All human experimental protocols in the present study (no. 2728, 2729, 3758, 3759) were approved by the Ethics Review Committee of Tokyo Medical University (Tokyo, Japan). RA patients received stable doses of methotrexate (6–10 mg/week) before joint replacement surgery. Written informed consent was obtained from all patients prior to the collection of joint tissue samples. All procedures involving animals were performed in accordance with institutional and national guidelines for animal experimentation, and were approved by the Institutional Animal Care and Use Committee of Tokyo Medical University (no. S-28038, S-28040).

Mice were kept in SPF under conditions (20–26°C temperature; 40–65% humidity) on a 12 h light/12 h dark cycle. F-1 Foods (5.1% fat, 21.3% protein) were purchased from Funabashi farm (Chiba, Japan). Mice had free access to water bottles. Tamoxifen (Tam)-inducible Syvn1 knockout mice (CAG-Cre-ER; Syvn1^flox/flox) was generated previously (14). To isolate MEFs, embryos were isolated at E13.5, and the head and internal (including reproductive) organs were removed. The remaining tissue was physically dissociated and incubated in trypsin at 37°C for 15 min. Cells were resuspended in DMEM and plate the cells in 10 cm tissue culture dishes. On the next day, medium was changed and cells were expanded for two passages before freezing.

The Syvn1 (NM_032431) and PGC-1β (NM_133249) plasmids are described in the literature (7,14,15). The following antibodies were used: Anti-HA (3F10) (Roche Molecular Biochemicals, Indianapolis, IN, USA). Polyclonal antiserum against GST was generated by immunizing rats with purified GST. Anti-PGC-1β antibody has been described previously (14). Walnut extracts were prepared from the branches of walnut trees by the standard ethanol extraction method (16). Briefly, the air-dried walnut branches were milled into fine powder in the blender and the fibrous powder of walnut branches was extracted twice, on each occasion with 60% ethyl alcohol at room temperature for 24 h. The combined ethanol extract was filtered, and the filtrate was concentrated to dryness under reduced pressure in a rotary evaporator. The ethanol extract was freeze-dried. Without any further purification, the plant crude ethanol extract was used in our study. Aliquot portions of the ethanol extract was dissolved in DMSO for use of our experiments.

Rheumatoid synovial cells were obtained by standard methods (17). Briefly, the tissue was minced into small pieces and digested with collagenase (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The single-cell suspension was incubated overnight, and then floating cells were removed, and adherent cells were cultured in dishes. RSCs and MEFs, which were derived from Tam-inducible Syvn1 knockout mice (CAG-Cre-ER; Syvn1^flox/flox), were cultured in Dulbecco's modified Eagle's medium (DMEM) as previously described (14). RSC proliferation was measured with DMSO or walnut extract treatment (1, 3.3, 10, 33.3 µg/ml) for 3 days using the Cell Counting Kit-8 (Dojindo, Tokyo, Japan). MEFs were treated with DMSO or Tamoxifen (2.5 µM) for 2 days, and were then treated with DMSO or walnut extract (50 µg/ml) for 3 days. Electron microscopic analysis was then performed.

The GST pull-down assay was performed as previously described (14,18). Briefly, GST-Syvn1ΔTM and MBP-PGC-1β (1–367) were expressed and purified using glutathione sepharose beads and amylose beads, respectively (GE Healthcare Life Sciences, Little Chalfont, UK). GST-Syvn1ΔTM was incubated with MBP-PGC-1β bound to resin in 1 ml buffer A (20 mM Tris-HCl, pH 8.0; 100 mM NaCl; 1 mM ethylenediaminetetraacetic acid (EDTA); 1 mM dithiothreitol (DTT); 0.1% Nonidet P-40 (NP-40); 5% glycerol; 1 mM Na₃VO₄; 5 mM NaF; 1 µg/ml aprotinin; and 1 µg/ml leupeptin) for 4 h at 4°C. After washing the beads with buffer A, bound proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blotting.

In vitro ubiquitination assays were performed as previously described (14). Briefly, GST-PGC-1β (1–367) was incubated with 0.75 µg HA-Ub, 125 ng E1 (Biomol International, Plymouth Meeting, PA, USA), 150 ng UbcH5c, and 150 ng MBP-Syvn1ΔTM in reaction buffer (50 mM Tris-HCl, pH 7.5; 5 mM MgCl₂; 0.6 mM DTT; and 2 mM ATP) at 37°C for 2 h. Glutathione sepharose was added to the solution, after which the mixture was washed with GST wash buffer (50 mM Tris-HCl, pH 7.5; 0.5 M NaCl; 1% Triton X-100; 1 mM EDTA; 1 mM DTT; and protease inhibitors). Ubiquitinated PGC-1β was analyzed by western blotting using anti-PGC-1β antibodies.

In vivo ubiquitination assays were performed as previously described (14). Briefly, 293T cells were transfected with HA-PGC-1β, FLAG Ub, or Syvn1 expression plasmids. Cells were treated with DMSO or walnut extract (50 µg/ml) for 3 days. Cells were lysed in lysis buffer (50 mM HEPES, pH 7.9; 150 mM KCl; 1 mM phenylmethanesulfonyl fluoride, 1% Triton X-100; 10% glycerol; and protease inhibitors). Lysates were mixed with 1 µg anti-HA antibody conjugated to protein G-sepharose beads. After a 4-h incubation at 4°C, beads were washed three times with lysis buffer. Bound proteins were fractionated by SDS-PAGE and analyzed by immunoblotting.

For analysis of mitochondria using MitoTracker Red (Molecular Probes, Eugene, OR, USA), MEFs were treated with DMSO or walnut extract (50 µg/ml) for 3 days. Mitochondria were stained with the MitoTracker Red probe for 30 min at 37°C according to the manufacturer's protocol. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The intensity of staining by mitotracker was measured (n=8).

Tam-inducible Syvn1 knockout MEFs were treated with DMSO (WT MEFs) or Tam (Syvn1 knockout MEFs) for 2 days, and then WT MEFs and Syvn1 knockout MEFs were treated with DMSO or walnut extract (50 µg/ml) for 3 days. Total RNA from MEFs treated with DMSO or walnut extract was purified by using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions and reverse transcribed by using ReverTra Ace with random primers (Toyobo, Osaka, Japan). RT-qPCR was performed by using LightCycler 480 Probes Master (Roche Diagnostics, Mannheim, Germany) and the Step One Plus Detection System (Applied Biosystems; Life Technologies Japan, Tokyo, Japan). The thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 20 sec and extension at 72°C for 1 sec. Expression levels were determined relative to that of 18sRNA (RSCs) or ACTB (MEFs). Primers and probes used in the present study are shown in Table I. Relative expression was determined using the 2^−∆∆Cq method (19).

siRNAs for Syvn1 were previously described (14). Transfection with siRNAs (20 µM) was performed by using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Total RNA from RSCs was purified 2 days after transfection using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions, and reverse transcribed using ReverTra Ace with random primers (Toyobo).

All data are expressed as the mean ± standard deviation and were analyzed using Excel Statistics 2012 version 1.00 (Social Survey Research Information Co., Ltd., Tokyo, Japan). Differences between two groups were examined by Student's t-test. One-way analysis of variance with Tukey-Kramer post hoc analysis was used to determine correlations in datasets containing multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Syvn1 is a crucial factor involved in RSC proliferation (7,13,20). To identify Syvn1 inhibitors in natural products, we tested the effects of natural products on RSC proliferation with or without Syvn1. At first, we performed knockdown experiments with control siRNA (siControl) or siRNA for Syvn1 (siSyvn1). RT-qPCR showed that siSyvn1 induced 60% repression of Syvn1 expression (Fig. 1A). Walnut extract inhibited proliferation in a concentration-dependent manner in two RSC lines treated with siControl (Fig. 1B). Whereas, the inhibitory effect was attenuated in siSyvn1-treated cells (Fig. 1B). In the case of patient 1, walnut extract did not significantly have any effect (n.s.). In the case of patient 2, walnut extract had still inhibited cell growth, however, the strength of the effect was reduced as compared to control siRNA-treated cells.

Syvn1 negatively regulates PGC-1β activity via direct interaction with PGC-1β in vitro and in vivo (14). To determine whether walnut extract inhibits the interaction of Syvn1 with PGC-1β, we performed in vitro binding assays using glutathione S-transferase-tagged Syvn1 lacking the transmembrane domain (GST-Syvn1ΔTM) and maltose binding protein-tagged PGC-1β (amino acids 1–367) (MBP-PGC-1β (1–367)). As previously reported (14), MBP-PGC-1β (1–367) directly bound to GST-Syvn1ΔTM (Fig. 2A). MBP-PGC-1β (1–367) did not bind to GST, and GST-Syvn1ΔTM did not bind to MBP (Fig. 2A). Walnut extract inhibited the interaction of Syvn1 and PGC-1β in a concentration-dependent manner (Fig. 2B).

Syvn1 ubiquitinates PGC-1β in vitro and in vivo, negatively regulating PGC-1β abundance (14). To investigate ubiquitination of PGC-1β by Syvn1 in the presence of walnut extract, we performed an in vitro assay of ubiquitination with MBP-Syvn1ΔTM and GST-PGC-1β (1–367) in the presence of ATP, hemagglutinin-tagged ubiquitin (HA-Ub), E1, and E2 (UbcH5c) (14). As previously reported (14), Syvn1 induced polyubiquitination of PGC-1β in vitro, and polyubiquitination of PGC-1β was not observed in the absence of ATP or Syvn1. Walnut extract inhibited PGC-1β polyubiquitination (Fig. 3A). To examine the effect of walnut extract in vivo, we performed in vivo ubiquitination assay. FLAG-tagged Ub and HA-PGC-1β were coexpressed with Syvn1 in HEK 293T cells and cells were treated with DMSO or walnut extract (50 µg/ml) for 3 days. The ubiquitination of PGC-1β was observed in Syvn1-expressing cells (DMSO-treated cells). The treatment with walnut extract decreased the ubiquitination of PGC-1β in Syvn1-expressing cells (Fig. 3B). Walnut extract did not inhibit autoubiquitination of Syvn1 (Fig. 3C). The effect of walnut extract was also examined in vivo. FLAG-tagged Ub (FLAG Ub) and Syvn1/HA were coexpressed in HEK 293T cells and cells were treated with DMSO or walnut extract (50 µg/ml) for 3 days. Autoubiquitination of Syvn1 was not inhibited in walnut extract-treated cells (Fig. 3D).

PGC-1β is a coactivator of several transcription factors, including PPARα, and is implicated in various biological processes, including mitochondrial biogenesis (21,22). LS-102 exposure increases the number of mitochondria in cultured cells (14). To investigate the effect of walnut extract on regulation of mitochondria by PGC-1β, we performed mitochondrial staining using MitoTracker with mouse embryonic fibroblasts (MEFs) (14). MitoTracker staining showed increased mitochondria in MEFs treated with walnut extract compared with MEFs treated with DMSO (Fig. 4A). We used electron microscopy and counted mitochondria. The cells treated with walnut extract had significantly more mitochondria than the cells treated with dimethyl sulfoxide (DMSO) did (Fig. 4B and C). In addition, the number of mitochondria in Syvn1 knockout MEFs treated with DMSO (KO+DMSO) increased compared to that in wildtype MEFs treated with DMSO. However, walnut extract produced no additional effect on the number of mitochondria in the Syvn1 KO MEFs (KO+Walnut extract). Furthermore, the expression of PGC-1β target genes, medium chain acyl-coenzyme A dehydrogenase (MCAD) and mitochondrial ATP synthase β subunit (ATP5b), was also induced in MEFs treated with walnut extract and in Syvn1 KO MEFs treated with DMSO (Fig. 4D). However, walnut extract produced no additional effect on the induction of MCAD and ATP5b in the Syvn1 KO MEFs (Fig. 4D).