Migration Of Fibroblast-like Synoviocytes Research Articles

Hsa_circ_0088036 promotes the proliferation and migration of fibroblast-like synoviocytes by sponging miR-140-3p and upregulating SIRT 1 expression in rheumatoid arthritis

Circular RNAs (circRNAs) have been demonstrated to play crucial roles in the development and progression of various types of cancers by serving as microRNA sponges to regulate the expression of target genes. Although in-depth studies of circRNAs have been conducted, their functional and pathological significance in autoimmune diseases, including rheumatoid arthritis (RA), remains unclear. Our previous study verified that hsa_circ_0088036 (circ0088036) is significantly elevated in peripheral blood mononuclear cells from patients with RA. The present study aimed to explore the roles of circ0088036 in the pathogenesis of RA. The circ0088036/miR-140−3p/silent information regulator 1 (SIRT 1) axis was predicted by bioinformatics tools. Circ0088036 was found to be aberrantly upregulated in fibroblast-like synoviocytes (FLSs) in RA compared with FLSs in osteoarthritis (OA). Functionally, upregulated circ0088036 promoted the proliferation and migration of RA-FLSs. Mechanistically, circ0088036 acted as a miR-140−3p sponge to upregulate SIRT 1 expression, subsequently promoting RA progression. In conclusion, this study revealed that circ0088036 may play an essential role in promoting synovial pathogenesis via the circ0088036/miR-140−3p/SIRT 1 axis in RA, providing new insight into circRNAs during RA progression.

Inhibition of BMP3 increases the inflammatory response of fibroblast-like synoviocytes in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a persistent autoimmune disease. Fibroblast-like synoviocytes (FLS) are a key component of invasive pannus and a pathogenetic mechanism in RA. Expression of bone morphogenetic protein 3 (BMP3) mRNA is reportedly decreased in the arthritic synovium. We previously showed that BMP3 expression is significantly downregulated in the synovial tissues of RA patients and models of adjuvant-induced arthritis (AIA). In the present study, we explored the association between BMP3 and FLS migration and secretion of proinflammatory factors in RA. We found that inhibition of BMP3 expression using BMP3 siRNA increased the proinflammatory chemokines and migration of FLS stimulated with TNF-α. Inhibition of BMP3 expression also increased expression of IL-6, IL-1β, IL-17A, CCL-2, CCL-3, VCAM-1, MMP-3, and MMP-9, but not TIMP-1, in AIA and RA FLS. Correspondingly, induction of BMP3 overexpression through intra-articular injection of ad-BMP3 diminished arthritis severity in AIA rats. We also found that BMP3 may inhibit activation of TGF-β1/Smad signaling. These data indicate that BMP3 may suppress the proliferation and migration of FLS via the TGF-β1/Smad signaling pathway.

Paeoniflorin-6′-O-benzene sulfonate down-regulates CXCR4-Gβγ-PI3K/AKT mediated migration in fibroblast-like synoviocytes of rheumatoid arthritis by inhibiting GRK2 translocation

ObjectiveThis study aims to explore the effect of paeoniflorin-6′-O-benzene sulfonate (CP-25) on the migration of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) and the mechanism focused on CXCR4-Gβγ-PI3K/AKT signaling. MethodsHuman synovial tissues were collected from RA and osteoarthritis (OA) patients. Immunohistochemistry (IHC) and Western blot were used to detect the protein expression of CXCR4, GRK2, Gβγ, PI3K, p-PI3K, AKT and p-AKT. Transwell was adopted to analyse the migration of fibroblast-like synoviocytes (FLS). Co-immunoprecipitation (Co-IP) and laser scanning confocal microscopy (LSCM) were used to detect the combination of GRK2 and Gβγ, the combination of PI3K and Gβγ. ResultsThe expression level of CXCR4, GRK2, Gβγ, p-p85 and p-AKT were increased in RA synovial tissue according to the results of IHC and Western blot. In vitro, the migration of FLS was increased after stimulation of CXCL12, inhibition of Gβγ suppressed the migration and phosphorylation of p85 and AKT induced by CXCL12 in FLS, and CP-25 had the same effect as inhibition of Gβγ. The membrane expression of GRK2, Gβγ, PI3K and the combination of GRK2 and Gβγ, the combination of PI3K and Gβγ in FLS were increased after the stimulation of CXCL12, and CP-25 had an ability in reducing the membrane expression and the combination of these proteins. ConclusionExcessive migration of FLS in RA was associated with over-activation of PI3K/AKT signaling, and the activity of Gβγ was involved in the over-activation of PI3K/AKT. CP-25 down-regulated CXCR4-Gβγ-PI3K/AKT signals by inhibiting GRK2-Gβγ complex membrane translocation.

Resolvin D1 suppresses pannus formation via decreasing connective tissue growth factor caused by upregulation of miRNA-146a-5p in rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation and joint stiffness, finally leading to tissue destruction. Connective tissue growth factor (CTGF) is a critical factor in RA progression, which promotes fibroblast-like synoviocyte (FLS) proliferation, pannus formation, and the damage of cartilage as well as bone. Resolvin D1 (RvD1) can promote inflammation resolution in acute inflammatory diseases, and recently, effects of RvD1 on chronic inflammatory diseases also attracted attention. This study aimed to examine the effect of RvD1 on pannus formation in RA and the underlying mechanism.MethodsSerum levels of RvD1 and CTGF were determined in RA patients and healthy persons by UPLC-MS/MS and ELISA respectively. The levels of CTGF and inflammatory factors were assessed by qRT-PCR and ELISA. MicroRNA expression profile was determined by miRNA microarray. The effects of CTGF, RvD1, and miR-146a-5p on angiogenesis were evaluated with tube formation and chick chorioallantoic membrane (CAM) assays. Collagen-induced arthritis (CIA) mice were constructed to detect the effects of RvD1 and miR146a-5p on RA. STAT3 activation was determined by Western blotting.ResultsRvD1 levels decreased while CTGF levels increased in RA patients’ serum, and an inverse correlation of the concentrations of RvD1 and CTGF in the serum of RA patients was synchronously observed. In CIA mice, RvD1 suppressed angiopoiesis and decreased the expression of CTGF. Simultaneously, RvD1 significantly decreased CTGF and pro-inflammation cytokines levels in RA FLS. Furthermore, CTGF suppressed angiopoiesis and RvD1 inhibited the proliferation and migration of RA FLS and angiopoiesis. MiRNA microarray and qRT-PCR results showed that RvD1 upregulated miRNA-146a-5p. The transfection experiments demonstrated that miRNA-146a-5p could decrease inflammatory factors and CTGF levels. Moreover, miRNA-146a-5p decreased the proliferation of FLS and angiogenesis in vivo. MiRNA-146a-5p also suppressed angiogenesis and downregulated the expression of CTGF in CIA mice. Finally, Western blot results revealed that miRNA-146a-5p inhibited the activation of STAT3.ConclusionRvD1 is prone to alleviate RA progression through the upregulation of miRNA-146a-5p to suppress the expression of CTGF and inflammatory mediators, thereby decreasing pannus formation and cartilage damage.

Monocyte chemoattractant protein‑1 promotes the proliferation, migration and differentiation potential of fibroblast‑like synoviocytes via the PI3K/P38 cellular signaling pathway.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints and joint destruction. Monocyte chemoattractant protein 1 (MCP‑1) is highly expressed in the joints of patients suffering from RA. The present study aimed to evaluate the effects of MCP‑1 on the phenotype of fibroblast‑like synoviocytes (FLSs) and their differentiation potential towards vascular endothelial cells. The expression of MCP‑1 in collagen‑induced arthritis (CIA) rats was investigated by PCR, ELISA and immunohistology. Cell proliferation induced by MCP‑1 was measured using a Cell Counting Kit‑8 (CCK‑8) and 5‑Bromo‑2‑deoxyuridine ELISA assay. In addition, the effects of MCP‑1 on the migration of FLSs was examined using a Transwell assay. Activation of PI3K and P38 were investigated by western blotting following MCP‑1 treatment. The vascular endothelial cell markers, tumor necrosis factor alpha (TNF‑α) and interleukin‑1 beta (IL‑β), were also examined by western blotting. LY294002 [PI3K inhibitor, (LY)] and SB203580 [P38 inhibitor, (SB)] were used to examine the proliferative and pro‑differentiation effect of PI3K and P38. The present findings showed that the expression level of MCP‑1 in the synovium of CIA rats was significantly higher compared with controls. The present invitro study suggested that MCP‑1 increased the FLSs cell numbers with a maximal effect at200ng/ml, and induced the maximal phosphorylation of PI3K at 15min and P38 at 30min. In addition, MCP‑1 stimulation significantly increased the migration of FLSs. Furthermore, MCP‑1‑induced the expression of vascular endothelial growth factor and CD31 in FLSs. Suppression of PI3K and P38 was found to reduce MCP‑1 induced FLSs proliferation and migration, and decreased the expression levels of angiogenesis markers increased following MCP‑1 treatment. MCP‑1 was also found to increase the expression levels of both TNF‑α and IL‑β. Therefore, MCP‑1 could promote the proliferation and migration of FLSs, and was found to increase the expression levels of various angiogenesis markers via PI3K/P38, suggesting a role for this pathway in synovium hyperplasia in RA.

Cadherin-11 cooperates with inflammatory factors to promote the migration and invasion of fibroblast-like synoviocytes in pigmented villonodular synovitis.

Rationale: Pigmented villonodular synovitis (PVNS) is a destructive benign tumor-like hyperplastic disease that occurs in synovial tissue. Fibroblast-like synoviocytes (FLS) are the predominant cell type comprising the structure of the PVNS synovial lining layer. Due to a high recurrence rate, high invasion, migration, and cartilage destruction ability, PVNS causes substantial damage to patients and the efficacy of surgical resection is not satisfactory. Therefore, exploring the pathogenesis and identifying novel therapeutic targets for PVNS are urgently required. Currently, the pathogenesis of PVNS remains unclear, and there is uncertainty and controversy regarding whether PVNS is an inflammatory or a neoplastic disease. Cadherin-11 is a classical molecule that mediates hemophilic cell-to-cell adhesion in FLS and plays an important role in the normal synovium lining layer formation. This study aimed to explore the role of inflammation and cadherin-11 in PVNS pathogenesis and determine the effects of cadherin-11 as a molecular target for PVNS treatment.Methods: FLS were primarily cultured from PVNS patients during arthroscopic synovectomy. The level of cytokines in the PVNS synovial fluid was evaluated using a human antibody array. Cadherin-11 expression of PVNS FLS was detected by qPCR, Western blots, tissue immunohistochemistry, and cell immunofluorescence. Cadherin-11 was down-regulated by siRNA or up-regulated with a plasmid, with or without inflammatory factor stimulation, and PI3K/Akt was inhibited with LY294002. The capacity of migration and invasion of PVNS FLS was tested using Transwell and wound-healing assays. Activation of the nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways was detected by Western blots. Chondrocyte damage by PVNS FLS was assessed with a co-culture assay.Results: Inflammatory factors (IL-1β and TNF-α) in the synovial fluid of PVNS patients were significantly up-regulated. Cadherin-11 was highly expressed in the FLS of PVNS patients, and positively correlated with recurrence, extra-articular migration, and cartilage destruction of PVNS. Knocking down of cadherin-11 inhibited the migration and invasion of PVNS FLS. Moreover, inflammatory factors up-regulated the expression of cadherin-11, which activated the NF-κB and MAPK signaling pathways and led to cartilage destruction. Inhibition of cadherin-11 blocked IL-1β- and TNF-α-induced activation of the above pathways, migration and invasion of PVNS FLS, and damage of chondrocyte. In addition, the elevation of cadherin-11 expression, together with the migration and invasion, of PVNS FLS was down-regulated by the inhibition of the PI3K/Akt signaling pathway.Conclusions: Cadherin-11 plays an important role in the pathogenesis of PVNS and forms a positive feedback loop with inflammatory factors, which further activates the NF-κB and MAPK pathways to trigger an inflammatory cascade. Cadherin-11-mediated inflammation results in PVNS with high recurrence, invasiveness, and strong cartilage destruction ability, and eventually promotes the transformation of PVNS from the initial inflammatory disease to neoplastic disease. Thus, inhibition of cadherin-11 together with its related inflammatory reaction, represents a new therapeutic strategy for PVNS.

All-Trans Retinoic Acid Inhibits Migration and Invasiveness of Rheumatoid Fibroblast-Like Synoviocytes.

Fibroblast-like synoviocytes (FLSs) are pivotal in inflammation and joint damage of rheumatoid arthritis (RA). They acquire an active and aggressive phenotype, displaying increased migration and invasiveness and contributing to perpetuate synovial inflammation and destruction of cartilage and bone. The main current therapies of RA are focused against inflammatory factors and immune cells; however, a significant percentage of patients do not successfully respond. Combined treatments with drugs that control inflammation and that reverse the pathogenic phenotype of FLS could improve the prognosis of these patients. An unexplored area includes the retinoic acid, the main biologic retinoid, which is a candidate drug for many diseases but has reached clinical use only for a few. Here, we explored the effect of all-trans retinoic acid (ATRA) on the aggressive phenotype of FLS from patients with RA. RA FLSs were treated with ATRA, tumor necrosis factor (TNF), or TNF+ATRA, and cell migration and invasion were analyzed. In addition, a microarray analysis of expression, followed by gene-set analysis and quantitative polymerase chain reaction validation, was performed. We showed that ATRA induced a notable decrease in FLS migration and invasion that was accompanied by complex changes in gene expression. At supraphysiological doses, many of these effects were overridden or reverted by the concomitant presence of TNF. In conclusion, these results have demonstrated the therapeutic potential of retinoic acid on RA FLS provided TNF could be counterbalanced, either with high ATRA doses or with TNF inhibitors. SIGNIFICANCE STATEMENT: All-trans retinoic acid (ATRA) reduced the rheumatoid arthritis (RA) fibroblast-like synoviocyte migration and invasiveness and down-regulated gene expression of cell motility and migration genes. At supraphysiological doses, some of these effects were reverted by tumor necrosis factor. Therefore, ATRA could be an RA drug candidate that would require high doses or combined treatment with anti-inflammatory drugs.

PI3K/Akt inhibitor partly decreases TNF-\u03b1-induced activation of fibroblast-like synoviocytes in osteoarthritis

BackgroundThe Cadherin-11 and PI3K/Akt pathway are increasingly recognized as the potential therapeutic target of osteoarthritis (OA) synovitis. The study aimed to investigate the role of PI3K/Akt signaling pathway in the expression of Cadherin-11 and migration and invasive capacity of fibroblast-like synoviocytes (FLS) of OA patients under stimulation of TNF-α and to explore the effect of the PI3K/Akt inhibitor and Cadherin-11 antibody in the therapy of the collagenase-induced osteoarthritis (CIOA) mice.MethodsFLS were primarily cultured from synovium of osteoarthritic patients during total knee arthroplasty. Under the simulation of TNF-α, with or without PI3K/Akt inhibitor LY294002, Cadherin-11 expression was detected by real-time PCR and Western blot, as well as the migration and invasive capacity changes of OA FLS. Cadherin-11 antibody was injected intraarticularly or LY294002 was injected intraperitoneally in CIOA mice to evaluate the changes of synovitis score, cartilage damage, and Cadherin-11 expression.ResultsTNF-α stimulation increased Cadherin-11 expression at mRNA and protein level in OA FLS and also increased the phosphorylation-dependent activation of Akt. PI3K inhibitor LY294002 attenuated TNF-α-induced overexpression of Cadherin-11 and decreased the invasive capacity of OA FLS. Intraperitoneal injection of PI3K inhibitor LY294002 could decrease the Cadherin-11 protein expression in synovium of CIOA mice, although it has no significant inhibitory effect on synovitis and cartilage damage. Intraarticular injection of Cadherin-11 antibody attenuated the synovitis and cartilage damage in the CIOA joints and decreased Cadherin-11 expression in the synovial lining.ConclusionsPI3K/Akt pathway was associated with TNF-α-induced activation of OA FLS, which may involve in the pathogenesis of osteoarthritis. Anti-Cadherin-11 therapy in CIOA mice could attenuate the pathological changes of OA joints.

Over-Expression of PTEN Attenuates the Inflammation of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis

Background: Rheumatoid arthritis (RA) is characterized by a tumor-like expansion of the synovium and the subsequent destruction of adjacent articular cartilage and bone. Recent studies have shown that phosphatase and tension homolog deleted on chromosome 10 (PTEN) might contribute to the survival of fibroblast-like synoviocytes (FLS) and the production of pro-inflammatory cytokines in RA. Methods: The expression was determined in RA and adjuvant-induced arthritis (AIA) synovial tissues by immunohistochemistry. FLSs were treatment with bpv, PTEN-RNAi or over-expression plasmid in RA and AIA. FLSs migration was assessed. The ad-PTEN was also injected into the knee of AIA in vivo. Chromatin Immunoprecipitation (ChIP) and Methylation-special PCR (MSP) assay were used to study the expression of PTEN mRNA in DNA methylation. Results: Down-regulated level of PTEN expression was observed in RA and AIA. Inhibition PTEN expression by bpv or PTEN-RNAi could promote the expression of pro-inflammatory cytokines, chemokines and migration of FLS with TNF-α in RA and AIA. Consistently, over-expression of PTEN reduced their low-expression of pro-inflammatory cytokines, chemokines and migration. Intra-articular injection of ad-PTEN in AIA knees dramatically reduced inflammatory and paw swelling in vivo. The ChIP and MSP assay has clearly detected the DNA methylation of PTEN was increased in FLS with TNF-α. Moreover, intraperitoneally injected 5-Aza in AIA also suppressed the inflammatory and paws swelling in vivo. Conclusions: Our findings suggest that over-expression PTEN attenuates the formation of pro-inflammatory cytokines, chemokines and migration of FLS, and it may be regulated by DNA methylation in the pathogenesis of RA. Funding Statement: This study is supported by the National Natural Science Foundation of China (No. 81770609). Declaration of Interests: All authors declare no conflict interests. Ethics Approval Statement: All experimental protocols used on the animals were approved by the institutions’ subcommittees on animal care of Anhui Medical University (approval number: 20160253).

MicroRNA-421 promotes inflammatory response of fibroblast-like synoviocytes in rheumatoid arthritis by targeting SPRY1.

The aim of this study was to investigate whether microRNA-421 could participate in the proliferative, migratory and inflammatory changes of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis by targeting SPRY1. The expressions of microRNA-421 and SPRY1 in synovial tissues and FLS were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. The binding condition between microRNA-421 and SPRY1 was verified by the Dual-Luciferase reporter gene assay. MicroRNA-421 mimics and inhibitor were constructed and transfected. The levels of extracellular interleukin-1 (IL-1), IL-6, and COX2 in FLS after microRNA-421 mimics or inhibitor transfection were detected by enzyme-linked immunosorbent assay (ELISA). The regulatory effect of microRNA-421 on the proliferation and migration of FLS was detected using cell counting kit-8 (CCK-8) and transwell assay, respectively. Furthermore, collagen-induced RA mouse model was constructed to confirm the specific effect of microRNA-421 on regulating RA development. MicroRNA-421 was highly expressed in the synovial tissues of RA patients. SPRY1 expression in FLS was negatively regulated by microRNA-421. Moreover, the overexpression of microRNA-421 significantly promoted proliferative, invasive potentials and inflammatory response of FLS. In vivo, RA mouse model indicated that downregulated microRNA-421 and upregulated SPRY1 were observed in mice injected with cortisone and microRNA-421 inhibitor when compared with those of controls. MicroRNA-421 promotes the inflammatory response of fibroblast-like synoviocytes in rheumatoid arthritis by downregulating the SPRY1 expression.

Pedunculoside attenuates pathological phenotypes of fibroblast-like synoviocytes and protects against collagen-induced arthritis

Objectives: The discovery of alternative and well-tolerated anti-arthritic drugs, especially from natural products, is becoming an area of active research. Pedunculoside (PE) is a novel triterpene saponin extracted from the dried bark of Ilex rotunda Thunb. Limited published papers have reported its pharmacological properties, including anti-inflammatory, anti-myocardial ischaemia, anti-liver injury, and hypocholesterolaemic activities. However, the effect of PE on rheumatoid arthritis (RA) remains unknown. Here, we investigated the anti-arthritic effect of PE in both in vitro and in vivo models.Method: The inhibitory effects of PE on proliferation, migration, and production of inflammatory mediators in primary fibroblast-like synoviocytes (FLSs) were examined by a 5-ethynyl-2ʹ-deoxyuridine incorporation assay, wound-healing assay, and real-time polymerase chain reaction, respectively. Cellular signalling mechanisms were analysed by Western blot. The in vivo studies were performed using a collagen-induced arthritis (CIA) rat model. Multiple methods, including arthritis scoring, enzyme-linked immunoassay, radiography, and histopathological assessment, were used to evaluate the therapeutic effects of PE on CIA rats.Results: The in vitro studies revealed that PE significantly inhibited proliferation and migration of FLSs. PE also decreased the production of pro-inflammatory cytokines, including interleukin-1β (IL-1β), IL-6, IL-8, and tumour necrosis factor-α (TNF-α). Western blot results suggested that PE suppressed TNF-α-stimulated activation of p38 and extracellular signal-regulated kinase. The in vivo studies showed that PE treatment significantly inhibited synovial inflammation and bone destruction in CIA rats.Conclusion: These results demonstrate that PE exerts an inhibitory role in FLSs and CIA rats, and therefore may have therapeutic value for the treatment of RA.

Transforming growth factor β1 promotes fibroblast-like synoviocytes migration and invasion via TGF-β1/Smad signaling in rheumatoid arthritis.

Migration and invasion are important characteristics of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs), which are involved in joint damage and contribute to rheumatoid arthritis (RA) pathology. However, the underlying mechanisms remain unclear. Because epithelial-mesenchymal transition (EMT) is a key mechanism related to migration and invasion in cancer cells, we investigated the relationship between EMT and RA-FLSs and explored whether the transforming growth factor β1 (TGF-β1)/Smad signaling pathway is involved. In vivo, fibroblast-like synoviocytes (FLSs) were isolated from the synovium of RA or osteoarthritis (OA) patients and cultured for 4-8 passages. EMT markers were detected by immunofluorescence and Western blotting. RA-FLSs were treated with TGF-β1 or Smad2/3 small interfering RNA (siRNA), EMT markers were detected, and migration and invasion were assessed by Transwell assays. EMT markers could be detected in FLSs; when compared with osteoarthritis fibroblast-like synoviocytes (OA-FLSs), E-cadherin and vimentin decreased, while N-cadherin and α-smooth muscle actin (α-SMA) increased in RA-FLSs. Furthermore, TGF-β1 enhanced migration and invasion by inducing EMT via activating Smad2/3 in RA-FLSs. Phosphorylation of Smad2/3 was accompanied by degradation of Smad3. Silencing Smad2/3 blocked EMT and inhibited the migration and invasion induced by TGF-β1. Matrix metalloproteinase 9 (MMP9) and vimentin were not affected when cells were treated with TGF-β1 or Smad2/3 siRNA. The TGF-β1/Smad signaling pathway is involved in EMT and contributes to migration and invasion in RA-FLSs. Interestingly, vimentin decreased in RA-FLSs, but there is no correlation between vimentin and TGF-β1/Smad signaling pathway. Thus, further research on vimentin should be conducted.

BMP9 inhibits the proliferation and migration of fibroblast-like synoviocytes in rheumatoid arthritis via the PI3K/AKT signaling pathway

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease; its pathogenesis remains unclear. Fibroblast-like synoviocytes (FLSs) play a vital role in the pathogenesis of RA. BMP9, a member of the bone morphogenetic protein (BMP) family, has been reported to play a critical role in both normal physiological processes and the pathology of various diseases. In this study, we explored the function and underlying mechanisms of BMP9 in the proliferation and migration of RA FLSs. We found that BMP9 expression was significantly downregulated in the synovial tissues of RA patients, compared with those of OA patients; BMP9 expression was also low in adjuvant-induced arthritis (AA) samples. Additionally, inhibition of BMP9 expression by BMP9 siRNA increased the proliferation of AA FLSs, and the expression of c-Myc, Cyclin D1, MMP-2, and MMP-9, but not TIMP-1, in AA FLSs. However, AA FLSs transfected with the overexpression vector PEX-3-BMP9 showed reduced proliferation and expression of c-Myc, Cyclin D1, MMP-2, and MMP-9, but not TIMP-1. Further studies indicate that BMP9 may induce the activation of the PI3K/AKT signaling pathway. Thus, these data indicate that BMP9 may play a critical role in the proliferation and migration of FLSs through the activation of the AKT signaling pathway.

Peficitinib Inhibits the Chemotactic Activity of Monocytes via Proinflammatory Cytokine Production in Rheumatoid Arthritis Fibroblast-Like Synoviocytes

Background: This study was performed to examine the effects of the Janus kinase (JAK) inhibitor peficitinib on fibroblast-like synoviocytes (FLS) obtained from patients with rheumatoid arthritis (RA). Methods: To examine the expression of JAK1, JAK2, and JAK3 in RA synovial tissue (ST) and FLS, immunohistochemistry was performed. We investigated the effects of peficitinib on interleukin 6 and IL-6 receptor responses in RA FLS. Phosphorylation of STAT was determined by western blot. To examine the functional analysis of peficitinib, we performed a proliferation and chemotaxis assays with FLS using THP-1 and peripheral blood mononuclear cells (PBMC). The inflammatory mediator expression of FLS was estimated by enzyme-linked immunosorbent assay. Results: JAK1, JAK2, and JAK3 were expressed in RA STs and FLS. Phosphorylation of STAT1, STAT3, and STAT5 in RA FLS was suppressed by peficitinib in a concentration-dependent manner. Peficitinib-treated RA FLS-conditioned medium reduced THP-1 and PBMC migration (p < 0.05) and proliferation of RA FLS (p < 0.05). Peficitinib suppressed the secretion of MCP-1/CCL2 in the RA FLS supernatant (p < 0.05). Conclusion: Peficitinib suppressed the JAK-STAT pathway in RA FLS and also suppressed monocyte chemotaxis and proliferation of FLS through inhibition of inflammatory cytokines.

Over-Expression of PTEN Suppresses the Proliferation and Migration of Fibroblast-Like Synoviocytes in Adjuvant-Induced Arthritis.

Rheumatoid arthritis (RA) is characterized by a tumor-like expansion of the synovium and the subsequent destruction of adjacent articular cartilage and bone. Recent studies have shown that phosphatase and tension homolog deleted on chromosome 10 (PTEN) might contribute to the surviva of fibroblast-like synoviocytes (FLSs) and the production of pro-inflammatory cytokines in RA. The purpose of this study was to explore the functions and underlying mechanisms of PTEN in the proliferation and migration of FLSs. FLSs were obtained from adjuvant-induced arthritis (AIA) and normal rats. The expression levels of PTEN, c-Myc, cyclin D1, PCNA, and MMP-9 were detected by quantitative-real-time-PCR and western blot assay. A BrdU proliferation assay, cell cycle analysis, and a wound-healing assay were used to study the role of PTEN in FLSs treated with PTEN inhibitor bpv, specific small interfering RNA targeting PTEN (PTEN-RNAi) or a PTEN over-expression vector (PTEN-GV141). Chromatin immunoprecipitation and methylation-special PCR assays were used to study the expression of PTEN mRNA in the presence of DNA methylation. PTEN expression was downregulated in AIA FLSs in comparison to normal rats. Moreover, inhibition of PTEN expression by bpv or PTEN-RNAi could promote the proliferation and migration of FLSs, and increase the expression of c-Myc, cyclin D1, PCNA, and MMP-9 in AIA FLSs, but had no effect on TIMP-1 expression.In addition, transfection of AIA FLSs with PTEN-GV141 reduced their proliferation and migration. Further study indicated that DNA methylation could regulate PTEN expression in AIA. Our findings suggest that PTEN might play a pivotal role in the proliferation and migration of FLSs through the activation of the AKT signaling pathway. Additionally, PTEN expression may be regulated by DNA methylation in the pathogenesis of AIA.

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

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. 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. 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. Abatacept inhibits FLS migration and MMPs expressions via inhibiting MAPK pathway, thereby inhibiting RA development.

DDR2–CYR61–MMP1 Signaling Pathway Promotes Bone Erosion in Rheumatoid Arthritis Through Regulating Migration and Invasion of Fibroblast-Like Synoviocytes

Regulation of matrix metalloproteinases (MMPs) by collagen in the fibroblast-like synoviocytes (FLSs) plays a critical role in joint destruction in rheumatoid arthritis (RA). Our previous study indicated that discoidin receptor 2 (DDR2) mediated collagen upregulation of MMPs. However, the precise underlying mechanism remains unclear. We report here that CYR61, a secreted, extracellular matrix-associated signaling protein which is capable of regulating a broad range of cellular activities, including cell adhesion, migration, proliferation, and apoptosis, is significantly upregulated in collagen II-stimulated RA FLS. Further studies found that collagen II-activated phosphorylated-DDR2 induces CYR61 through activation of transcription factor activator protein 1 (AP-1). The elevated CYR61, in turn, accelerates MMP1 production via ETS1 (ETS proto-oncogene 1). In addition, CYR61 significantly promotes FLS invasion and migration. Blockade of CYR61 by an adenovirus expressing CYR61 shRNA (Ad-shCYR61) in vivo remarkably ameliorated the severity of arthritis, reduced inflammatory cytokine secretion, and attenuated bone erosion as detected by micro-computed tomography (μCT), in collagen-induced arthritis (CIA) rats. Taken together, we uncovered the Collagen II-DDR2-AP-1-CYR61-ETS1-MMP1 loop in RA FLS. In which, CYR61 acts as a hinge to promote cartilage damage through regulating FLS invasion, migration, and MMP1 production and the inflammatory cascade in RA. Thus, CYR61 may be a promising diagnostic and therapeutic target for RA treatment. © 2016 American Society for Bone and Mineral Research.

Identification of Novel Genes in Osteoarthritic Fibroblast-Like Synoviocytes Using Next-Generation Sequencing and Bioinformatics Approaches.

Synovitis in osteoarthritis (OA) the consequence of low grade inflammatory process caused by cartilage breakdown products that stimulated the production of pro-inflammatory mediators by fibroblast-like synoviocytes (FLS). FLS participate in joint homeostasis and low grade inflammation in the joint microenvironment triggers FLS transformation. In the current study, we aimed to identify differentially expressed genes and potential miRNA regulations in human OA FLS through deep sequencing and bioinformatics approaches. The 245 differentially expressed genes in OA FLS were identified, and pathway analysis using various bioinformatics databases indicated their enrichment in functions related to altered extracellular matrix organization, cell adhesion and cellular movement. Moreover, among the 14 dysregulated genes with potential miRNA regulations identified, src kinase associated phosphoprotein 2 (SKAP2), adaptor related protein complex 1 sigma 2 subunit (AP1S2), PHD finger protein 21A (PHF21A), lipoma preferred partner (LPP), and transcription factor AP-2 alpha (TFAP2A) showed similar expression patterns in OA FLS and OA synovial tissue datasets in Gene Expression Omnibus database. Ingenuity Pathway Analysis identified the dysregulated LPP participated in cell migration and cell spreading of OA FLS, which was potentially regulated by miR-141-3p. The current findings suggested new perspectives into understanding the novel molecular signatures of FLS involved in the pathogenesis of OA, which may be potential therapeutic targets.

Role of small ubiquitin-like modifier proteins-1 (SUMO-1) in regulating migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is featured by erosive cartilage and bone destruction. The enhancing aggressive property of fibroblast-like synoviocytes (FLSs) plays a critical role in this process. Small ubiquitin-like modifier (SUMO) proteins, including SUMO-1, SUMO-2, SUMO-3 and SUMO-4, participate in regulating many cellular events such as survival, migration and signal transduction in some cell lines. However, their roles in the pathogenesis of RA are not well established. Therefore, we evaluated the role of SUMO proteins in RA FLSs migration and invasion. We found that expression of both SUMO-1 and SUMO-2 was elevated in FLSs and synovial tissues (STs) from patients with RA. SUMO-1 suppression by small interference RNA (siRNA) reduced migration and invasion as well as MMP-1 and MMP-3 expression in RA FLSs. We also demonstrated that SUMO-1 regulated lamellipodium formation during cell migration. To explore further into molecular mechanisms, we evaluated the effect of SUMO-1 knockdown on the activation of Rac1/PAK1, a critical signaling pathway that controls cell motility. Our results indicated that SUMO-1-mediated SUMOylation controlled Rac1 activation and modulated downstream PAK1 activity. Inhibition of Rac1 or PAK1 also decreased migration and invasion of RA FLSs. Our findings suggest that SUMO-1 suppression could be protective against joint destruction in RA by inhibiting aggressive behavior of RA FLSs.

Therapeutic Potential of Mesenchymal Cell-Derived miRNA-150-5p-Expressing Exosomes in Rheumatoid Arthritis Mediated by the Modulation of MMP14 and VEGF.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial tissue inflammation and joint destruction associated with the activation of angiogenesis. Exosomes, which play a role in cell-to-cell communication as carriers of genetic information, transfer microRNAs (miRNAs or miRs) between cells and have been studied as delivery vehicles for therapeutic molecules. The aim of the current study was to investigate the therapeutic effect of mesenchymal stem cell (MSC)-derived miR-150-5p exosomes on joint destruction in RA. The expression and secretion of miR-150-5p, matrix metalloproteinase (MMP) 14, and vascular endothelial growth factor (VEGF) in RA patients and fibroblast-like synoviocytes (FLS) were examined by quantitative RT-PCR, ELISA, and Western blotting. Immunohistochemistry was used to assess angiogenesis. MSCs were transfected with an miR-150-5p expression plasmid, and MSC-derived exosomes were harvested. The effect of MSC-derived miR-150-5p exosomes (Exo-150) on MMP14 and VEGF expression was examined. The effects of Exo-150 on cell migration and invasion in cytokine-stimulated FLS from RA patients were examined by HUVEC tube formation and transwell assays. The effect of Exo-150 in vivo was examined in a collagen-induced arthritis mouse model. Exo-150 decreased migration and invasion in RA FLS and downregulated tube formation in HUVECs by targeting MMP14 and VEGF. Injection of Exo-150 reduced hind paw thickness and the clinical arthritic scores in collagen-induced arthritis mice. Exo-150 reduced joint destruction by inhibiting synoviocyte hyperplasia and angiogenesis. Exosomes facilitate the direct intracellular transfer of miRNAs between cells and represent a potential therapeutic strategy for RA.