Laminin-dependent inflammatory response in synovial fibroblasts of rheumatoid arthritis patients

Elevated expression of matrix-metalloproteinases (MMP) contributes to cartilage destruction in rheumatoid arthritis. We report on a novel pathway of inflammatory activation of synovial fibroblasts that is induced by TGF-beta and laminin (extracellular matrix) and leads to increased expression of the proteases MMP-3 and MMP-10. Neither costimulation by the central inflammatory cytokines TNF-alpha and IL-1beta nor NFkB signalling is needed for this pathway.     

Microsatellite analysis in rheumatoid arthritis synovial fibroblasts

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic disease characterised by irreversible destruction of the affected joints. As aggressive transformed-appearing synovial fibroblasts are commonly found at the site of invasion of the rheumatoid synovium into the adjacent cartilage and bone, the presence of microsatellite instability (MSI) and expression of mismatch repair enzymes as a possible mechanism in the alteration of these cells was examined. METHODS: DNA was extracted from the synovial fibroblasts and blood of 20 patients with long term RA undergoing joint replacement, and the presence of MSI was studied at 10 microsatellite loci. In addition, immunohistochemistry was performed to evaluate the expression of the two major mismatch repair enzymes (hMLH1 and hMSH2) in rheumatoid synovium. RESULTS: MSI could not be detected in any of the fibroblast cell populations derived from the 20 different rheumatoid synovial samples. In addition, strong expression of mismatch repair enzymes could be seen in numerous cells, including fibroblasts, throughout the synovium. CONCLUSIONS: Applying the currently used and established markers for MSI, the data show for the first time that MSI does not appear to have an important role in alteration of rheumatoid synovial fibroblasts into an aggressive phenotype. On the other hand, strong mismatch repair enzyme synthesis in rheumatoid synovium supports the hypothesis of continuing DNA repair, presumably due to long term, inflammation induced DNA damage.     

Trex‐1 deficiency in rheumatoid arthritis synovial fibroblasts

Objective

To explore whether the increased expression of long interspersed nuclear element 1 (LINE‐1; L1) messenger RNA (mRNA) and protein in rheumatoid arthritis synovial fibroblasts (RASFs) is associated with decreased expression of Trex‐1, an exonuclease involved in the metabolization of L1 DNA:RNA hybrids.

Methods

Chromatin immunoprecipitation was used to detect L1‐related p40 protein (L1‐ORF1p) binding sequences in RASFs. Luciferase activity was measured in the synovial fibroblasts following cotransfection of the episomal plasmid with pJM105 expressing L1‐ORF1p and pGL3‐TS3 carrying the target sequence for L1‐ORF1p. This luciferase reporter assay was used to compare the activity between RASFs and osteoarthritis synovial fibroblasts (OASFs) and to assess correlations of luciferase activity with the expression of Trex‐1 measured by flow cytometry. The expression of Trex‐1 mRNA and protein was also compared using real‐time polymerase chain reaction, immunohistochemistry, and Western blot analyses. The role of Trex‐1 in the L1‐ORF1p–mediated luciferase activity assay was studied using interfering RNAs (iRNA) and a Trex‐1 expression vector.

Results

Increased luciferase activity occurred after cotransfection of synovial fibroblasts with pJM105 and pGL3‐TS3. L1‐ORF1p activity was increased in RASFs as compared with OASFs, and this was correlated inversely with the expression of Trex‐1. Levels of Trex‐1 mRNA and protein were lower in RASFs than in OASFs. After transfection of the L1 expression plasmid, Trex‐1 mRNA levels increased in OASFs, but not in RASFs. The addition of iRNA against Trex‐1, however, resulted in an enhancement of L1‐ORF1p activity in OASFs to the levels measured in RASFs. Overexpression of Trex‐1 inhibited 5‐azacytidine–induced expression of p38δ MAPK, a gene carrying the TS3 sequence.

Conclusion

The deficiency of Trex‐1 in RASFs allows a longer half‐life of gene products encoded by active endogenous L1 retrotransposons. This pathway may play a role in diseases in which the cells exhibit a “spontaneous” aggressive behavior.

     

DNA hypomethylation in rheumatoid arthritis synovial fibroblasts

Objective

Rheumatoid arthritis synovial fibroblasts (RASFs) are phenotypically activated and aggressive. We undertook this study to investigate whether the intrinsic activation of RASFs is due to global genomic hypomethylation, an epigenetic modification.

Methods

Global genomic hypomethylation was assessed by immunohistochemistry, flow cytometry, and L1 promoter bisulfite sequencing. The levels of Dnmt1 were determined in synovial tissue and cultured SFs by Western blotting before and after treatment with cytokines and growth factors. Normal SFs were treated for 3 months with a nontoxic dose of the DNA hypomethylation drug 5‐azacytidine (5‐azaC), and changes in gene expression were revealed using complementary DNA arrays. The phenotypic changes were confirmed by flow cytometry.

Results

In situ and in vitro, RASF DNA had fewer 5‐methylcytosine and methylated CG sites upstream of an L1 open‐reading frame than did DNA of osteoarthritis SFs, and proliferating RASFs were deficient in Dnmt1. Using 5‐azaC, we reproduced the activated phenotype of RASFs in normal SFs. One hundred eighty‐six genes were up‐regulated >2‐fold by hypomethylation, with enhanced protein expression. These included growth factors and receptors, extracellular matrix proteins, adhesion molecules, and matrix‐degrading enzymes. The hypomethylating milieu induced irreversible phenotypic changes in normal SFs, which resembled those of the activated phenotype of RASFs.

Conclusion

DNA hypomethylation contributes to the chronicity of RA and could be responsible for the limitation of current therapies.

     

RNAi抑制类风湿关节炎成纤维细胞环氧合酶-2合成及对炎症因子的影响

目的体外合成和筛选特异性阻断人滑膜成纤维细胞（RASF）环氧合酶-2（hCOX-2）的小分子干扰RNA（siRNA），同时了解COX-2抑制对炎症因子表达水平的影响。方法设计4条针对人COX-2mRNA siRNA（1#-4#siRNA），1条随机序列作为对照。分成A～H组，A组为空白阴性对照，B～F组处理依次为随机siRNA、1#-4#siRNA。应用LipofectAMINE 2000将上述siRNA转染入RASF，各培养孔加入100nmol／L的佛波酯。转染48h后，分别应用反转录聚合酶链式反应（RT-PCR）和Western Blot检测hCOX-2mRNA和蛋白表达水平。采用酶联免疫吸附（ELISA）方法检测各组上清液中前列腺素E2（PGE2）和白细胞介素（IL）-1β、IL-6、肿瘤坏死因子（TNF）-α、血管内皮生长因子（VEGF）水平。结果4#siRNA转染的RASF表达hCOX-2mRNA和蛋白水平明显低于其他siRNA干预组和阴性对照，且其上清液PGE2和IL-1β、IL-6、TNF-α、VEGF水平较其他各组明显下降。结论4#siRNA能有效抑制COX-2mRNA表达和COX-2蛋白的合成，且上清液中PGE2和IL-1β、IL-6、TNF-α及VEGF水平最低。     

Effects of miR-150-5p on the growth and SOCS1 expression of rheumatoid arthritis synovial fibroblasts

Clinical Rheumatology - miR-150-5p has been implicated in the regulation and onset of immune diseases. We investigated the effects of miR-150-5p on the functions of RA synovial fibroblasts (RASFs)....     

Relationship between IgG2b-inducing activity in rheumatoid arthritis synovial fluid and other well-known cytokines and inflammatory mediators

In the present study, we sought to identify the T cell-replacing factor which selectively induces IgG2b antibody formation in lipopolysaccharide-activated mouse spleen cells in vitro and in vivo, and which is present in the synovial fluid (SF) of rheumatoid arthritis (RA) patients. The protein A plaque assay was used to measure IgM, IgG1, IgG2b, and IgG3 plaque-forming cells. An enzyme-linked immunosorbent assay was used to measure interleukin-6 (IL-6) levels in RA SF. We found that IgG2b induction by RA SF is not caused by IL-6, IL-1, or any other inflammatory cytokines or mediators, such as transforming growth factor beta, platelet-derived growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, elastase, collagenase, and phospholipase A2. IgG2b-inducing factor in RA SF has unique biological properties compared with those of the interleukins and inflammatory mediators known to be present in RA SF.     

Adiponectin stimulates prostaglandin E2 production in rheumatoid arthritis synovial fibroblasts

Objective

Adipokines may influence inflammatory and/or immune responses. This study was undertaken to examine whether adiponectin affects the production of prostaglandin E₂ (PGE₂) by rheumatoid arthritis synovial fibroblasts (RASFs).

Methods

Synovial tissue was obtained from patients with RA who were undergoing joint replacement surgery. Fibroblast‐like cells from the third or fourth passage were used as RASFs. Expression of adiponectin receptor messenger RNA (mRNA) and protein was detected. PGE₂ (converted from arachidonic acid) was measured by enzyme‐linked immunosorbent assay (ELISA). Expression of mRNA and protein for cyclooxygenase 2 (COX‐2) and membrane‐associated PGE synthase 1 (mPGES‐1), key enzymes involved in PGE₂ synthesis, was detected in RASFs. The effects of RNA interference (RNAi) targeting the adiponectin receptor genes and the receptor signal inhibitors were examined. The influence of adiponectin on NF‐κB activation in RASFs was measured with an ELISA kit.

Results

Adiponectin receptors were detected in RASFs. Adiponectin increased both COX‐2 and mPGES‐1 mRNA and protein expression by RASFs in a time‐ and concentration‐dependent manner. PGE₂ production by RASFs was also increased by the addition of adiponectin, and this increase was inhibited by RNAi for the adiponectin receptor gene, or coincubation with the receptor signal inhibitors. Enhancement of NF‐κB activation by adiponectin as well as by interleukin‐1β was observed in RASFs.

Conclusion

Our findings indicate that adiponectin induces COX‐2 and mPGES‐1 expression, resulting in the enhancement of PGE₂ production by RASFs. Thus, adiponectin may play a role in the pathogenesis of synovitis in RA patients.

     

MiR-155在类风湿关节炎滑膜成纤维细胞中的表达及功能研究

目的 检测miR-155在类风湿关节炎(RA)患者滑膜成纤维细胞(SFs)中的表达,探讨miR-155对RASFs细胞因子分泌、细胞增殖、侵袭能力的影响及其机制.方法 留取RA患者及骨关节炎(OA)患者各9份膝关节置换术后滑膜组织,分离培养滑膜成纤维细胞,取第3～5代细胞用于实验.①提取总RNA,利用实时定量聚合酶链反应(RT-PCR)方法测定miR-155在RASFs和OASFs中固有性表达水平.②Lipo2000脂质体分别转染化学合成的miR-155类似物,miR-155抑制剂及无关序列小RNA对照.③转染48 h后通过酶联免疫吸附试验(E12SA)检测细胞上清基质金属蛋白酶(MMP)-3的分泌;通过3H掺入法检测细胞增殖;通过细胞侵袭试验(transwell)法检测细胞侵袭能力;通过RT-PCR法检测miR-155下游靶标IKBKE的mRNA表达水平.2组比较采用独立样本t检验,多组比较采用方差分析.结果 ①RA患者SFs中miR-155的表达明显高于OA组[分别为(1.79±1.94)和(0.11±0.17),P<0.05];②miR-155可抑制RASFs分泌MMP-3、细胞增殖和侵袭能力;③RASFs过表达miR-155后IKBKE的mRNA水平明显下调(P<0.05).结论 RASFs miR-155的表达上调,可能与滑膜局部的炎症环境相关;miR-155抑制RASFs增殖和降低侵袭能力可能是RASFs分泌MMP-3减少的原因之一,miR-155抑制RASFs分泌MMP-3可能与miR-1.55下调靶标IKBKE mRNA表达水平相关.     

Cordycepin inhibits IL-1{beta}-induced MMP-1 and MMP-3 expression in rheumatoid arthritis synovial fibroblasts

Objective. MMP is a key enzyme in the degradation of extracellularmatrices, and its expression plays important roles in inflammatorydiseases. Cordycepin (3'-deoxyadenosine), a bioactive compoundof Cordyceps militaris, has been shown to exhibit many pharmacologicalactivities, such as anti-cancer, anti-inflammatory and anti-infectionactivities. In this study, we aimed at the inhibitory effectof cordycepin on IL-1β-induced MMP-1 and MMP-3 expressionas well as the molecular basis using RA synovial fibroblasts(RASFs). Methods. RASFs were isolated from synovial tissue obtained from12 patients with RA and cultured in monolayer. Expression ofMMP-1 and MMP-3 was evaluated using western blotting and real-timePCR. Chemokines were analysed by ELISA. The phosphorylationof mitogen-activated protein kinase was measured by westernblotting. Electrophoretic mobility shift assay was performedto evaluate binding activities of DNA to nuclear factor-B (NF-B)and activator protein-1 (AP-1). Results. Cordycepin inhibited IL-1β-induced MMP-1 and MMP-3expressions in RASFs in a dose-dependent manner. Among variouschemokines [such as monocyte chemoattractant protein-1 (MCP-1),GRO-, regulated upon activation, normal T-cell expressed andpresumably secreted (RANTES) and epithelial neutrophil activatingpeptide 78 (ENA-78)], cordycepin specifically blocked IL-1β-inducedENA-78 production in RASF. Moreover, cordycepin significantlyinhibited IL-1β-induced p38/JNK and AP-1 activation, butnot extracellular signal-regulated kinase (ERK) and NF-B activation. Conclusions. Cordycepin is a potent inhibitor of IL-1β-inducedchemokine production and MMP expression and strongly blocksthe p38/JNK/AP-1 signalling pathway in RASFs. KEY WORDS: Cordycepin, Interleukin-1β, Matrix metalloproteinase, p38 mitogen-activated protein kinase, Activator protein-1     

LIGHT induces cell proliferation and inflammatory responses of rheumatoid arthritis synovial fibroblasts via lymphotoxin beta receptor

OBJECTIVE: To investigate the effects of LIGHT (lymphotoxin-like, exhibits inducible expression and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes) on the proliferation and gene expression of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). METHODS: We measured LIGHT levels in RA synovial fluids (SF) by ELISA, and compared them with those in osteoarthritis (OA) SF. Levels of LIGHT and its receptors in RA-FLS and synovium were assessed using real-time quantitative polymerase chain reaction (PCR). RA-FLS proliferation was examined by a bromodeoxyuridine assay. Expression of intercellular adhesion molecule-1 (ICAM-1) and several chemokines, such as interleukin 8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha), was examined by real-time quantitative PCR, ELISA, and flow cytometry. The effects of LIGHT on nuclear factor-kappaB (NF-kappaB) activation were investigated using immunofluorescence and Western blotting. RESULTS: LIGHT was upregulated in both SF and synovium of RA patients compared with OA patients. Herpes virus entry mediator (HVEM) and lymphotoxin beta receptor (LTbetaR), but not LIGHT, were detected in RA-FLS. LIGHT significantly promoted RA-FLS proliferation and induced expression of MCP-1, IL-8, MIP-1alpha, and ICAM-1 by RA-FLS. As well, LTbetaR small interfering RNA (siRNA), but not HVEM siRNA, inhibited these effects of LIGHT. LIGHT induced IkappaBa degradation and NF-kappaB translocation, and a NF-kappaB inhibitor suppressed the effects of LIGHT on RA-FLS. CONCLUSION: Our findings suggest that LIGHT signaling via LTbetaR plays an important role in the pathogenesis of RA by affecting key processes such as the proliferation and activation of RA-FLS. Regulation of LIGHT-LTbetaR signaling may represent a new therapeutic target for RA treatment.     

Functional analysis of choline transporters in rheumatoid arthritis synovial fibroblasts

Objectives: In this study, we examined the functional characteristics of choline uptake and sought to identify the transporters in rheumatoid arthritis synovial fibroblasts (RASFs).

Methods: The expression of choline transporters was evaluated by quantitative real-time PCR, western blotting, and immunocytochemistry. Time course, Na⁺-dependency, and kinetics of [³H]choline uptake were investigated. Effects of cationic drugs on the uptake of [³H]choline, cell viability, and caspase-3/7 activity were also examined. Finally, we investigated the influence of choline uptake inhibitor, hemicholinium-3 (HC-3), and choline deficiency on cell viability and caspase-3/7 activity.

Results: Choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein were highly expressed in RASFs and were localized to the plasma membrane. [³H]Choline uptake occurred via a Na⁺-independent and pH-dependent transport system. The cells have two different [³H]choline transport systems, high- and low-affinity. Various organic cations, HC-3 and choline deficiency inhibited both [³H]choline uptake and cell viability, and enhanced the caspase-3/7 activity. The functional inhibition of choline transporters could promote apoptotic cell death. In RASFs, [³H]choline uptake was significantly increased compared with that in OASFs without a change in gene expression.

Conclusions: These results suggest that CTL1 (high-affinity) and CTL2 (low-affinity) are highly expressed in RASFs and choline may be transported by a choline/H^+?antiport system. Identification of this CTL1- and CTL2-mediated choline transport system should provide a potential new target for RA therapy.     

Correction to: Effects of miR-150-5p on the growth and SOCS1 expression of rheumatoid arthritis synovial fibroblasts

Clinical Rheumatology - The first name of the co-author of the above article was presented incorrect in the published version. The author name “Miangliang Qiu” should read...