Dataset Information

Cyr61 synthesis is induced by interleukin-6 and promotes migration and invasion of fibroblast-like synoviocytes in rheumatoid arthritis.

ABSTRACT:

Background

Interleukin-6 (IL-6) is involved in fibroblast-like synoviocyte (FLS) activation and promotes pannus formation and bone and cartilage destruction in rheumatoid arthritis (RA). Cysteine-rich 61 (Cyr61) protein regulates cell proliferation, migration, and differentiation. The aim of this study was to investigate the role of Cyr61 in RA-FLS migration and invasion after IL-6 stimulation.

Methods

Western blotting, immunohistochemistry, reverse transcription-polymerase chain reaction, and real time-polymerase chain reaction were used to examine protein and mRNA levels of Cyr61, matrix metalloproteinases (MMPs), and other signalling proteins. Knockdown of gene expression was performed with siRNA, and RNA sequencing was performed for differential gene analysis. Migration and invasion were assessed by wound healing and Boyden chamber assays.

Results

Cyr61 levels were elevated in FLSs from RA patients compared to those in osteoarthritis patients. Control and IL-6-treated FLSs showed differential gene expression. IL-6 stimulated protein synthesis of Cyr61, which was attenuated by the extracellular signal-related kinase 1/2 (ERK 1/2) inhibitor, PD98059, and knockdown of early growth response 3 (EGR3), but not of JUN. IL-6-induced Cyr61 protein synthesis increased expression of MMP2. Cyr61 promoted FLS migration and invasion in an autocrine manner. Knockdown of CYR61 and a neutralising antibody attenuated Cyr61 synthesis and IL-6-induced FLS migration.

Conclusions

By modulating the ERK/EGR3 pathway, IL-6 stimulated Cyr61 production and in turn increased invasiveness of FLS. Our data suggest that Cyr61 might be a potential target to prevent the progression of joint damage in RA.

SUBMITTER: Choi C

PROVIDER: S-EPMC7685583 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Publications

Cyr61 synthesis is induced by interleukin-6 and promotes migration and invasion of fibroblast-like synoviocytes in rheumatoid arthritis.

Choi Changmin C Jeong Wooseong W Ghang Byeongzu B Park Yonggeun Y Hyun Changlim C Cho Moonjae M Kim Jinseok J

Arthritis research & therapy 20201123 1

<h4>Background</h4>Interleukin-6 (IL-6) is involved in fibroblast-like synoviocyte (FLS) activation and promotes pannus formation and bone and cartilage destruction in rheumatoid arthritis (RA). Cysteine-rich 61 (Cyr61) protein regulates cell proliferation, migration, and differentiation. The aim of this study was to investigate the role of Cyr61 in RA-FLS migration and invasion after IL-6 stimulation.<h4>Methods</h4>Western blotting, immunohistochemistry, reverse transcription-polymerase chain ...[more]

PMID: 33228785

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Project description:IntroductionAnti-malarial drug artesunate can suppress inflammation and prevent cartilage and bone destruction in collagen-induced arthritis model in rats-suggesting it may be a potent drug for rheumatoid arthritis (RA) therapy. We aimed to investigate its effect on the invasive property of fibroblast-like synoviocytes (FLS) from patients with RA.MethodsSynovial tissues were obtained by closed needle biopsy from active RA patients, and FLS were isolated and cultured in vitro. RA-FLS were treated with artesunate at various concentrations, while methotrexate or hydroxychloroquine was employed as comparator drugs. Cell viability, proliferation, cell cycle, apoptosis, migration, invasion, and pseudopodium formation of RA-FLS were assessed by CCK-8 assays, EdU staining, Annexin V-FITC/PI staining, transwell assays, or F-actin staining, respectively. Further, relative changes of expressed proteases were analyzed by Proteome profiler human protease array and verified by quantitative real-time PCR (qPCR), Western blot, and ELISA. The expression of signaling molecules of MAPK, NF-κB, AP-1, and PI3K/Akt pathways were measured by qPCR and Western blot. PDK-1 knockdown by specific inhibitor AR-12 or siRNA transfection was used to verify the pharmacological mechanism of artesunate on RA-FLS.ResultsArtesunate significantly inhibited the migration and invasion of RA-FLS in a dose-dependent manner with or without TNF-α stimulation. The effect was mediated through artesunate inhibition of MMP-2 and MMP-9 production, and pre-treatment with exogenous MMP-9 reversed the inhibitory effect of artesunate on RA-FLS invasion. Artesunate had a stronger inhibitory effect on migration and invasion of RA-FLS as well as greater anti-inflammatory effect than those of hydroxychloroquine. Similar inhibitory effect was detected between artesunate and methotrexate, and synergy was observed when combined. Mechanistically, artesunate significantly inhibited PDK-1 expression as well as Akt and RSK2 phosphorylation-in a similar manner to PDK-1-specific inhibitor AR-12 or PDK-1 knockdown by siRNA transfection. This inhibition results in suppression of RA-FLS migration and invasion as well as decreased MMP-2 and MMP-9 expression.ConclusionsOur study demonstrates artesunate is capable of inhibiting migration and invasion of RA-FLS through suppression of PDK1-induced activation of Akt and RSK2 phosphorylation-suggesting that artesunate may be a potential disease-modifying anti-rheumatic drug for RA.

Project description:INTRODUCTION:It is well known that neutrophils play very important roles in the development of rheumatoid arthritis (RA) and interleukin (IL)-8 is a critical chemokine in promoting neutrophil migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in RA promotes FLS proliferation and Th17 cell differentiation, thus Cyr61 is a pro-inflammatory factor in RA pathogenesis. In this study, we explored the role of Cyr61 in neutrophil migration to the joints of RA patients. METHODS:RA FLS were treated with Cyr61 and IL-8 expression was analyzed by real-time PCR and ELISA. The migration of neutrophils recruited by the culture supernatants was determined by the use of a chemotaxis assay. Mice with collagen-induced arthritis (CIA) were treated with anti-Cyr61 monoclonal antibodies (mAb), or IgG1 as a control. Arthritis severity was determined by visual examination of the paws and joint destruction was determined by hematoxylin-eosin (H&E) staining. Signal transduction pathways in Cyr61-induced IL-8 production were investigated by real-time PCR, western blotting, confocal microscopy, luciferase reporter assay or chromatin immunoprecipitation (ChIP) assay. RESULTS:We found that Cyr61 induced IL-8 production by RA FLS in an IL-1? and TNF-? independent pathway. Moreover, we identified that Cyr61-induced IL-8-mediated neutrophil migration in vitro. Using a CIA animal model, we found that treatment with anti-Cyr61 mAb led to a reduction in MIP-2 (a counterpart of human IL-8) expression and decrease in neutrophil infiltration, which is consistent with an attenuation of inflammation in vivo. Mechanistically, we showed that Cyr61 induced IL-8 production in FLS via AKT, JNK and ERK1/2-dependent AP-1, C/EBP? and NF-?B signaling pathways. CONCLUSIONS:Our results here reveal a novel role of Cyr61 in the pathogenesis of RA. It promotes neutrophil infiltration via up-regulation of IL-8 production in FLS. Taken together with our previous work, this study provides further evidence that Cyr61 plays a key role in the vicious cycle formed by the interaction between infiltrating neutrophils, proliferated FLS and activated Th17 cells in the development of RA.

Project description:BackgroundFibroblast-like synoviocytes (FLSs), which can migrate and directly invade the cartilage and the bone, are crucial players in joint damage in rheumatoid arthritis (RA). Nevertheless, the detailed mechanisms underlying the aberrant activation of RA FLSs remain unclear. Several studies have attempted to explore the relationship between long non-coding RNAs (lncRNAs) and RA pathology; however, the role of lncRNAs in RA is unknown. The present study aimed to determine the functions of tumor necrosis factor-α and heterogeneous nuclear ribonucleoprotein L-related immunoregulatory lincRNA (THRIL) in RA FLSs migration and invasion.MethodsSmall interfering RNA targeting THRIL or lentivirus overexpressing THRIL was used to knockdown or overexpress THRIL. Quantitative reverse transcription polymerase chain reaction (PCR) was employed for the detection of RNA expression. The proliferation rate of RA FLSs was measured using a 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Migration and invasion were detected using a transwell chamber. Downstream targets were identified using a human cell cycle real-time PCR array and a human cell motility real-time PCR array.ResultsA significant decrease in THRIL expression was found in RA FLSs compared with cells from healthy control (HC)patients. THRIL is mainly localized in the nucleus. Knockdown of THRIL increased the proliferation, migration, and invasion of RA FLSs. In contrast, THRIL overexpression had the opposite effect. THRIL knockdown increased interleukin-1β (IL-1β)-triggered expression of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13. THRIL overexpression led to a significant decrease in MMP-13 expression in response to stimulation with IL-1β. Furthermore, we observed that the expression levels of cyclin-dependent kinase 1 (CDK1) and G2 and S phase-expressed-1 (GTSE1), both of which are associated with cellular mobility and proliferation, were downregulated with THRIL overexpression.ConclusionsReduced expression of lncRNA THRIL represses the proliferation, migration, and invasion of RA FLSs, suggesting that lncRNA THRIL might be a potential target for RA therapy.

Dataset Information

Cyr61 synthesis is induced by interleukin-6 and promotes migration and invasion of fibroblast-like synoviocytes in rheumatoid arthritis.

Background

Methods

Results

Conclusions

Publications

Cyr61 synthesis is induced by interleukin-6 and promotes migration and invasion of fibroblast-like synoviocytes in rheumatoid arthritis.

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