miR-483-3p Promotes IL-33 Production from Fibroblast-Like Synoviocytes by Regulating ERK Signaling in Rheumatoid Arthritis.

Our previous studies have identified miR-483-3p to be highly expressed in synoviocytes from patients with rheumatoid arhtirits (RA); however, its effects on inflammation of RA fibroblast-like synoviocytes (FLSs) have remained unclear. The expression of miR-483-3p and cytokines in RA FLSs was detected using quantitative real-time polymerase chain reaction. Enzyme-linked immunosorbent was conducted to determine interleukin (IL)-33 production from RA FLSs. Western blotting was employed to quantify the levels of p-ERK and total ERK. Overexpressed miR-483-3p significantly increased the mRNA and protein expression of IL-33, but not of IL-27 or IL-34, in RA FLSs, whereas miR-483-3p suppression showed the opposite effects. Furthermore, miR-483-3p upregulation activated the ERK signaling pathway. The ERK signaling inhibitor PD98059 partly reversed the elevation of IL-33 levels mediated by miR-483-3p overexpression. Our results reveal that miR-483-3p promotes IL-33 expression by regulating the ERK signaling pathway in RA FLSs. Thus, miR-483-3p may be a potential effective target for RA treatment.

USP13 mediates PTEN to ameliorate osteoarthritis by restraining oxidative stress, apoptosis and inflammation via AKT-dependent manner.

Osteoarthritis is a chronic, systemic and inflammatory disease. However, the pathogenesis and understanding of RA are still limited. Ubiquitin-specific protease 13 (USP13) belongs to the deubiquitinating enzyme (DUB) superfamily, and has been implicated in various cellular events. Nevertheless, its potential on RA progression has little to be investigated. In the present study, we found that USP13 expression was markedly up-regulated in synovial tissue samples from patients with RA, and was down-regulated in human fibroblast-like synoviocytes (H-FLSs) stimulated by interleukin-1ß (IL-1ß), tumor necrosis factor alpha (TNF-α) or lipopolysaccharide (LPS). We then showed that over-expressing USP13 markedly suppressed inflammatory response, oxidative stress and apoptosis in H-FLSs upon IL-1ß or TNF-α challenge, whereas USP13 knockdown exhibited detrimental effects. In addition, USP13-induced protective effects were associated with the improvement of nuclear factor erythroid 2-related factor 2 (Nrf-2) and the repression of Casapse-3. Furthermore, phosphatase and tensin homolog (PTEN) expression was greatly improved by USP13 in H-FLSs upon IL-1ß or TNF-α treatment, whereas phosphorylated AKT expression was diminished. In response to IL-1ß or TNF-α exposure, nuclear transcription factor κB (NF-κB) signaling pathway was activated, whereas being significantly restrained in H-FLSs over-expressing USP13. Mechanistically, USP13 directly interacted with PTEN. Of note, we found that USP13-regulated cellular processes including inflammation, oxidative stress and apoptotic cell death were partly dependent on AKT activation. Furthermore, USP13 over-expression effectively inhibited osteoclastogenesis and osteoclast-associated gene expression. The in vivo experiments finally confirmed that USP13 dramatically repressed synovial hyperplasia, inflammatory cell infiltration, cartilage damage and bone loss in collagen-induced arthritis (CIA) mice via the same molecular mechanisms detected in vitro. Taken together, these findings suggested that targeting USP13 may provide feasible therapies for RA.

Diosgenin Inhibits Excessive Proliferation and Inflammatory Response of Synovial Fibroblasts in Rheumatoid Arthritis by Targeting PDE3B.

Rheumatoid arthritis (RA) is a chronic inflammation that can lead to loss of range of joint abnormalities in severe cases. Diosgenin has anti-inflammatory effects. This paper discussed the effect and mechanism of diosgenin on excessive proliferation and inflammatory response of synovial cells in RA. CCK-8 detected the cell viability, TUNEL assay detected the apoptosis of cells and western blot detected the expression of apoptosis-related proteins. Wound healing was used to detect cell migration and western blot detected the expression of migration-related proteins. ELISA kits were used to detect the levels of inflammatory cytokines in cells. Diosgenin can inhibit the proliferation and migration of RA synovial cells. At the same time, diosgenin could reduce the inflammatory response of RA synovial cells, during which the expression of PDE3B was significantly decreased. By overexpressing PDE3B, we found that diosgenin inhibited the proliferation, migration, and inflammatory response of RA synovial cells by downregulating PDE3B. Diosgenin can inhibit excessive proliferation and inflammatory response of synovial fibroblasts by targeting PDE3B.

Long noncoding RNA ZFAS1 silencing alleviates rheumatoid arthritis via blocking miR-296-5p-mediated down-regulation of MMP-15.

Rheumatoid arthritis (RA), a chronic inflammatory disease, deprives patients' walking ability and reduces their life quality worldwide. Though recent studies have indicated the role of long noncoding RNA (lncRNA) ZFAS1 in several diseases, however, its role in RA remains uncharacterized. The present study aimed to unravel the the effect of ZFAS1 on RA. Herein, the RA mouse model and the human RA synoviocyte MH7A cell lines stimulated with TNF-α were established. ZFAS1 was next determined to be highly expressed in the mice with RA-like symptoms and TNF-α-stimulated MH7A cells while inhibiting ZFAS1 was demonstrated to promote proliferation and suppress apoptosis of MH7A cells. Furthermore, ZFAS1 knockdown exerted anti-inflammation effect in vitro and in vivo and reduced the arthritis index value. Moreover, RNA immunoprecipitation and dual-luciferase reporter assays identified the binding of ZFAS1 to microRNA (miR)-296-5p as well as the binding of miR-296-5p to matrix metalloproteinase-15 (MMP-15). Of note, ZFAS1 could bind miR-296-5p to up-regulate the expression of MMP-15. Our results from in vitro and in vivo experiments demonstrated silencing ZFAS1 mitigated RA-like symptoms such as inflammation and hyperplasia via miR-296-5p-dependent inhibition of MMP-15. Taken altogether, our study confirmed that ZFAS1 involved in RA progression by competitively binding to miR-296-5p and regulating MMP-15 expression.

Lnc RNA ZFAS1 regulates the proliferation, apoptosis, inflammatory response and autophagy of fibroblast-like synoviocytes via miR-2682-5p/ADAMTS9 axis in rheumatoid arthritis.

BACKGROUNDS: Rheumatoid arthritis (RA) is a frequent autoimmune disease. Emerging evidence indicated that ZNFX1 antisense RNA1 (ZFAS1) participates in the physiological and pathological processes in RA. However, knowledge of ZFAS1 in RA is limited, the potential work pathway of ZFAS1 needs to be further investigated. METHODS: Levels of ZFAS1, microRNA (miR)-2682-5p, and ADAM metallopeptidase with thrombospondin type 1 motif 9 (ADAMTS9) were estimated using quantitative real-time polymerase chain reaction (qRT-PCR) assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to explore the ability of cell proliferation in fibroblast-like synoviocytes (FLS-RA). Cell apoptosis was measured via flow cytometry. Also, levels of ADAMTS9, apoptosis-related proteins, cleaved-caspase-3 (active large subunit), and autophagy-related proteins were identified adopting Western blot. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the productions of inflammatory cytokines. Beside, the interrelation between miR-2682-5p and ZFAS1 or ADAMTS9 was verified utilizing dual-luciferase reporter assay. RESULTS: High levels of ZFAS1 and ADAMTS9, and a low level of miR-2682-5p were observed in RA synovial tissues and FLS-RA. Knockdown of ZFAS1 led to the curbs of cell proliferation, inflammation, autophagy, and boost apoptosis in FLS-RA, while these effects were abolished via regaining miR-2682-5p inhibition. Additionally, the influence of miR-2682-5p on cell phenotypes and inflammatory response were eliminated by ADAMTS9 up-regulation in FLS-RA. Mechanically, ZFAS1 exerted its role through miR-2682-5p/ADAMTS9 axis in RA. CONCLUSION: ZFAS1/miR-2682-5p/ADAMTS9 axis could modulate the cell behaviors, inflammatory response in FLS-RA, might provide a potential therapeutic target for RA treatment.

MiR-1193 represses the proliferation and induces the apoptosis of interleukin-1ß-treated fibroblast-like synoviocytes via targeting JAK3.

OBJECTIVES: To explore the roles of miR-1193/Janus kinase 3 (JAK3) axis and the potential mechanism in rheumatoid arthritis (RA). METHODS: The dysregulated genes and microRNAs (miRNAs) were screened using the datasets of GSE12021 and GSE72564, while the upstream miRNA of JAK3 was forecasted by TargetScan. Then the MH7A cells were treated with interleukin-1ß (IL-1ß) to induce local inflammation. Double luciferase report assay was used to estimate the association between JAK3 and miR-1193. Flow cytometry and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assays were taken to analyze the apoptosis and proliferation of MH7A cells with IL-1ß-induced inflammation, respectively. The relative expression of genes and proteins were detected by quantitative real-time polymerase chain reaction and western blot analyses. Finally, rescue experiments were employed to explore the effects of miR-1193/JAK3 axis on IL-1ß-induced inflammation. RESULTS: JAK3 was notably up-regulated in RA, and was targeted and negatively regulated by miR-1193 which was lowly expressed in RA tissues and IL-1ß-treated cells. MiR-1193 mimic significantly inhibited while miR-1193 inhibitor promoted the proliferation of MH7A cells with IL-1ß-induced inflammation. Furthermore, overexpression of JAK3 presented auxo-action while depletion of JAK3 exhibited inhibitory effect on the proliferation of MH7A cells with IL-1ß-induced inflammation, which could be weakened by miR-1193 mimic and miR-1193 inhibitor, respectively. Analogously, JAK3 recovered the cell proliferation that inhibited by miR-1193 mimic and inhibited cell apoptosis enhanced by miR-1193 mimic. CONCLUSION: Up-regulation of miR-1193 suppressed the proliferation and expedited the apoptosis of MH7A cells with IL-1ß-induced inflammation through targeting JAK3, which might provide novel understanding on the mechanism underlying RA.

The JNK pathway represents a novel target in the treatment of rheumatoid arthritis through the suppression of MMP-3.

BACKGROUND AND AIM: The pathophysiology of rheumatoid arthritis (RA) is characterized by excess production of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) by neutrophils and macrophages in synovium. Additionally, these cytokines promote the production of reactive oxygen species (ROS), and increased production of matrix metalloproteinases (MMPs), including MMP-3, in synoviocytes that result in joint destruction. There is limited information on how proteolytic enzymes such as MMP-3 can be regulated. We evaluated the effect of the antioxidant N-acetylcysteine (NAC) on RA and identified the relationship between the c-Jun N terminal kinase (JNK) pathway and MMP-3. We hypothesized that elucidating this relationship would lead to novel therapeutic approaches to RA treatment and management. METHODS: We investigated the effect of administering a low dose (1000 µM or less) of an antioxidant (NAC) to human rheumatoid fibroblast-like synoviocytes (MH7A cells). We also investigated the response of antioxidant genes such as nuclear factor erythroid -derived 2-related factor 2 (Nrf2) and Sequestosome 1 (p62). The influence of MMP-3 expression on the JNK pathway leading to joint destruction and the mechanisms underlying this relationship were investigated through primary dispersion culture cells collected from the synovial membranes of RA patients, consisting of rheumatoid arthritis-fibroblast-like synoviocytes (RA-FLS). RESULTS: Low-dose NAC (1000 µM) increased the expression of Nrf2 and phospho-p62 in MH7A cells, activating antioxidant genes, suppressing the expression of MMP-3, and inhibiting the phosphorylation of JNK. ROS, MMP-3 expression, and IL-6 was suppressed by administering 30 µM of SP600125 (a JNK inhibitor) in MH7A cells. Furthermore, the administration of SP600125 (30 µM) to RA-FLS suppressed MMP-3. CONCLUSIONS: We demonstrated the existence of an MMP-3 suppression mechanism that utilizes the JNK pathway in RA-FLS. We consider that the JNK pathway could be a target for future RA therapies.

Ameliorating effects of Gö6976, a pharmacological agent that inhibits protein kinase D, on collagen-induced arthritis.

Toll-like receptor (TLR) signaling can contribute to the pathogenesis of arthritis. Disruption of TLR signaling at early stages of arthritis might thereby provide an opportunity to halt the disease progression and ameliorate outcomes. We previously found that Gö6976 inhibits TLR-mediated cytokine production in human and mouse macrophages by inhibiting TLR-dependent activation of protein kinase D1 (PKD1), and that PKD1 is essential for proinflammatory responses mediated by MyD88-dependent TLRs. In this study, we investigated whether PKD1 contributes to TLR-mediated proinflammatory responses in human synovial cells, and whether Gö6976 treatment can suppress the development and progression of type II collagen (CII)-induced arthritis (CIA) in mouse. We found that TLR/IL-1R ligands induced activation of PKD1 in human fibroblast-like synoviocytes (HFLS). TLR/IL-1R-induced expression of cytokines/chemokines was substantially inhibited in Gö6976-treated HFLS and PKD1-knockdown HFLS. In addition, serum levels of anti-CII IgG antibodies, and the incidence and severity of arthritis after CII immunization were significantly reduced in mice treated daily with Gö6976. Synergistic effects of T-cell receptor and TLR, as well as TLR alone, on spleen cell proliferation and cytokine production were significantly inhibited in the presence of Gö6976. Our results suggest a possibility that ameliorating effects of Gö6976 on CIA may be due to its ability to inhibit TLR/IL-1R-activated PKD1, which might play an important role in proinflammatory responses in arthritis, and that PKD1 could be a therapeutic target for inflammatory arthritis.

Abatacept alleviates rheumatoid arthritis development by inhibiting migration of fibroblast-like synoviocytes via MAPK pathway.

OBJECTIVE: To investigate whether Abatacept could regulate the occurrence and progression of rheumatoid arthritis (RA) by mediating cell migration of fibroblast-like synoviocytes (FLS) via mitogen-activated protein kinase (MAPK) pathway. PATIENTS AND METHODS: Levels of MMP1, MMP3 and MMP13 in RA-FLS treated with Abatacept or MAPK pathway inhibitor were detected by quantitative Real-time-polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The regulatory effect of Abatacept on MAPK pathway was detected by Western blot. Transwell assay was performed to access the role of Abatacept in regulating cell migration of RA-FLS. RESULTS: Abatacept treatment remarkably downregulated levels of MMP1, MMP3 and MMP13 in FLS, which were confirmed by qRT-PCR and ELISA. Migratory ability of FLS was inhibited by Abatacept treatment. Western blot results suggested that Abatacept treatment downregulated MAPK pathway-related genes in FLS. The effects of Abatacept on MMPs expressions and cell migration were partially reversed by SB203580 treatment, the MAPK pathway inhibitor. CONCLUSIONS: Abatacept inhibits FLS migration and MMPs expressions via inhibiting MAPK pathway, thereby inhibiting RA development.

PIM-1 kinase is a novel regulator of proinflammatory cytokine-mediated responses in rheumatoid arthritis fibroblast-like synoviocytes.

Objectives: This study investigated the expression of proviral-integration site for Moloney murine leukaemia virus (PIM) -1 kinase in RA synovium and RA fibroblast-like synoviocytes (FLSs) along with its impact on RA-FLS aggressiveness. Methods: The expression of PIM kinases was assessed in synovial tissues by immunohistochemistry and double IF. After PIM-1 inhibition using either small-interfering RNA or the chemical inhibitor AZD1208, we performed proliferation and migration assays and measured the levels of MMPs and IL-6 released from RA-FLSs under stimulation with proinflammatory cytokines (TNF-α, S100A4 and IL-6/soluble IL-6 receptor). Additionally, PIM-1-associated downstream signalling pathways were analysed by immunoblotting. Results: Three isoforms of PIM kinases were immunodetected in the synovial tissues from patients with RA or OA. Specifically, PIM-1 and PIM-3 were upregulated in RA synovium and PIM-1 was expressed in T cells, macrophages and FLSs. Additionally, upon stimulation of RA-FLSs with TNF-α, S100A4 and IL-6/sIL-6R, PIM-1 and PIM-3, but not PIM-2, were significantly inducible. Moreover, PIM-1 knockdown or AZD1208 treatment significantly suppressed basal or cytokine-induced proliferation and migration of RA-FLS and the secretion of MMPs from stimulated RA-FLSs. PIM-1 knockdown significantly affected the phosphorylation levels of extracellular signal-regulated kinase and cAMP responsive element binding protein in RA-FLSs. Conclusion: PIM-1 was upregulated in RA synovial tissues and RA-FLSs and its inhibition significantly reduced the proliferation, migration and MMP production of RA-FLSs in vitro. These findings suggest PIM-1 as a novel regulator of the aggressive and invasive behaviour of RA-FLSs and indicate its potential as a target for RA treatment.

CCL3 participates in the development of rheumatoid arthritis by activating AKT.

OBJECTIVE: To investigate whether CC chemokine 3 (CCL3) could exert a certain effect on rheumatoid arthritis (RA) by regulating inflammatory responses and provide a new direction for the treatment of RA. PATIENTS AND METHODS: Totally 47 RA patients (10 males and 37 females) with complete clinical data were included. Meanwhile, 27 healthy volunteers with same age and gender were recruited as healthy controls. The mRNA and protein level of CCL3 in the peripheral blood mononuclear cells (PBMCs) of RA patients and normal controls were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. The inflammatory infiltration of synovial tissue was observed by hematoxylin and eosin (HE) staining. Immune fluorescence was used to further analyze the level of CCL3 in T and B cells of synovial tissue in RA patients. Simultaneously, real-time flow cytometry was applied to detect the level of CCL3 in T and B cells of PBMCs in the normal control group and the RA group. Western blot was used to detect the level of pAKT in RA-FLS treated with different concentrations of recombinant human CCL3. Besides, enzyme-linked immunosorbent assay (ELISA) was applied to detect the levels of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and receptor activator of nuclear factor kappa-B ligand (RANKL) in the culture supernatant of RA-FLS stimulated by different doses of recombinant human CCL3. RESULTS: The level of CCL3 in peripheral blood and synovial fluid of RA patients was markedly higher than that of normal controls. Inflammatory cells were infiltrated in synovial tissue of RA patients. Meanwhile, CCL3 was mainly expressed in CD4+ T cells. CCL3 treatment in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) could activate the PI3K/AKT signaling pathway to different degrees and increase the expression of cytokines including interleukin-6 (IL-6), IL-1ß, TNF-α, and RANKL. These results indicated that CCL3 might participate in the progression of RA by activating AKT. CONCLUSIONS: We showed that CCL3 enhanced the expression level of pro-inflammatory cytokines such as IL-6, IL-1ß, TNF-α, and RANKL by activating the PI3K/AKT signaling pathway. Besides, CCL3 could up-regulate CD4+T cells to mediate the inflammatory response of RA. These findings might provide new directions for the prevention of RA.

Hexokinase 2 as a novel selective metabolic target for rheumatoid arthritis.

OBJECTIVES: Recent studies indicate that glucose metabolism is altered in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Hexokinases (HKs) catalyse the first step in glucose metabolism, and HK2 constitutes the principal HK inducible isoform. We hypothesise that HK2 contributes to the synovial lining hypertrophy and plays a critical role in bone and cartilage damage. METHODS: HK1 and HK2 expression were determined in RA and osteoarthritis (OA) synovial tissue by immunohistochemistry. RA FLS were transfected with either HK1 or HK2 siRNA, or infected with either adenovirus (ad)-GFP, ad-HK1 or ad-HK2. FLS migration and invasion were assessed. To study the role of HK2 in vivo, 108 particles of ad-HK2 or ad-GFP were injected into the knee of wild-type mice. K/BxN serum transfer arthritis was induced in HK2F/F mice harbouring Col1a1-Cre (HK2Col1), to delete HK2 in non-haematopoietic cells. RESULTS: HK2 is particular of RA histopathology (9/9 RA; 1/8 OA) and colocalises with FLS markers. Silencing HK2 in RA FLS resulted in a less invasive and migratory phenotype. Consistently, overexpression of HK2 resulted in an increased ability to migrate and invade. It also increased extracellular lactate production. Intra-articular injection of ad-HK2 in normal knees dramatically increased synovial lining thickness, FLS activation and proliferation. HK2 was highly expressed in the synovial lining after K/BxN serum transfer arthritis. HK2Col1 mice significantly showed decreased arthritis severity, bone and cartilage damage. CONCLUSION: HK2 is specifically expressed in RA synovial lining and regulates FLS aggressive functions. HK2 might be an attractive selective metabolic target safer than global glycolysis for RA treatment.

Bcl-XL and Mcl-1 upregulation by calreticulin promotes apoptosis resistance of fibroblast-like synoviocytes via activation of PI3K/Akt and STAT3 pathways in rheumatoid arthritis.

OBJECTIVES: Fibroblast-like synoviocytes (FLS) play key roles in synovium hyperplasia and pannus formation in rheumatoid arthritis (RA). The present study was undertaken to explore the mechanisms that calreticulin (CRT) promoted anti-apoptosis of RA FLS. METHODS: The expression of CRT and anti-apoptotic proteins Bcl-XL and Mcl-1 in RA synovium were detected by immunohistochemistry. The expression of Bcl-XL and Mcl-1 in RA FLS by CRT were determined. The phosphorylation of Akt and STAT3 was detected by western blot. The effect of CRT on proliferation of RA FLS was examined by MTT assay. The ability of CRT to inhibit RA FLS apoptosis was assessed by flow cytometry. RESULTS: Increased expressions of CRT, Bcl-XL and Mcl-1 were detected in RA synovium compared with osteoarthritis (OA). Moreover, CRT expression correlated positively with Bcl-XL and Mcl-1 in RA, respectively. In vitro, CRT induced upregulation of Bcl-XL and Mcl-1 protein levels in RA FLS, in dose/time dependent manners. Upregulated expression of Bcl-XL and Mcl-1 induced by CRT were inhibited by PI3K/Akt or STAT3 pathways inhibitors in RA FLS, respectively. The increased phosphorylation levels of Akt and STAT3 were also detected with CRT incubation, in dose/time dependent manners. Additionally, CRT rescued apoptosis of RA FLS mediated by FasL. CONCLUSIONS: This study showed that upregulation of Bcl-XL and Mcl-1 expression in RA FLS by CRT were PI3K/Akt and STAT3 signal pathways dependent, and promoted the anti-apoptosis of RA FLS. Therefore, this may represent a therapeutic target for the treatment of RA.

Platelets Contribute to the Accumulation of Matrix Metalloproteinase Type 2 in Synovial Fluid in Osteoarthritis.

Inflammation plays a role in the initiation and progression of osteoarthritis (OA), a chronic degenerative joint disorder. Platelets are inflammatory cells, contain and release matrix metalloproteinases (MMPs) and favour the release of these enzymes, key effectors of cartilage and subchondral bone degradation, by other cells; however, their role in OA has not been investigated yet. Our aims were (1) to assess the presence of platelets and of MMP-2 in synovial fluid (SF) of OA patients; (2) to evaluate the contribution of platelets to MMP-2 release by fibroblast-like synoviocytes (FLS); and (3) to investigate if hyaluronic acid (HA) interferes with these processes. SF was collected from 27 OA patients before and after treatment with intra-articular HA (20 mg/2 mL). Moreover, FLS were co-cultured with platelets, and the release of MMP-2 in supernatants was measured. Our results show that platelets are present in OA SF and show markers of activation. OA SF also contains relevant amounts of MMP-2. Co-incubation of platelets with FLS favours the release of MMP-2 by the interaction of platelet surface P-selectin with FLS CD44 by a mechanism involving the activation of pAkt and pSrc in FLS. Administration of HA to OA patients decreased the infiltration of platelets in SF and reduced the levels of MMP-2. The addition of HA in vitro inhibited the release of MMP-2 by FLS triggered by the interaction with platelets. In conclusion, our data show that platelets may contribute to joint degeneration in OA by favouring the accumulation of MMP-2 in SF.

Blockade of IL-17 alleviated inflammation in rat arthritis and MMP-13 expression.

OBJECTIVE: Rheumatoid arthritis (RA) is one systemic auto-immune disorder featured as chronic synovitis and can destruct joint cartilage. Fibroblast-like synoviocyte (FLS) secretes various factors affecting chondrocyte matrix and degradation. This study thus investigated the effect of interleukin-17A (IL-17A) on FLS and osteoclast. MATERIALS AND METHODS: Type II collagen-induced arthritis (CIA) rats were assigned to CIA model, CIA + IgG1 isotype, and CIA + Anti-Rat IL-17A groups. Tissue volume and arthritis index (AI) evaluated arthritis condition. ELISA and flow cytometry measured IL-17A content and Th17 cell percentage in joint cavity fluid. Matrix metallopeptidase 13 (MMP-13) and collagen type II alpha 1 (COL2A1) expression in synovial tissues were compared. FLS-osteoclast co-culture system was treated with IL-17A + IgG1 Isotype or CIA + Anti-Rat IL-17A. MMP-13 and COL2A1 expression were compared. RESULTS: CIA model rats had significantly higher IL-17A and Th17 cell ratio in joint cavity fluid. Injection of Anti-Rat IL-17A decreased AI and tissue volume in model rats, decreased MMP-13 while increased COL2A1 expression in synovial or cartilage tissues. IL-17A treatment remarkably up-regulated MMP-13 mRNA or protein expression in chondrocytes. Anti-IL-17A weakened effects of IL-17A on FLS or chondrocytes. CONCLUSIONS: IL-17A inhibits COL2A1 mRNA and protein expression of chondrocyte in the co-culture system via inducing MMP-13 expression in FLS, thus enhancing collagen degradation and playing a role in RA-related cartilage injury.

Glutaminase 1 plays a key role in the cell growth of fibroblast-like synoviocytes in rheumatoid arthritis.

BACKGROUND: The recent findings of cancer-specific metabolic changes, including increased glucose and glutamine consumption, have provided new therapeutic targets for consideration. Fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients exhibit several tumor cell-like characteristics; however, the role of glucose and glutamine metabolism in the aberrant proliferation of these cells is unclear. Here, we evaluated the role of these metabolic pathways in RA-FLS proliferation and in autoimmune arthritis in SKG mice. METHODS: The expression of glycolysis- or glutaminolysis-related enzymes was evaluated by real-time polymerase chain reaction (PCR) and Western blotting, and the intracellular metabolites were evaluated by metabolomic analyses. The effects of glucose or glutamine on RA-FLS cell growth were investigated using glucose- or glutamine-free medium. Glutaminase (GLS)1 small interfering RNA (siRNA) and the GLS1 inhibitor compound 968 were used to inhibit GLS1 in RA-FLS, and compound 968 was used to study the effect of GLS1 inhibition in zymosan A-injected SKG mice. RESULTS: GLS1 expression was increased in RA-FLS, and metabolomic analyses revealed that glutamine metabolism was increased in RA-FLS. RA-FLS proliferation was reduced under glutamine-deprived, but not glucose-deprived, conditions. Cell growth of RA-FLS was inhibited by GLS1 siRNA transfection or GLS1 inhibitor treatment. Treating RA-FLS with either interleukin-17 or platelet-derived growth factor resulted in increased GLS1 levels. Compound 968 ameliorated the autoimmune arthritis and decreased the number of Ki-67-positive synovial cells in SKG mice. CONCLUSIONS: Our results suggested that glutamine metabolism is involved in the pathogenesis of RA and that GLS1 plays an important role in regulating RA-FLS proliferation, and may be a novel therapeutic target for RA.

Rheumatoid Arthritis Fibroblast-like Synoviocyte Suppression Mediated by PTEN Involves Survivin Gene Silencing.

Survivin is a proto-oncogene biomarker known for its anti-apoptotic and cell cycle regulating properties induced by the activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. In the context of non-cancer pathology, such as rheumatoid arthritis (RA), survivin has emerged as a feature associated with severe joint damage and poor treatment response. Phosphatase and tensin homolog (PTEN) is a phosphatase antagonizing all classes of PI3K. The interplay between survivin oncogenic mechanisms and proliferation suppression networks in RA has remained largely elusive. This study investigated the effect of PTEN on survivin gene expression in rheumatiod arthritis fibroblast-like synoviocyte (RA-FLS). We showed for the first time that the suppression of RA-FLS was mediated by PTEN involving survivin silencing. Considering that survivin suppressants are currently available in clinical trials and clinical use, their effects in RA-FLS support a probably RA therapy to clinical practice.

Role of protein arginine methyltransferase 5 in inflammation and migration of fibroblast-like synoviocytes in rheumatoid arthritis.

To probe the role of protein arginine methyltransferase 5 (PRMT5) in regulating inflammation, cell proliferation, migration and invasion of fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA). FLSs were separated from synovial tissues (STs) from patients with RA and osteoarthritis (OA). An inhibitor of PRMT5 (EPZ015666) and short interference RNA (siRNA) against PRMT5 were used to inhibit PRMT5 expression. The standard of protein was measured by Western blot or immunofluorescence. The excretion and genetic expression of inflammatory factors were, respectively, estimated by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR). Migration and invasion in vitro were detected by Boyden chamber assay. FLSs proliferation was detected by BrdU incorporation. Increased PRMT5 was discovered in STs and FLSs from patients with RA. In RA FLSs, the level of PRMT5 was up-regulated by stimulation with IL-1ß and TNF-α. Inhibition of PRMT5 by EPZ015666 and siRNA-mediated knockdown reduced IL-6 and IL-8 production, and proliferation of RA FLSs. In addition, inhibition of PRMT5 decreased in vitro migration and invasion of RA FLSs. Furthermore, EPZ015666 restrained the phosphorylation of IκB kinaseß and IκBα, as well as nucleus transsituation of p65 as well as AKT in FLSs. PRMT5 regulated the production of inflammatory factors, cell proliferation, migration and invasion of RA FLS, which was mediated by the NF-κB and AKT pathways. Our data suggested that targeting PRMT5 to prevent synovial inflammation and destruction might be a promising therapy for RA.