Inhibitory Effect of a Novel Antirheumatic Drug T-614 on the IL-6-Induced RANKL/OPG, IL-17, and MMP-3 Expression in Synovial Fibroblasts from Rheumatoid Arthritis Patients

T-614 (also named as iguratimod), a novel antirheumatic drug, could attenuate joint inflammation and articular damage in rheumatoid arthritis (RA) patients, providing a new therapy for RA. Here, we tested the role T-614 on the IL-6-induced receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG), IL-17, and MMP-3 expression in synovial fibroblasts from rheumatoid arthritis (RASFs) patients. T-614 decreased RANKL expression and RANKL/OPG ratio in IL-6-induced RASFs. We confirmed this effect by a decrease of the mRNA and protein RANKL and mRNA RANKL/OPG in RASFs exposed in vitro to T-614 or MTX. Markedly decreased levels of IL-17, retinoid-related orphan receptor C (RORc), and MMP-3 mRNA expression were also observed in IL-6-induced RASFs in the presence of T-614 or MTX compared with those in its absence. Furthermore, T-614 blocked expression of p-ERK1/2 protein without affecting ERK1/2 expression, indicating that the way that T-614 regulated RANKL expression might be ERK1/2 pathway. Our results suggest that T-614 yields a strong improvement in arthritis via exact suppression of RANKL/OPG, IL-17, and MMP-3 expression in RASFs.


Introduction
Rheumatoid arthritis (RA) is a complicated autoimmune disease characterized by persistent synovitis and, thereby, bone erosion. Although the etiology is not fully understood, a combination of genetic and environmental risk factors contributes to breaching of the immune tolerance, playing an important role in RA pathogenesis. The goal of RA treatment is remission or low disease activity, ultimately slowing or preventing the progression of joint destruction.
T-614 (also named iguratimod), a novel disease-modifying antirheumatic drug, has been widely used in clinics in China and Japan. T-614 could inhibit the production of various inflammatory cytokines, including interleukin-1, interleukin-6, interleukin-8, and TNF and dramatically improve symptoms in RA patients [1] and collagen-induced arthritis (CIA) mice [2]. Moreover, T-614 could markedly suppress disease progression and bone erosion in CIA mice [3]; however, the exact mechanism remains unclear.
2 BioMed Research International degradation in RA [9]. MMP-3 is also involved in bone destruction in RA patients by providing space for RASFs to invade through removing the extracellular matrix [10]. Here, we study the effect of T-614 on the RANKL, OPG, IL-17, and MMP-3 expression in a simulative inflammatory context by RASFs stimulated with IL-6 and conducted a comparative analysis of the antiarthritic of T-614 and MTX, the classic disease-modifying antirheumatic drugs (DMARDs) used for RA therapy.

Cell Culture and Reagents.
Synovial tissues were obtained from active RA patients who were undergoing surgery for knee replacement surgery. All RA patients fulfilled the American College of Rheumatology revised criteria for the diagnosis of RA [11]. Synovial tissues were harvested and incubated with collagenase type I (1 mg/mL) for 2 hours at 37 ∘ C. After digestion, RASFs were washed extensively and cultured in DMEM supplemented with 10% FCS in a humidified 5% CO 2 atmosphere. After overnight culture, nonadherent cells were removed, and adherent cells were cultured in DMEM supplemented with 10% FCS. RASFs were used between passages 3 and 7.  2.6. Statistical Analysis. Data are presented as mean ± SD. Statistical analysis was performed using ANOVA test. A probability < 0.05 was defined as significant.

RASFs Cultured In Vitro.
A homogeneous population (<1% CD11b, <2.5% CD14+, <1%CD3+, and <1% CD19+) was identified as RASFs [10]. A majority of RASFs at the first or second generation were polygonal or fusiform, with prominent nucleoli and more mitotic figures. The morphology of RASFs at the fourth generation was displayed in Figure 1, which was used in the next experiments.

Cell Viability Assay.
To test the effect of T-614 on cell viability, RASFs were treated with different concentrations of T-614 (0, 20, 50, 100, and 150 g/mL). After 72 h, CCK-8 was added to cells to assay cell viability. As shown in Figure 2, T-614 could inhibit the cell ability in a dose dependent manner; only the concentration of T-614 less than 20 g/mL did not markedly affect the growth of RASFs and was then used in current study.

T-614 Inhibited RANKL/OPG Expression in IL-6-Stimulated RASFs.
In the pathological condition of RA, RASFs in the inflamed joints could cause the exaggerated expression of multiple proinflammatory cytokines including TNF-, IL-6, IL-17, and IL-1 , all of which resulted in an increased RANKL expression in local joint. Given that previous studies reported that only IL-1 or IL-6 plus sIL-6R (but not IL-6 alone) is capable of inducing RANKL expression in vitro after RASFs treatment with different cytokines [12], thus, we stimulated RASFs with IL-6/sIL-6R to induce RANKL expression in current study. As expected, a robust increased RANKL and a modest decreased OPG expression were observed in RASFs upon IL-6 stimulation (Figures 3(a), 3(b), 3(c), and 3(d)).

T-614 Inhibited RANKL Expression via ERK1/2 Signal.
It is reported that IL-6 exerted its action via JAK/STAT (Janus kinase/signal transducer and activator of transcription) and MAPK (mitogen-activated protein kinase) cascade by binding to its receptors [15]. Previous studies also showed that activation of ERK was necessary for RANKL expression in RASFs [5,16,17]. To investigate the mechanism of T-614 on suppression of RANKL expression in IL-6-stimulated RASFs, we tested the p-ERK1/2 and ERK1/2 protein expression in RASFs upon IL-6 and T-614 stimulation. The western blot suggested that IL-6 could induce ERK1/2 phosphorylation in RASFs. The peak of ERK1/2 phosphorylation was at 1 h after the addition of IL-6 into cells ( Figure 5(a)). T-614 and MTX could markedly decrease the phosphorylation of p-ERK1/2 ( < 0.05; Figures 5(b) and 5(c)), indicating that the inhibitory role of T-614 on RANKL protein expression might be through ERK1/2 pathway.

Discussions
The novel disease-modifying antirheumatic drug, T-614, has a reported effect on preventing the inflammatory and destructive processes of RA by inhibiting the production of immunoglobulins and various inflammatory cytokines (IL-1, IL-6, IL-8, and TNF-), exerting a unique mechanism for RA therapy [18]. Here, we confirmed the anti-inflammatory role of T-614; moreover, our data suggested that T-614 could downregulate the ratio of RANKL/OPG via blocking ERK activation, suggesting a novel mechanism of T-614 on inhibition of bone destruction in RA.  Osteoclast is the cell ultimately responsible for destruction in RA. A mass of evidences indicates that RANKL plays an important role in regulating osteoclast development [19]. RANKL is known to be produced by a number of different cell types including T cells, B cells, dendritic cells, macrophages, and synovial fibroblasts in RA [20][21][22]. OPG acts as its natural decoy receptor by blocking the RANK/RANKL interaction and the elevated ratio of RANKL/OPG may represent a high state of osteoclastogenesis and high activity of bone degradation. Given that RASFs were one of the major cells that express RANKL [23], here, RASFs were stimulated with IL-6 to induce RANKL expression. We found, for the first time, that T-614 has a potent ability to decrease the ratio of RANKL/OPG, which suggested that the therapeutic effect of T-614 on preventing disease progression, at least in part, attributed to its regulation role on RANKL/OPG axis hence contributing to suppress osteoclastogenesis [24].
IL-17, also produced by RASFs [7], plays a crucial role in inflammation and bone erosion in RA patients and in CIA mice [12,13]. IL-17 could also upregulate the expression of RANKL in osteoblasts and synovial fibroblasts and then amplify the bone destruction in collagen-induced arthritis (CIA) mice. RORc is the transcription factor for IL-17. Consistent with recent report that T-614 could suppress IL-17 signal in RASFs [5], our data proved that T-614 could significantly downregulate the production of IL-17 and RORc in IL-6-induced RASFs. Moreover, T-614 showed a greater inhibitory effect on IL-17 and RORc expression than MTX.
MMP-3 has been suggested to play a pivotal role in the cartilage destruction in RA. Patients with joint injury have been found to have persistently increased proMMP-1 and proMMP-3 levels in synovial fluid, which mainly came from RASFs [25]. Serum MMP3 was correlated with IL-8, IL-6, IFN-, CRP and cartilage breakdown in 128 patients [26] and have been suggested to be a good laboratory index to evaluate the joint injury status and therapeutic effect [27]. In our study, IL-6 increased the production of MMP-3 in RASFs, confirming that inflammatory environment of joints was the major cause of high expression of MMP-3. Here, our data demonstrated that T-614 could decrease MMP-3 production in IL-6-induced RASFs, implying that T-614 could reduce disease activity and bone erosion of RA. Substantial evidence has suggested that ERK phosphorylation was involved in RANKL [5,16,17], IL-17 [28], and MMP-3 [29] expression in RASFs. In order to investigate the potential pathway of T-614 on RAKL/OPG expression in RASFs, we studied the effect of T-614 on ERK1/2 signaling. Our data showed that the phosphorylation of ERK1/2 was triggered after the stimulation of RASFs by IL-6 and reached peak at 1 h after stimulation with IL-6. The level of ERK1/2 phosphorylation could be abolished by T-614 and MTX. The high expression of RANKL protein in IL-6-induced RASFs could be cancelled by ERK inhibitor, PD98059, confirming that T-614 inhibited RANKL expression via ERK1/2 signal. Taken together, our data suggested that T-614 could inhibit RANKL expression in RASFs via ERK1/2 pathway.

Conclusions
T-614 has been confirmed as a highly effective drug for RA therapy and has been widely used in clinics in China and Japan. The present study demonstrates, for the first time, that T-614 could decrease the RANKL expression and downregulate the ratio of RANKL/OPG in RASFs via blocking ERK1/2 phosphorylation; we also confirmed that T-614 could suppress IL-17 and MMP-3 expression in IL-6-induced RASFs. This study also identified that the immunosuppressive effects in RANKL and IL-17 expression of T-614 on RASFs were stronger than MTX; our data provide a novel insight into the mechanisms of antiarthritic effect in T-614. Tumor necrosis factor CIA: Collagen-induced arthritis sIL-6R: Soluble IL-6 receptor.