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Available online at www.sciencedirect.com

Original article

Inhibition of glycogen synthase kinase-3␤ suppresses inflammatory responses in rheumatoid arthritis fibroblast-like synoviocytes and collagen-induced arthritis Yong-Jin Kwon a , Chong-Hyeon Yoon b , Sang-Won Lee a , Yong-Beom Park a , Soo-Kon Lee a , Min-Chan Park a,∗ a b

Division of Rheumatology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea Division of Rheumatology, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, South Korea

a r t i c l e

i n f o

Article history: Accepted 12 September 2013 Available online xxx Keywords: Rheumatoid arthritis Glycogen synthase kinase-3␤ Fibroblast-like synoviocytes Collagen-induced arthritis

a b s t r a c t Objectives: Glycogen synthase kinase (GSK)-3␤, a serine/threonine protein kinase, has been implicated as a regulator of the inflammatory response. This study was performed to evaluate the effect of selective GSK-3␤ inhibitors in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and collagen-induced arthritis (CIA). Method: FLS from RA patients were treated with selective GSK-3␤ inhibitors, including lithium chloride, 6-bromoindirubin-3 -oxime (BIO), or 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8). The effects of GSK-3␤ inhibition on pro-inflammatory mediators were determined by real-time PCR and ELISA. The levels of NF-␬B, phosphorylated JNK, c-jun, ATF-2 and p-38 proteins were evaluated by western blot analysis. The in vivo effects of GSK-3␤ inhibitors were examined in mice with CIA. Results: Treatment of RA FLS with GSK-3␤ inhibitors induced dose-dependent reductions in gene expression and the production of pro-inflammatory mediators. The levels of NF-␬B, phosphorylated JNK, c-jun, ATF-2 and p-38 were decreased following treatment with GSK-3␤ inhibitors. GSK-3␤ inhibitors treatment attenuated clinical and histological severities of CIA in mice. Infiltration of T-cells, macrophages, and tartrate-resistant acid phosphatase positive cells was decreased in joint sections of CIA mice by GSK3␤ inhibitors treatment. Serum levels of IL-1␤, IL-6, TNF-␣ and IFN-␥ in CIA mice were also significantly decreased in dose-dependent manners by treatment with GSK-3␤ inhibitors. Conclusion: Treatment with GSK-3␤ inhibitors suppressed inflammatory responses in RA FLS and CIA mice. These findings suggest that the inhibition of GSK-3␤ can be used as an effective therapeutic agent for RA. © 2013 Published by Elsevier Masson SAS on behalf of the Société Française de Rhumatologie.

1. Introduction Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial proliferation, leading to cartilage damage and joint destruction. A variety of transcriptional factors play a role in these processes. Nuclear factor-kappa B (NF-␬B), which is overexpressed in RA synovium, regulates the expression of various pro-inflammatory mediators, including tumor necrosis factor (TNF)-␣, interleukin (IL)-1␤, IL-6, and interferon (IFN)-␥, as well as cellular adhesion molecules [1–3]. Mitogen-activated protein kinases (MAPKs), including c-jun Nterminal kinase (JNK) and p38 also play significant pathogenic roles in RA. JNK MAPK regulates the production of matrix

∗ Corresponding author. Department of Internal Medicine, Yonsei University College of Medicine, Gangnam Severance Hospital, 211 Eonjuro, Gangnam-gu, Seoul 135-720, South Korea. Tel.: +82 2 2019 3310; fax: +82 2 2019 3508. E-mail address: [email protected] (M.-C. Park).

metalloproteinase (MMP) by synovial fibroblasts and drives osteoclast differentiation in RA [4–6]. p38 MAPK regulates IL-1stimulated the production of IL-6 and IL-8 and the induction of cyclooxygenase (COX)-2 and MMP-1 and -3 in fibroblasts [7,8]. p38 MAPK also regulates osteoclast differentiation by downregulating tartrate-resistant acid phosphatase (TRAP) mRNA levels [9]. Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine kinase known as a regulator of glycogen metabolism. Although GSK-3 was originally identified based on its ability to inactivate glycogen synthase and modulate blood glucose levels [10,11], recent studies have shown that this kinase participates in numerous and diverse biological functions, including signaling from the cell membrane to nucleus, gene transcription, translation, cytoskeletal organization, cell cycle progression, and survival [12–14]. GSK-3 exists in two isoforms in mammalian cells; GSK-3␣ and GSK-3␤, which are encoded by different genes and share 97% similar sequence within their catalytic domains but differ from one another outside this region, with GSK-3␣ possessing an extended N-terminal tail [15].

1297-319X/$ – see front matter © 2013 Published by Elsevier Masson SAS on behalf of the Société Française de Rhumatologie. doi:10.1016/j.jbspin.2013.09.006

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Recently, GSK-3␤ has emerged as an important regulator of numerous signaling pathways and is involved in a wide range of cellular processes ranging from glycogen metabolism to the regulation of cell survival, angiogenesis, and inflammatory responses. Pharmacologic inhibition of GSK-3␤ activity suppresses the production of pro-inflammatory cytokines [16] and reduces organ damages caused by endotoxemia [17]. GSK-3␤ inhibition also suppresses the development of experimental colitis [18], lung injury [19], bronchial asthma [20], and non-septic shock in mice [21]. Although the mechanisms underlying the anti-inflammatory effects of GSK3␤ inhibition are not fully understood, several studies have shown that NF-␬B can serve as substrate for GSK-3␤ and it has been shown that GSK-3␤ is involved in multiple steps of events, leading to the activation of NF-␬B, including the prevention of I␬B degradation, the inhibition of p65 nuclear translocation, and the prevention of p65 phosphorylation [22–26]. Consequentially, the inhibition of GSK-3␤ activity leads to the suppression of NF-␬B activation, in turn, resulting in potent anti-inflammatory effects. Synovial inflammation observed in RA is strongly associated with various pro-inflammatory mediators and transcriptional factors, which have been shown to be associated with GSK-3␤ activity; however, studies on the role of GSK-3␤ in the pathophysiology of RA are lacking. The present study investigated the effects of well-known selective inhibitors of GSK-3␤, such as lithium chloride [27,28], 6-bromoindirubin-3 -oxime (BIO) [16,29,30], and 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) [17–20,31], on inflammatory responses in FLS from patients with RA and collagen-induced arthritis (CIA) in mice.

2. Methods 2.1. Isolation and culture of RA FLS Synovial tissues were obtained from patients with RA who fulfilled the 1987 revised criteria of the American College of Rheumatology [32] during total knee replacement surgery or arthroscopic synovectomy. Tissues were digested with 4 mg/mL of collagenase II (Sigma, St Louis, MO) in serum-free Dulbecco’s modified Eagle’s medium (DMEM) for at least 4 h at 37 ◦ C. Cell suspensions were passed through a nylon mesh, and FLS were then collected by centrifugation at 800 × g for 5 min and were resuspended in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY). Harvested cells were cultured in 75 cm2 culture flasks (Costar, Cambridge, MA) with DMEM supplemented with 1% penicillin/streptomycin and 10% FBS at 37 ◦ C in a humidified atmosphere of 5% CO2 . When the cells had grown to confluence they were detached with 0.25% trypsin, split at a ratio of 1:3, and re-cultured in DMEM under the same conditions. Then, FLS were cultured in DMEM supplemented with 10% FBS. Cells obtained from the 4th to 6th passages were used in experiments. The use of patient biological samples and experimental procedures for this study was approved by the Ethics Committee of Gangnam Severance Hospital, Seoul, South Korea, and all study subjects provided their signed informed consents.

2.2. Antibodies and reagents Antibodies and reagents used were as follows: NF-␬B, phosphorylated JNK, c-Jun, ATF-2 and p-38 for western blotting (Cell Signaling Technology, Beverly, MA); mouse antibodies against ␤actin (Santa Cruz Biotechnology, Santa Cruz, CA); recombinant human IL-1␤, IL-6, chemokine ligand (CCL)-2, CCL-7, COX-2, and MMP-9 (R&D Systems, Minneapolis, MN); recombinant mouse TNF-␣, IL-1␤, IL-6, and IFN-␥ (Santa Cruz Biotechnology); lithium

chloride and BIO (Molecular Biology Grade, Merck, Germany); TDZD-8 (Sigma). 2.3. Assessment of cell viability Cultured RA FLS were plated in triplicates at 2 × 104 cells per well in 96-well plates and were allowed to adhere for 24 h. Media were then replaced by media containing either DMSO or various concentrations of lithium chloride (1, 5, 10, 20, 50 mM), BIO (1, 10, 50, 100, 200 nM), or TDZD-8 (1, 5, 10, 20, 50 ␮M). The doses of each GSK-3␤ inhibitors were chosen based on the methods of previous studies [27–31]. After 24 and 48 h of incubation, the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma) was performed according to the manufacturer’s instructions to assess the effect of selective GSK-3␤ inhibitors on cell viability. The absorbance was read at 570/690 nm by SpectraMax 340 (Molecular Devices Co.). Based on the MTT assay results, the concentrations of GSK-3␤ inhibitors that do not adversely affect cell viability were employed in the subsequent experiments. 2.4. RNA isolation and reverse transcriptase-polymerase chain reaction (PCR) RA FLS were treated with lithium chloride (1, 5, 10 mM), BIO (1, 10, 50 nM), TDZD-8 (1, 5, 20 ␮M) or the DMSO vehicle for 24 h, and with TNF-␣ (10 ng/mL) where indicated. Total RNA from RA FLS was isolated using TRIzol and treated with DNase I (Invitrogen, Carlsbad, CA) and the concentration was determined with a NanoDrop (R&D Systems). Total RNA was reverse-transcribed into complimentary DNA (cDNA) using of TaKaRa Kit (Takara Korea Biomedical, Seoul, South Korea). The reaction mixture included MgCl2 , reverse transcriptase buffer, diethyl pyrocarbonate-treated water, Oligo dT, AMV, inhibitor, and sample RNA. The amplifications were accomplished by following condition: 42 ◦ C for 30 min, 95 ◦ C for 5 min, and 4 ◦ C to the end. PCR products were electrophoresed on a 2% agarose gels and stained with ethidium bromide. 2.5. Quantitative real-time PCR Real-time PCR was performed using 1 ␮L complementary DNA (cDNA) per well, TaqMan Master Mix (Applied Biosystems, Foster City, CA), and 250 nM each of sense and antisense primers. Primers and probes were used to detect IL-1␤ (Hs00174087 m1), IL-6 (Hs00985641 m1), CCL-2 (Hs00234140 m1), CCL-7 (Hs00171147 m1), COX-2 (Hs00153133 m1), MMP-9 (Hs00234579 m1), and ␤-actin (Hs99999903 m1). All the samples were run in duplicate and the results were evaluated using the CT method and the calculated number of copies was normalized to the number of beta actin mRNA copies in the same sample. 2.6. Western blot analysis After stimulation with 10 ng/mL of TNF-␣, RA FLS (70–80% confluency) were incubated with lithium chloride (10 mM), BIO (50 nM), or TDZD-8 (20 ␮M) for 24 h. Supernatants were collected and concentrated with Microcon YM-10 columns (Millipore, Bedford, MA). The same amount of total proteins (5 ␮g) was loaded into 3–8% SDS–polyacrylamide gels (Invitrogen) and electrophoresed under reducing conditions. Proteins were transferred to a PVDF membrane (Millipore). Membranes were blocked with 5% non-fat milk overnight at 4 ◦ C, then, incubated with each primary antibody (anti-NF-␬B, anti-phosphorylated JNK, anti-c-jun, antiATF-2, and anti-phosphorylated p38) in PBS with 0.1% Tween 20 for 1 h at room temperature. HRP-conjugated goat anti-mouse IgG

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was used as the secondary antibody and incubated for 1 h at room temperature. Immunoreactive protein bands were detected by enhanced chemiluminescence (ECL Plus; Amersham Biosciences, Piscataway, NJ) and visualized using Kodak X-OMAT films.

GA). All cells per high-power field (HPF) were counted, and the percentage of positively stained cells was determined. Ten representative HPFs per paw were evaluated.

2.7. Induction of collagen-induced arthritis (CIA) and treatment

2.10. Enzyme-linked immunosorbent assay (ELISA)

All animal procedures were conducted in accordance with the Institutional Animal Care and Use Committee of Yonsei University College of Medicine guidelines. Male DBA/1 J mice (6–8 weeks of age) were purchased from SLRC Laboratory Animals (Shanghai, China). Bovine type II collagen (CII; Sigma) was dissolved to a concentration of 2 mg/mL in 0.01 M acetic acid at 4 ◦ C, with constant overnight mixing. Complete Freund’s adjuvant (CFA) was prepared by the addition of Mycobacterium tuberculosis H37 Ra (Difco, Detroit, MI) at 5 mg/mL. CII was emulsified with an equal volume of CFA. Mice were immunized intradermally at the base of the tail with 100 ␮L of emulsion on day 1 and day 21. Mice were subsequently injected intraperitoneally with lithium chloride (5 and 20 mg/kg), BIO (1 and 10 mg/kg), TDZD-8 (1 and 10 mg/kg) or vehicle (10% DMSO) once daily between day 24 and day 44 post-immunization. The doses of each GSK-3␤ inhibitors for in vivo experiment were chosen based on the methods of previous studies [19,21,28]. Sham-treated mice were treated on day 1 and day 21 with 100 ␮L of 0.01 M acetic acid. Sham-treated mice were also injected intraperitoneally with same doses of lithium chloride, BIO, TDZD-8, or vehicle once daily between day 24 and day 44 post-immunization. Ten mice were assigned to each group. Mice were sacrificed on day 46; the hind paws and knee joints were removed for histological examination, and the serum was saved for quantification of cytokines, CII-specific antibody titer, and toxicity.

The concentrations of pro-inflammatory mediators, including IL-1␤, IL-6, CCL-2, CCL-7, COX-2, and MMP-9, in culture supernatants of RA FLS and serum concentrations of IgG anti-CII antibodies and pro-inflammatory cytokines, including TNF-␣, IL1␤, and IL-6, in CIA mice were determined using commercially available ELISA kits (R&D Systems Inc.) according to the manufacturers’ protocol.

2.8. Clinical and histological assessment of CIA An investigator blinded to the treatment regimen performed all the clinical examinations. The following grading system was used: • • • • •

grade 0, no swelling; grade 1, slight swelling and erythema; grade 2, moderate swelling and edema; grade 3, extreme swelling and pronounced edema; grade 4, joint rigidity.

Mice were evaluated for arthritis on a daily basis and scores for all four paws were summed for each mouse, and the mean arthritis score for each group was calculated. For histological analysis, mice were anaesthetized and sacrificed 46 days after primary immunization. The hind paws and knee joints were fixed in 4% formalin, decalcified, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H–E) for detection of synovial inflammation. To determine the loss of proteoglycans from articular cartilage, toluidine blue staining was performed. Joint sections were also stained for tartrate-resistant acid phosphatase (TRAP) to detect osteoclasts and assess bone erosion. 2.9. Immunohistochemical analysis Different cell populations were analyzed in the synovial membrane by immunohistochemistry to detect infiltration of T-cells (anti-CD3) and macrophages (anti-F4/80). Briefly, hind paw sections were pretreated with citrate buffer, pH 6, for macrophages or proteinase K for T-cells. Subsequently, the sections were incubated with biotinylated species-specific secondary antibodies (Vector) and avidin-biotin-peroxidase complex, using 3, 3 -diaminobenzine as a chromogen. Positively stained cells were counted using a Nikon microscope and the OsteoMeasure system (OsteoMetrics, Decatur,

2.11. Statistical analysis The results are expressed as the mean ± SEM. For experiments involving histology or immunohistochemistry, the results are representative of at least three experiments performed on different days. Differences were assessed by one-way and two-way repeated-measures analysis of variance (ANOVA), followed by the Bonferroni post-hoc test for comparison of multiple groups or Mann–Whitney U-test for comparisons of arthritis index medians. P values less than 0.05 were considered statistically significant.

3. Results 3.1. Effects of GSK-3ˇ inhibitors on cell viability of RA FLS To investigate the effect of GSK-3␤ inhibitor treatments on cell viability, RA FLS were incubated with varying concentrations of lithium chloride, BIO, and TDZD-8 for 24 and 48 h. As shown in Fig. 1, treatment with lithium chloride (1, 5, 10 mM), BIO (1, 10 nM), and TDZD-8 (1, 5, 20 ␮M) showed no significant suppression of cell viability, compared with vehicle-treated control cells. Based on these results, we employed GSK-3␤ inhibitor concentrations that did not adversely affect cell viability during given incubation times for subsequent experiments.

3.2. Effects of GSK-3ˇ inhibitors on pro-inflammatory mediators in RA FLS To examine the effects of GSK-3␤ inhibition on inflammatory responses in RA, we first examined the expression of various proinflammatory genes after treatment of RA FLS with lithium chloride (1, 5, 10 mM), BIO (1, 10, 50 nM), or TDZD-8 (1, 5, 20 ␮M) or DMSO vehicle for 24 h with or without 10 ng/mL of TNF-␣ stimulation. The expressions of IL-1␤, IL-6, CCL-2, CCL-7, MMP-9, and COX-2 mRNA compared to those of ␤-actin were augmented in RA FLS by TNF-␣ stimulation. Treatment with GSK-3␤ inhibitors significantly suppressed mRNA expressions of IL-1␤, IL-6, CCL-2, CCL-7, COX-2, and MMP-9 in dose-dependent manners (Fig. 2A). We next performed ELISA to evaluate the effect of GSK-3␤ inhibitors on the levels of pro-inflammatory mediator productions in culture supernatants of RA FLS. As shown in Fig. 2B, all GSK-3␤ inhibitor treatments significantly decreased the productions of IL-6, CCL-2, and CCL-7 in RA FLS. Although some GSK-3␤ inhibitor treatments did not show statistically significant reductions in IL-1␤, COX-2, and MMP-9 levels, we could find decrements in their levels in RA FLS by GSK-3␤ inhibitor treatments. Taken together, these findings indicate anti-inflammatory activity of GSK3␤ inhibitors in RA FLS.

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Fig. 1. Effects of GSK-3␤ inhibitor treatments on cell viability of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). RA FLS were treated with varying concentrations of lithium chloride (A), BIO (B), TDZD-8 (C) or DMSO vehicle for 24 and 48 h, and cell viability was assessed by MTT assay. Values were expressed as % of control and means ± SD. Values are mean ± SEM of 3 independent experiments. * P < 0.05 versus control treated with vehicle.

Fig. 2. Effects of GSK-3␤ inhibitor treatments on pro-inflammatory mediators in RA FLS. A. RA FLS were treated with various concentrations of GSK-3␤ inhibitors and mRNA expressions of IFN-␥, IL-1␤, IL-6, CCL-2, CCL-7, COX-2 and MMP-9 were determined by real-time RT-PCR. B. The concentrations of IL-1␤, IL-6, CCL-2, CCL-7, COX-2, and MMP-9 in culture supernatant of RF FLS were determined using ELISA after treatment with lithium chloride (10 mM), BIO (50 nM), and TDZD-8 (20 ␮M) or DMSO. Data are shown as the mean ± SEM of 3 independent experiments. * P < 0.05 and ** P < 0.01 versus control treated with vehicle.

3.3. Effects of GSK-3ˇ inhibitors on NF-B, JNK and p38 signaling pathways After stimulation with TNF-␣, the effect of GSK-3␤ inhibitor treatment on NF-␬B and JNK signaling was assessed by examining the levels of NF-␬B and phosphorylated forms of JNK, c-jun, ATF-2, and p-38 by western blotting. The levels of NF-␬B, phosphorylated forms of JNK, c-jun, ATF-2, and p-38 were reduced by treatment with lithium chloride, BIO, and TDZD-8 (Fig. 3).

3.4. Effect of GSK-3ˇ inhibitors on the severity of CIA CIA developed rapidly in mice immunized with CII. Clinical signs of the disease including periarticular erythema and edema first appeared in the hind paws between 22 and 24 days after CII immunization, and the incidence of CIA was 100% by day 33. Erythema and swelling in the hind paws increased in frequency and severity over time, reaching maximum arthritis indices between 33 and 35 days after CII immunization in the vehicle-treated mice. Daily intraperitoneal injection of lithium chloride, BIO, or TDZD-8 decreased the clinical severity of CIA through day 44 (Fig. 4).

Fig. 3. Effect of GSK-3␤ inhibitor treatments on NF-␬B, phosphorylated JNK, c-jun, ATF-2 and p-38 protein productions in RA FLS. RA FLS were treated with lithium chloride, BIO and TDZD-8 for 24 h and western blot analysis was performed to assess the levels of NF-␬B, phosphorylated form of JNK, c-jun, ATF-2 and p-38 protein. NF␬B, phosphorylated JNK, c-jun, ATF-2 and p-38 were decreased in the RA FLS treated with GSK-3␤ inhibitors compared with control.

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Fig. 4. Effects of GSK-3␤ inhibitor treatments on the clinical severity of CIA. Severity of hind paws erythema and swelling increased as time passes. Treatments with lithium chloride, BIO, or TDZD-8 showed significant decreases in the arthritis score of CIA mice and these changes were in dose-dependent manners (* P < 0.05 versus CIA mice treated with vehicle). Values are means ± SEM of 10 animals per group.

Histological evaluation of paws from CII-immunized mice revealed extensive pannus formation with synovial hyperplasia, infiltration of inflammatory cells, cartilage destruction, and bone erosion. Treatment with GSK-3␤ inhibitors suppressed these pathological changes compared to vehicle-treated mice (Fig. 5A). Joint sections were stained with toluidine blue to assess cartilage damage and with TRAP to detect osteoclasts. As shown in Fig. 5B, unstained cartilage in mice treated with GSK-3␤ inhibitors indicated less proteoglycan loss from cartilage compared to vehicle-treated mice. In addition, joint sections from CIA mice treated with GSK-3␤ inhibitors showed significant reductions in TRAP-positive cells and bony erosions compared to those from vehicle-treated CIA mice (Fig. 5C).

Fig. 6. Effects of GSK-3␤ inhibitor treatment on serum levels of anti-CII IgG and pro-inflammatory cytokines in CIA mice. A. Levels of anti-CII IgG in serum of vehicletreated and GSK-3␤ inhibitors-treated mice. Serum samples were collected on day 46, anti-CII IgG was measured by ELISA. GSK-3␤ inhibitors treatment demonstrated a significant decrease in anti-CII IgG level. B. GSK-3␤ inhibitors-treated CIA mice showed significantly reduced serum TNF-␣, IL-1␤, IL-6 and IFN-␥ levels. Data are shown as the mean ± SEM of 3 independent experiments. * P < 0.05 and ** P < 0.01 versus control treated with vehicle.

Infiltration of T-cells and macrophages in joint secretions from CIA mice was determined by immunohistochemical staining for CD3 and F4/80, respectively. The numbers of T-cells and macrophages were decreased in CIA mice treated with GSK-3␤ inhibitors compared to vehicle-treated CIA mice (Fig. 5D and E). 3.5. Effect of GSK-3ˇ inhibitors on CII-specific immune responses and inflammatory cytokine productions in CIA mice Anti-CII IgG was not detected in serum from sham-treated mice, but levels of anti-CII IgG were markedly increased in mice with CIA. Treatment with lithium chloride, TDZD-8, or BIO resulted in significant decrease of anti-CII IgG levels in a dose-dependent manner (Fig. 6A). In addition, we also found significantly reduced levels of TNF-␣, IL-1␤, IL-6, and IFN-␥ in serum from CIA mice treated with GSK-3␤ inhibitors compared to vehicle-treated CIA mice (Fig. 6B). Furthermore, decreases in these cytokines were dependent on the dose of GSK-3␤ inhibitors administered. 4. Discussion

Fig. 5. Effects of GSK-3␤ inhibitor treatment on synovial inflammation and joint destruction in mice with CIA. A. Hematoxylin and erosion stained sections of hind paws from vehicle-treated and GSK-3␤ inhibitors-treated. Arrows in the photomicrograph from the mouse with vehicle-treated CIA indicate proliferating synovium with inflammation. B. Proteoglycan loss as determined using toluidine blue-stained sections. Proteoglycan-depleted areas (arrows) are unstained as compared to proteoglycan-rich dark blue-stained areas. C. Arthritic bone erosions were analyzed with TRAP stained sections. Osteoclasts appear as purple stained multinucleated cells. D. Immunohistochemical analysis of T-cells (CD3+ ) and (E) macrophages (F4/80+ ) in the inflamed synovuim of vehicle-treated and GSK-3␤ inhibitors-treated mice were performed. Positively labeled cells appear brown. Original magnification × 10.

GSK-3␤ has recently emerged as a key regulatory switch in the modulation of inflammatory responses, and the anti-inflammatory effects of GSK-3␤ inhibition have been shown in vitro and in several in vivo models of inflammation [16–21]. Most studies on the mechanisms underlying the anti-inflammatory effects of GSK-3␤ inhibition have focused on the interaction of GSK-3␤ with NF-␬B [11,12,22–26]. Hence, pharmacologic inhibition of GSK-3␤ activity can exert anti-inflammatory potentials by inhibiting NF-␬B transcriptional activity in many disorders, in which NF-␬B plays a crucial pathophysiological role. The present study herein was undertaken to elucidate the effects of GSK-3␤ inhibition on inflammatory responses of FLS from RA patients and an experimentally induced murine model of RA. We evaluated the effects of synthetic GSK-3␤ inhibitors on RA FLS and found that treatment with GSK3␤ inhibitors significantly suppressed various pro-inflammatory

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mediators, including pro-inflammatory cytokines, chemokines, COX-2, and MMP-9 in RA FLS. In RA, FLS and various inflammatory cells can produce proinflammatory cytokines, such as IL-1␤ and TNF-␣ [33]. In response to these cytokines, RA FLS regulate the synthesis of chemokines, MMPs, and COX-2, which further promote inflammatory responses and bone/cartilage destruction by altering the balance between extracellular matrix production and degradation. Many proinflammatory cytokines and MMPs are regulated by several transcriptional factors, including NF-␬B and MAPKs [1–9]. To clarify the underlying mechanisms of GSK-3␤ inhibition in RA FLS, we evaluated the effects of GSK-3␤ inhibitors on the production of several significant transcriptional factors of RA and found that treatment with GSK-3␤ inhibitors downregulated NF-␬B production in RA FLS. This result is consistent with previous results indicating that NF-␬B-dependent anti-inflammatory effects of GSK3␤ inhibition [11,12,14–16]. In addition, we also found that the suppression of JNK and p38 MAPK signalling pathways can be another possible mechanism responsible for anti-inflammatory actions mediated by GSK-3␤ inhibition in RA FLS. There are controversies on the role of GSK-3␤ in JNK and p38 activation. It has been showed that homozygous disruption of GSK-3␤ sensitizes mouse embryonic fibroblasts to JNK activation, induced by lysophosphatidic acid and sphingosine-1-phosphate [34]. On the other hand, GSK-3␤ has also been shown to be a positive regulator of TNFinduced activation of JNK [35], and inhibition of GSK-3␤ by lithium chloride has no effect on LPS-induced JNK activation in human monocytes [36]. These differences may reflect the use of different cell types. In the JNK pathway, a stimulus activates MAP3K members and these in turn phosphorylate MAP2K members, which then phosphorylate JNK. Kim et al. demonstrated that GSK-3␤ is linked to and activates MAP3Ks, which leads to the stimulation of the JNK pathway [37]. Wang et al. showed that GSK-3␤ inhibition abrogates LPS-induced activation of JNK in microglial cells [38]. A previous study showed that p38 MAPK directly inactivates GSK-3␤ by phosphorylating Ser-389 at the C terminus of GSK-3␤ [39], indicating that p38 is associated with the GSK-3␤ signalling pathway. The present study is the first to document a regulatory role of GSK-3␤ in JNK and p38 activation in RA FLS, suggesting that the suppression of inflammatory responses by GSK-3␤ inactivation is due, at least in part, to the inhibition of downstream signalling molecules JNK and p38 MAPK. Previously Cuzzocrea et al. demonstrated a therapeutic effect of GSK-3␤ inhibition in CIA mice. They found that treatment with TDZD-8 improves clinical signs and histological joint damage in CIA mice. Several markers for nitrosative insults and proinflammatory genes are significantly reduced in joint sections and plasma from CIA mice by TDZD-8 treatment [40]. On the basis of this anti-arthritic effect of GSK-3␤ inhibition, we also evaluated the therapeutic effects of lithium chloride, BIO, and TDZD-8 in CIA mice to further confirm the anti-inflammatory effect of GSK-3␤ inhibition observed in the present in vitro study. In accordance with this previous result by Cuzzocrea et al., we found that clinical and histologic severities of CIA were markedly attenuated following treatment with GSK-3␤ inhibitors. We also found that the levels of IgG antibody to CII and several pro-inflammatory mediators were significantly diminished in sera from CIA mice treated with GSK-3␤ inhibitors. Taken together, these results clearly demonstrate that GSK-3 is an important component of the inflammatory response and GSK-3 inhibition can represent a new co-adjuvant therapy in a number of conditions associated with rheumatoid inflammation. In conclusion, the present study showed dose-dependent reductions of various pro-inflammatory mediators in FLS from RA patients and attenuation of clinical and pathological manifestations of experimentally induced arthritis in CIA mice by treatment with GSK-3␤ inhibitors. These results might be at least in part due to the

downregulation of the NF-␬B signaling pathway, but the expression of active forms of JNK, c-jun, ATF-2, and p-38 were also downregulated in RA FLS upon treatment with GSK-3␤ inhibitors. Although the exact mechanisms underlying the anti-inflammatory effect of GSK-3␤ inhibition in RA still remain unclear, our data suggest that NF-␬B is not the sole target of GSK-3␤ activity in RA. Findings from the present in vitro and in vivo studies demonstrated a novel role for GSK-3␤ inhibition as a negative regulator in rheumatoid inflammation and support a potential role for GSK-3␤ inhibitors in the treatment of RA. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This study was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (NRF-20090064825). The authors are grateful to Tae-Yeon Kim, Gangnam Severance Hospital Biomedical Research Center, for technical assistance. References [1] Handel ML, McMorrow LB, Gravallese EM. Nuclear factor-kappa B in rheumatoid synovium. Localization of p50 and p65. Arthritis Rheum 1995;38:1762–70. [2] Gerlag DM, Ransone L, Tak PP, et al. The effect of a T cell-specific NF-kappa B inhibitor on in vitro cytokine production and collagen-induced arthritis. J Immunol 2000;165:1652–8. [3] Marok R, Winyard PG, Coumbe A, et al. Activation of the transcription factor nuclear factor-kappaB in human inflamed synovial tissue. Arthritis Rheum 1996;39:583–91. [4] Thalhamer T, McGrath MA, Harnett MM. MAPKs and their relevance to arthritis and inflammation. Rheumatology (Oxford) 2008;47:409–14. [5] Han Z, Boyle DL, Chang L, et al. c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J Clin Invest 2001;108:73–81. [6] David JP, Sabapathy K, Hoffmann O, et al. JNK1 modulates osteoclastogenesis through both c-Jun phosphorylation-dependent and independent mechanisms. J Cell Sci 2002;115:4317–25. [7] Suzuki M, Tetsuka T, Yoshida S, et al. The role of p38 mitogen-activated protein kinase in IL-6 and IL-8 production from the TNF-alpha- or IL-1beta-stimulated rheumatoid synovial fibroblasts. FEBS Lett 2000;465:23–7. [8] Westra J, Limburg PC, de Boer P, et al. Effects of RWJ 67657, a p38 mitogen-activated protein kinase (MAPK) inhibitor, on the production of inflammatory mediators by rheumatoid synovial fibroblasts. Ann Rheum Dis 2004;63:1453–9. [9] Matsumoto M, Sudo T, Saito T, et al. Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-kappa B ligand (RANKL). J Biol Chem 2000;275:31155–61. [10] Frame S, Cohen P. GSK-3 takes centre stage more than 20 years after its discovery. Biochem J 2001;359:1–16. [11] Van Wauwe J, Haefner B. Glycogen synthase kinase-3 as drug target: from wallflower to center of attention. Drug News Perspect 2003;16:557–65. [12] Martinez A. Preclinical efficacy on GSK-3 inhibitors: towards a future generation of powerful drugs. Med Res Rev 2008;28:773–96. [13] Forde JE, Dale TC. Glycogen synthase kinase-3: a key regulator of cellular fate. Cell Mol Life Sci 2007;64:1930–44. [14] Kockeritz L, Doble B, Patel S, et al. Glycogen synthase kinase-3 – an overview of an over-achieving protein kinase. Curr Drug Targets 2006;7:1377–88. [15] Wada A. GSK-3 inhibitors and insulin receptor signaling in health, disease, and therapeutics. Front Biosci 2009;14:1558–70. [16] Martin M, Rehani K, Jope RS, et al. Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase-3. Nat Immunol 2005;6:777–84. [17] Dugo L, Collin M, Allen DA, et al. GSK-3beta inhibitors attenuate the organ injury/dysfunction caused by endotoxemia in the rat. Crit Care Med 2005;33:1903–12. [18] Whittle BJ, Varga C, Posa A, et al. Reduction of experimental colitis in the rat by inhibitors of glycogen synthase kinase-3beta. Br J Pharmacol 2006;147:575–82. [19] Cuzzocrea S, Crisafulli C, Mazzon E, et al. Inhibition of glycogen synthase kinase3beta attenuates the development of carrageenan-induced lung injury in mice. Br J Pharmacol 2006;149:687–702.

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Please cite this article in press as: Kwon Y-J, et al. Inhibition of glycogen synthase kinase-3␤ suppresses inflammatory responses in rheumatoid arthritis fibroblast-like synoviocytes and collagen-induced arthritis. Joint Bone Spine (2013), doi:10.1016/j.jbspin.2013.09.006