well in 24-well plates. Twenty-four hours after the preparation, decoy ODNs were transfected at 200 nM into the cells by using FUGENE6 (Roche) reagents. Twenty-four hours after transfection, SyG –199/+845 as a reporter plasmid and CMV-ß-galactosidase as an internal control were transfected. Thirty-six hours later, the cells were harvested and lysed with passive lysis buffer and the whole extracts were subjected to luciferase assay. Data are means ± standard deviations. (B) Twenty-four hours after preparation of NIH 3T3 cells, decoys for either EBS-1 or Scramble at 200 nM were transfected using FUGENE6 (Roche) reagents and incubated for 84 h. Western blotting using decoy for either EBS-1 or Scramble is shown in the upper panel. Photographs taken 84 h after transfection are shown in the lower panels at a magnification of x100. Mouse polyclonal antibody to Synoviolin (4) and mouse monoclonal antibody to ß-actin (clone AC-15; Sigma) were used in these experiments. (C) As in panel B, siRNAs for either GFP or synoviolin at 25 nM were transfected using Lipofectamine 2000 (Invitrogen) reagentsand incubated for 84 h. Western blotting is shown in the upper panel, and photographs taken 84 h after transfection are shown in the lower panels at a magnification of x100.

To confirm the results on synoviolin shown in Fig. 6B, first we investigated the effect of synoviolin knockdown in NIH 3T3 cells. siRNA for Synoviolin was applied to NIH 3T3 cells, and the results showed a decrease in cell number (Fig. 6C). These results suggest that a low transcript level of synoviolin promoted deterioration of cellular homeostasis and consequently resulted in fewer cells.

Next, we verified the relationship between transcriptional regulation of synoviolin via EBS-1 and apoptosis. Since GABP targets several genes that are considered important in apoptotic signaling (35, 42, 53), to determine the role of transcriptional regulation of synoviolin in the induction of the observed apoptosis, we carried out the following experiments. Using two types of stable cell lines, Western blotting with an antibody against Synoviolin confirmed that Synoviolin-HA driven by a CMV promoter overexpressed in cell lines, named syno, is not affected by EBS-1 decoy, because the CMV overexpression system is independent of EBS-1-mediated transcriptional regulation. On the other hand, pcDNA3-overexpressing cell lines showed that mock, endogenous Synoviolin was repressed by EBS-1 decoy (Fig. 7A). Furthermore, the rate of apoptosis induced by EBS-1 decoy transfer was comparable, and these comparisons indicated a significant inhibition of apoptosis of Synoviolin-overexpressing stable cells compared with mock cells (proportion in annexin V-positive Synoviolin-overexpressing stable cell line, 29.8%; that in pcDNA3 stable cell line, 51.1%) (Fig. 7B). Taken together, these results indicate that the transcriptional regulation of synoviolin via EBS-1 is a crucial factor in the control of resistance to apoptosis and hence promotion of cell survival.



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  		FIG. 7. Synoviolin expression protects against apoptosis induced by EBS-1 decoy. (A) Establishment of a stable cell line for Synoviolin overexpression and empty expression vector (pcDNA3) overexpression. For stable cell lines, either Synoviolin-HA/pcDNA3 or HA/pcDNA3 empty vector was transfected into NIH 3T3 cells by Lipofectamine 2000 (Invitrogen) reagents. After addition of selective medium (containing 0.5 µg/ml G418) on the following day, the media were changed every 3 days and the concentration of G418 increased gradually up to 1.0 µg/ml. After colony formation, a limited dilution was performed in order to prepare a clone for stable expression of Synoviolin-HA/pcDNA3 expression vector or HA/pcDNA3 empty vector. One of the established clones was used in the following experiments. To ascertain the expression from plasmids in cell lines, Western blotting was performed using antibody against HA tag. Furthermore, Western blotting using Synoviolin antibody was performed to ensure the extent of repression for Synoviolin. ß-Actin antibody was used as a control. (B) Synoviolin overexpression protects the cells against apoptosis induced by EBS-1 decoy. The percentage of apoptotic cells was determined after treatment with EBS-1 decoy. Eighty-four hours after EBS-1 transfection by FUGENE6 (Roche) reagents, the cells were harvested and collected in microtubes and then labeled with annexin V-FITC. Magnification, x100. The distribution patterns of live and apoptotic cells were determined by fluorescence-activated cell sorter analysis. Percent data represent the percentages of apoptotic cells.


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DISCUSSION
 
Most cells secrete various products, such as hormones, extracellular matrix proteins, and a variety of growth factors. The newly produced proteins enter the ER, where they are modified for protein folding and subjected to posttranslational modification. However, although chaperones help in the refolding of these proteins, many protein molecules (more than 80% for some proteins) translocated into the ER fail to achieve proper folding (1). Such proteins are exported from the ER lumen through translocon into the cytosol, where they are ubiquitinated and degraded. These processes are called ERAD (58, 64), in which Synoviolin functions as an E3 ligase to degrade such misfolded and unfolded proteins. Our previous study revealed that mice deficient in the synoviolin gene die before birth (60), and synoviolin-specific knockdown by RNA interference approaches results in deterioration of cell survival in cell cultures, suggesting that Synoviolin is involved in the ERAD 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 increased protein synthesis, the cells have specific signal responses called the unfolded protein response to deal with such perturbation in the ER (30, 31, 40, 64), which induces a set of genes such as Bip for refolding and synoviolin for degradation (29, 54, 62). However, under normal conditions, the unfolded protein response is not activated, and thus the constitutively expressed genes (which are known to cope with misfolded and unfolded proteins produced under nonstress conditions) are necessary in order to control ER quality. It is conceivable that maintenance of ER homeostasis requires constitutive expression of synoviolin. In the present study, we identified a cis-acting element, CGGAAGTG, termed EBS-1, which was found to be essential for maintenance of cellular homeostasis. Furthermore, down-regulation of synoviolin targeted by EBS-1 decoy induced apoptosis, and the induction of apoptosis was significantly rescued by overexpression of Synoviolin (Fig. 7). That is, transcriptional regulation for constitutive expression of synoviolin via EBS-1 might be required continuously for ER homeostasis by eliminating unfolded and misfolded proteins produced in the ER. Therefore, down-regulation of Synoviolin leads to disruption of ER homeostasis and consequently leads to apoptosis.

Our findings point to a fundamental mechanism underlying the transcriptional regulation of synoviolin via EBS-1 for the Synoviolin expression pattern. Using transgenic mice carrying the promoter of the synoviolin gene, our analysis showed ubiquitous distribution of synoviolin expression, although a stronger expression was observed in some tissues, such as the granular and Purkinje layers of the cerebellum and the renal pelvis (Fig. 4B). In embryos, for example, synoviolin was highly expressed in mesenchymal cells such as somites (data not shown), mesenchymal condensation of limb buds, and neural tube (Fig. 4C). The promoter region located 1.0 kb from the translation start site of the synoviolin gene was sufficient for its constitutive expression (Fig. 4B and C, SyL 1.0k wt). Furthermore, transcriptional regulation of synoviolin for constitutive expression in various tissues and the abundant expression in mesenchymal cells are dependent on the promoter region within 1 kbp from the translation start site (Fig. 4B and C, SyL 1.0k wt). Our studies using mutation of EBS-1 in transgenic mice revealed that EBS-1 is crucial for the regulation of Synoviolin expression in vivo, suggesting that EBS-1 is the core element for transcription of the synoviolin gene in vivo. These results indicate that EBS-1 is responsible for the Synoviolin expression pattern, namely, for constitutive expression as well as abundant expression.

The Ets binding site (EBS), GGAA/T, is a binding site for Ets family members that regulate a number of viral and cellular genes (47). In the present study, we demonstrated that EBS-1 is bound by GABP{alpha}, one of the Ets family members, and that the GABP{alpha}/ß complex regulates the synoviolin promoter via EBS-1. GABP{alpha} is expressed ubiquitously and targets housekeeping genes for constitutive expression (11, 13, 39, 43). On the other hand, GABP regulates lineage-restricted genes for differentiation in specific cells such as myeloid cells (9, 41) and neuromuscular-specific cells (8, 14, 15, 18, 32, 45), where GABP is targeted by phosphorylation events that lie downstream of specific signal transduction pathways such as c-Jun-N-terminal kinase (JNK) (24) and extracellular signal-regulated kinase (ERK) (5, 24) or by the physiological and functional interaction of GABP{alpha} and GABPß with each other and with other transcription factors and cofactors (9, 15, 41). Since GABP receives signaling for various biological settings and then regulates a set of genes for adaptive responses, such as mitochondrial function (11, 46), protein synthesis (16), and cell cycle events (25, 26, 43, 44, 48), it is known as an integrator of intracellular signaling pathway (42). Therefore, GABP might work as an integrator to regulate synoviolin via EBS-1 for constitutive expression and specific expression under certain conditions, such as development and maintenance of cell life. In addition, aberration of this GABP-dependent regulation of synoviolin, that is, the activated regulation or association with another transcriptional factor by which Synoviolin expression was increased, might lead certain cells to a pathological state such as proliferation of synovial cells.

Since EBS-1 is responsible for transcriptional regulation implicated in synoviolin constitutive ubiquitous expression in tissues and stronger expression in some tissues, we first assessed the implication of GABP in the constitutive expression of synoviolin. Taking the features of GABP for transcriptional regulation of target genes into consideration, GABP is known to regulate housekeeping genes for basic cellular activities (39, 42), which are expressed in virtually all cell types (2) and tend to be GC rich, TATA-less, and with no initiation element (11, 43). These features of GABP target genes are consistent with those of the synoviolin promoter structure (Fig. 1A). In addition, given that Synoviolin has an ubiquitous distribution (60), that deficiency of the synoviolin gene caused death in utero of homozygote mice (60), and that the RING finger domain of synoviolin is evolutionarily highly conserved from yeast to human, synoviolin is likely to play an important role in maintaining cell life, similar to that of housekeeping genes. Taken together, it is likely that GABP regulates constitutive expression of synoviolin via EBS-1. During protein synthesis, the ERAD system is required for the degradation of unfolded and misfolded proteins because many new proteins are misfolded and unfolded in the ER (1). Therefore, GABP probably transcriptionally 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 pathway, integrated by GABP, targets EBS-1 for constitutive expression of synoviolin, which is implicated in the ERAD system (Fig. 3).

With regard to the transcriptional regulation of synoviolin for more specific and stronger expression, Synoviolin is overexpressed in the synovia of mice with experimentally induced arthritis (4). In the present study, the synovia of transgenic mice carrying SyL 1.0k wt (–2055/+845) showed significant induction of synoviolin in response to the onset of arthritis (Fig. 5A and B). This result suggests that a certain transcriptional mechanism regulates this cell-specific expression of Synoviolin in response to arthritis induction. Our study indicated that the element responsible for the increased expression of Synoviolin in arthritis is within 1.0 kbp of the synoviolin promoter, including the EBS-1 core promoter element. However, in the case of arthritis, it remains unknown whether the increased expression of Synoviolin in the synovium is due to increased binding of GABP to EBS-1 (by modification or by association with other transcriptional factors) or to the change of binding to EBS-1 from GABP to other Ets family member transcription factors on the synoviolin promoter. Given that EBS-1 plays an important role in transcriptional regulation of synoviolin for cellular homeostasis and that GABP regulates the constitutive expression of synoviolin via EBS-1, it is possible that certain specific signals such as JNK and ERK (since GABP is known to lie in these signaling pathways) (5, 20, 23), are involved in the increased expression of Synoviolin via EBS-1. In particular, in the case of rheumatoid arthritis, JNK is known to be highly activated in RASCs and synovium (22). Therefore, aberrant signals of JNK could target certain transcriptional factors, e.g., GABP, which subsequently activate the binding to EBS-1 of the synoviolin promoter, leading to increased synoviolin expression in the synovium followed by high expression of synoviolin, and consequently leading to overgrowth of synovial cells. This hypothesis needs to be confirmed by detailed analysis of the signal transduction in arthritis.

Our results confirmed that EBS-1 is a crucial site for the regulation of Synoviolin expression, which is implicated in synovial outgrowth and onset of arthropathy. These results enhance our understanding of the mechanism of augmented expression of Synoviolin in the arthritic synovium. Furthermore, we demonstrated that the 1.0 kbp within the proximal promoter of synoviolin is responsible for the increased expression of Synoviolin in arthritis (Fig. 5A and B). Taking into consideration that EBS-1 is the core element for Synoviolin expression, the EBS-1 decoy could be potentially useful in the treatment of RA based on its effective suppression of Synoviolin expression and induction of apoptosis. Studies that examine the effect of EBS-1 decoy transfer in a mouse experimental model of arthritis are under way in our laboratories.

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


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ACKNOWLEDGMENTS
 
We thank all members of T. Nakajima's laboratory and E. Shono for helpful discussions; F. Issa for reading and editing the manuscript; and F. Amano, W. Ohkawa, S. Shinkawa, K. Suzuki, Y. Suzuki, N. Takagi, Y. Nakagawa, M. Hayashi, M. Hinata, and M. Yui for technical assistance. We also thank Barbara J. Graves and Nancy A. Speck for providing the murine GABP{alpha} and murine GABPß1 expression vectors.

This work was supported financially by LocomoGene Inc.; the Japanese Ministry of Education, Science, Culture and Sports; the Japanese Ministry of Health and Welfare; the Japan Science and Technology Corporation; the Human Health Science Foundation; the Memorial Yamanouchi Foundation; the Kato Memorial Trust for Nanbyo Research; Kanagawa Academy of Science and Technology research grants; the Japan Medical Association; the Nagao Memorial Fund; the Kanae Foundation for Life and Socio-Medical Science; the Japan Research Foundation for Clinical Pharmacology; the Kanagawa Nanbyo Foundation; the Japan College of Rheumatology; the Nakajima Foundation; the Mitsubishi Pharma Research Foundation; the New Energy and Industrial Technology Development Organization; Mochida Pharmaceutical Co., Ltd.; the Pharmaceuticals and Medical Devices Agency; the Kanagawa High-Technology Foundation; Kanagawa Academy of Science and Technology research grants; the Ministry of Education, Culture, Sports and Technology; the Japan Society for Promotion of Science; the Ministry of Health, Labor and Welfare; and the Kanto Bureau of Economy, Trade and Industry.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Genomic Science, Institute of Medical Science, St. Marianna University School of 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: nakashit{at}marianna-u.ac.jp. Back


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Molecular and Cellular Biology, August 2005, p. 7344-7356, Vol. 25, No. 16
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.16.7344-7356.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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