Expression In Fibroblast-like Synoviocytes Research Articles

MicroRNA-152 modulates the canonical Wnt pathway activation by targeting DNA methyltransferase 1 in arthritic rat model.

Rheumatoid arthritis (RA) is an autoimmune and progressive systemic disease of unknown etiology. Research shows that fibroblast-like synoviocytes (FLS) participate in the cartilage erosion, synovial hyperplasia, inflammatory cytokine secretion and suggests that fibroblast-like synoviocytes (FLS) display a crucial role in RA pathogenesis. Recent studies have suggested the role of the Wnt signaling pathway in the pathogenesis of RA. In previous study, we identified that increased methyl-CpG-binding protein 2 (MeCP2) reduced the secreted frizzled-related protein 4 (SFRP4) expression in FLS in Arthritic rat model and the DNA methyltransferase (DNMT) inhibitor 5-Aza-2'-deoxycytidine (5-azadC) could induce the SFRP4 expression, indicating that DNMT has a key role in the differential expression of SFRP4. MicroRNAs (MiRNAs), which are small non-coding RNAs, are involved in diverse biological functions, regulation of gene expression, pathogenesis of autoimmune disease and carcinogenesis. In light of the directly down-regulation of miR-152 on DNMT1 expression by targeting the 3' untranslated regions of its transcript in nickel sulfide (NiS)-transformed human bronchial epithelial cells, we investigated whether miR-152 is aberrantly expressed and targets DNMT1 in FLS in Arthritic rat model. Our results demonstrated that the expression of miR-152 was specifically down-regulated in Arthritic rat model, whereas up-regulation of miR-152 in FLS resulted in a marked reduction of DNMT1 expression. Further experiments revealed that increased miR-152 indirectly up-regulated the SFRP4 expression, a negative regulator of WNT signaling pathway, by targeting the DNMT1. Moreover, activation of miR-152 expression in FLS could inhibit the canonical Wnt pathway activation and result in a significant decrease of FLS proliferation. MiR-152 and DNA methylation may provide molecular mechanisms for the activation of canonical Wnt pathway in RA. Combination of miR-152 and DNMT1 may be a promising treatment strategy for RA patients in which SFRP4 is inactivated.

Functional proteomics revealed IL-1β amplifies TNF downstream protein signals in human synoviocytes in a TNF-independent manner

IL-1β is readily detectable in numerous joint inflammations. It can change the transcriptomic signature of fibroblast-like synoviocytes (FLS) of arthritis toward promoting migration and invasion that are relevant to arthritis progression. We hypothesize that IL-1β partially contributes to the onset of osteoarthritis (OA). We compared the tissue samples from OA and fracture subjects and found that IL-1β expression was significantly higher in the OA synovium, while TNF-α expression showed no significance. We demonstrated that IL-1β significantly increases the IL-6 and IL-8 secretions of human normal FLS; however, IL-1β does not induce TNF secretion. With metabolic labeling based proteomics and pathway analysis, we found that IL-1β significantly increases the TNF downstream protein expression in FLS even with complete absence of TNF and/or blocking of the NF-κB pathway. Among these proteins, we verified that p62 can differentiate the OA from fracture synovitis. In conclusion, we demonstrated that IL-1β can amplify the TNF downstream protein signals in human synoviocytes in a TNF-independent manner; in addition, p62 is a potential FLS biomarker for synovitis.

The role of transforming growth factor β signaling in fibroblast-like synoviocytes from patients with oligoarticular juvenile idiopathic arthritis: dysregulation of transforming growth factor β signaling, including overexpression of bone morphogenetic protein 4, may lead to a chondrocyte phenotype and may contribute to bony hypertrophy.

This study was designed to investigate the pathogenic contributions of fibroblast-like synoviocytes (FLS) to juvenile idiopathic arthritis (JIA) by identifying pathways with dysregulated gene expression in FLS from patients with oligoarticular JIA. FLS were derived from synovial fluid obtained by arthrocentesis from patients with JIA undergoing intraarticular steroid injections and from orthopedic control patients. Gene expression profiles of the JIA and control FLS were obtained using the Affymetrix platform, with application of Ingenuity Pathway Analysis and Gene Set Enrichment Analysis software to define gene sets in dysregulated pathways and networks of potential pathologic relevance in this disease. Biologically relevant differentially expressed genes were confirmed by RNA and protein analysis. Exploration of global gene expression profiles of the JIA FLS revealed important dysregulated pathways, including the transforming growth factor β (TGFβ) signaling, as well as endochondral bone formation, cartilage formation, and β-catenin networks. Importantly, bone morphogenetic protein 4 (BMP-4) was significantly overexpressed in the JIA FLS. FLS from patients with oligoarticular JIA exhibit a chondrocyte phenotype, as evidenced by expression of type II collagen and aggrecan. Dysregulation of the pathways involved in the pathogenesis of oligoarticular JIA were revealed through gene expression profiling. JIA FLS displayed dysregulated TGFβ signaling and exhibited a hypertrophic chondrocyte phenotype. These characteristics, along with contributions from the β-catenin network may have implications for endochondral bone formation and local growth disturbances in oligoarticular JIA. Overexpression of BMP-4 in FLS from patients with oligoarticular JIA in particular may play an important role in disease pathogenesis, with a direct effect on functional outcome and with implications for future treatment.

The roles of IFN-γ versus IL-17 in pathogenic effects of human Th17 cells on synovial fibroblasts

Objectives Th17 cells, while indispensable in host defense, may play pathogenic roles in many autoimmune diseases, including rheumatoid arthritis (RA). However, the mechanisms by which human Th17 cells drive autoimmunity have not been fully defined. We assessed the potential of the human Th17 CD4 T cell subset to induce expression of cell–cell interaction molecules and inflammatory mediators by fibroblast-like synoviocytes (FLS), and the roles of IFN-γ and IL-17 in these interactions.Methods Th1 or Th17 cells were induced from healthy adult donor CD4 T cells and were co-cultured with FLS for 48 h with/without neutralization of IFN-γ, IL-17A, or both. Alternatively, FLS were treated only with IFN-γ or IL-17 for 48 h. FLS expression of CD40, CD54, and MHC-II, as well as IL-6 and IL-8 secretion, were assessed by surface staining followed by flow cytometry and ELISA, respectively.Results Both Th1 and Th17 cells secreted IL-17 as well as IFN-γ, although IFN-γ production was much greater from Th1 cells. FLS expression of CD40, CD54, and MHC-II significantly increased upon co-culture with Th1 cells, while Th17 cells increased only the percentage of FLS that were CD54+. Both T cell subsets induced IL-6 and IL-8 secretion by RA FLS. Neutralization of IL-17A did not reduce FLS expression of CD40, MHC-II, or CD54, but did inhibit IL-6 and IL-8 secretion. Although IFN-γ was a weak inducer of IL-6 secretion and significantly inhibited IL-8 secretion from FLS when used as a single stimulus, neutralization of IFN-γ inhibited the secretion of both cytokines in Th17/FLS co-cultures with RA but not OA FLS.Conclusion FLS cell–cell interaction molecules and soluble inflammatory mediators are differentially regulated by IFN-γ and IL-17. The effects of IFN-γ may depend in part on the particular milieu of other co-existing cytokines and its potential to induce cell–cell interactions. The potential benefit of therapeutic neutralization of either IL-17 or IFN-γ could depend on the relative proportions of these cytokines in the synovial compartment of an RA patient.

Anti-invasive effects of celastrol in hypoxia-induced fibroblast-like synoviocyte through suppressing of HIF-1α/CXCR4 signaling pathway.

Rheumatoid arthritis (RA) joints are in a hypoxic condition. Hypoxia-induced migration and invasion of fibroblast-like synoviocytes (FLSs) are considered to play a critical role in the pathogenesis of RA. Among the key genes upregulated by hypoxia-inducible factor-1α (HIF-1α), CXC chemokine receptor 4 (CXCR4) plays an important role in FLS migration and invasion. Our previous studies have shown that celastrol exerts anti-arthritic effects by inhibiting FLS migration and invasion under normoxic conditions. However, the effect and molecular mechanisms underlying the effect of celastrol on hypoxia-induced FLS migration and invasion are poorly understood. In the present study, we assessed the effect of celastrol on hypoxia-induced FLS migration and invasion. Results showed that celastrol suppressed hypoxia-induced FLS migration and invasion. In addition, we also found that celastrol inhibited hypoxia-induced CXCR4 expression at both the mRNA and the protein levels in RA-FLSs. Meanwhile, it is revealed that celastrol inhibited the transcriptional activity of CXCR4 under hypoxic conditions by suppressing the binding activity of HIF-1α in the CXCR4 promoter, and blocked hypoxia-induced accumulation of nuclear HIF-1α. Furthermore, treatment with HIF-1α inhibitor reduced the hypoxia-induced expression and transcriptional activity of CXCR4. In conclusion, our results indicate that celastrol inhibits hypoxia-induced migration and invasion via suppression of HIF-1α mediated CXCR4 expression in FLSs under hypoxic conditions. These results provide a strong rationale for further testing and validation of the use of celastrol as a new alternative for using in the treatment of RA.

TWEAK Promotes Osteoclastogenesis in Rheumatoid Arthritis

Bone destruction is critical in the functional disability of patients with rheumatoid arthritis (RA). Osteoclasts, specialized bone-resorbing cells regulated by cytokines, such as receptor activator of NF-κB ligand (RANKL), are primarily implicated in bone destruction in RA. The aim of the study was to examine whether tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, has osteoclastogenic activity in patients with RA and in animal models, including mice with collagen-induced arthritis (CIA) and IL-1 receptor antagonist knockout (IL-1RaKO) mice. TWEAK was increased in the synovium, synovial fluid, and serum of patients with RA and in the synovium of CIA mice and IL-1RaKO mice. TWEAK induced RANKL expression in mixed joint cells and splenocytes from CIA mice, IL-1RaKO mice, and fibroblast-like synoviocytes from patients with RA. Both osteoclast precursor cells and osteoclasts express TWEAK receptor fibroblast growth factor-inducible 14. In addition, TWEAK enhanced invitro osteoclastogenesis without the presence of RANKL-providing cells and by inducing RANKL expression in fibroblast-like synoviocytes. Moreover, treatment with fibroblast growth factor-inducible 14-Fc inhibited RANKL-induced osteoclastogenesis, indicating that endogenous TWEAK also has osteoclastogenic activity. Our data demonstrated that TWEAK promotes osteoclastogenesis in RA, suggesting that therapeutic strategies targeting TWEAK could be effective for treatment of patients with RA, especially in preventing bone destruction.

Effect of pulchinenoside in regulating FLS SFRP2 expression of RA model rats

To study the effect of pulchinenoside (PULC) in modulating SFRP2 expression in fibroblast-like synoviocytes (FLS) of rheumatoid arthritis (RA) model rats. The effect of PULC in treating RA rats was evaluated by rat arthritis score and paw swelling score. The inhibitory effect of PULC on FLS proliferation was detected by MTT reagent. The effects of PULC gavage treatment in modulating gene expression of FLS SFRP2, critical gene beta-catenin of Wnt pathway and downstream effector genes C-myc of of Wnt pathway were detected by RT-PCR and Western blotting. PULC had a significant effect in treating RA rats and that SFRP2 expression was down-regulated in FLS. After PULC gavage treatment, FLS SFRP2 expression was obviously up-regulated, whereas beta-catenin and C-myc gene expressions were significantly down-regulated. PULC can inhibit abnormal proliferation of synovial membrane by modulating Wnt pathway of RA rats.

AB0166 Up-regulation of SOCS3 by calcineurin inhibitor, tacrolimus, suppresses IL-6/SIL-6R-mediated rankl expression in fibroblast-like synoviocytes

ObjectivesInteractions between interleukin-6 (IL-6)/gp130/Janus activated kinase (JAK)/signal transducer and activator of transcription-3 (STAT3) and suppressor of cytokine signaling (SOCS) are responsible for the expression of receptor activator of NF-kB ligand...

AB0046 The sp1 transcription factor is essential for the expression of gliostatin/thymidine phosphorylase in rheumatoid fibroblast-like synoviocytes

BackgroundGliostatin/thymidine phosphorylase (GLS) has angiogenic and arthritogenic activities, and GLS production has been observed in the active synovial membranes of rheumatoid arthritis (RA) patients. The human GLS gene promoter contains...

THU0028 Fibroblast-like synoviocytes inhibit TNF- and RANKL-induced human osteoclast precursor differentiation following exposure to IL-1BETA

BackgroundInflammatory cytokines such as TNF-alpha (TNF), IL-1beta (IL-1b) and IL-6 have been shown to contribute to osteoclastogenesis independently or in conjunction with M-CSF or RANKL, two key cytokines involved in...

Regulation of DNA Methylation in Rheumatoid Arthritis Synoviocytes

Rheumatoid arthritis (RA) is a chronic inflammatory disease in which fibroblast-like synoviocytes (FLS) exhibit an aggressive phenotype. Although the mechanisms responsible are not well defined, epigenetic determinants such as DNA methylation might contribute. DNA methyltransferases (DNMTs) are critical enzymes that establish and maintain DNA methylation. We evaluated whether proinflammatory cytokines might contribute to differential DNA methylation previously described in RA FLS through altered DNMT expression. FLS were obtained from RA and osteoarthritis (OA) synovium at the time of total joint replacement. Gene expression was determined by quantitative real-time PCR and protein expression by Western blot analysis. DNMT activity was measured with a functional assay, and global methylation was determined by an immunoassay that detects methylcytosine. Resting expression of DNMT1, -3a, and -3b mRNA were similar in RA and OA FLS. Western blot showed abundant DNMT1 and DNMT3a protein. Exposure to IL-1 decreased DNMT1 and DNMT3a mRNA expression in FLS. Dose responses demonstrated decreased DNMT expression at concentrations as low as 1 pg/ml of IL-1. DNMT mRNA levels decreased rapidly, with significant suppression after 2-8 h of IL-1 stimulation. IL-1 stimulation of OA FLS did not affect methylation of LINE1 sites but led to demethylation of a CHI3L1 locus that is hypomethylated in RA FLS. Chronic IL-1 stimulation also mimicked the effect of a DNMT inhibitor on FLS gene expression. Exposure to proinflammatory mediators reversibly alters DNA methylation in FLS by decreasing DNMT expression and function. These data suggest that IL-1 can potentially imprint cells in chronic inflammatory diseases.

The roles of IFN-γ versus IL-17 in pathogenic effects of human Th17 cells on synovial fibroblasts

Th17 cells, while indispensable in host defense, may play pathogenic roles in many autoimmune diseases, including rheumatoid arthritis (RA). However, the mechanisms by which human Th17 cells drive autoimmunity have not been fully defined. We assessed the potential of the human Th17 CD4 T cell subset to induce expression of cell-cell interaction molecules and inflammatory mediators by fibroblast-like synoviocytes (FLS), and the roles of IFN-γ and IL-17 in these interactions. Th1 or Th17 cells were induced from healthy adult donor CD4 T cells and were co-cultured with FLS for 48 h with/without neutralization of IFN-γ, IL-17A, or both. Alternatively, FLS were treated only with IFN-γ or IL-17 for 48 h. FLS expression of CD40, CD54, and MHC-II, as well as IL-6 and IL-8 secretion, were assessed by surface staining followed by flow cytometry and ELISA, respectively. Both Th1 and Th17 cells secreted IL-17 as well as IFN-γ, although IFN-γ production was much greater from Th1 cells. FLS expression of CD40, CD54, and MHC-II significantly increased upon co-culture with Th1 cells, while Th17 cells increased only the percentage of FLS that were CD54+. Both T cell subsets induced IL-6 and IL-8 secretion by RA FLS. Neutralization of IL-17A did not reduce FLS expression of CD40, MHC-II, or CD54, but did inhibit IL-6 and IL-8 secretion. Although IFN-γ was a weak inducer of IL-6 secretion and significantly inhibited IL-8 secretion from FLS when used as a single stimulus, neutralization of IFN-γ inhibited the secretion of both cytokines in Th17/FLS co-cultures with RA but not OA FLS. FLS cell-cell interaction molecules and soluble inflammatory mediators are differentially regulated by IFN-γ and IL-17. The effects of IFN-γ may depend in part on the particular milieu of other co-existing cytokines and its potential to induce cell-cell interactions. The potential benefit of therapeutic neutralization of either IL-17 or IFN-γ could depend on the relative proportions of these cytokines in the synovial compartment of an RA patient.

Regulatory effect of calcineurin inhibitor, tacrolimus, on IL-6/sIL-6R-mediated RANKL expression through JAK2-STAT3-SOCS3 signaling pathway in fibroblast-like synoviocytes

IntroductionThis study investigated whether the calcineurin inhibitor, tacrolimus, suppresses receptor activator of NF-κB ligand (RANKL) expression in fibroblast-like synoviocytes (FLS) through regulation of IL-6/Janus activated kinase (JAK2)/signal transducer and activator of transcription-3 (STAT3) and suppressor of cytokine signaling (SOCS3) signaling.MethodsThe expression of RANKL, JAK2, STAT3, and SOCS3 proteins was assessed by western blot analysis, real-time PCR and ELISA in IL-6 combined with soluble IL-6 receptor (sIL-6R)-stimulated rheumatoid arthritis (RA)-FLS with or without tacrolimus treatment. The effects of tacrolimus on synovial inflammation and bone erosion were assessed using mice with arthritis induced by K/BxN serum. Immunofluorescent staining was performed to identify the effect of tacrolimus on RANKL and SOCS3. The tartrate-resistant acid phosphatase staining assay was performed to assess the effect of tacrolimus on osteoclast differentiation.ResultsWe found that RANKL expression in RA FLS is regulated by the IL-6/sIL-6R/JAK2/STAT3/SOCS3 pathway. Inhibitory effects of tacrolimus on RANKL expression in a serum-induced arthritis mice model were identified. Tacrolimus inhibits RANKL expression in IL-6/sIL-6R-stimulated FLS by suppressing STAT3. Among negative regulators of the JAK/STAT pathway, such as CIS1, SOCS1, and SOCS3, only SOCS3 is significantly induced by tacrolimus. As compared to dexamethasone and methotrexate, tacrolimus more potently suppresses RANKL expression in FLS. By up-regulating SOCS3, tacrolimus down-regulates activation of the JAK-STAT pathway by IL-6/sIL-6R trans-signaling, thus decreasing RANKL expression in FLS.ConclusionsThese data suggest that tacrolimus might affect the RANKL expression in IL-6 stimulated FLS through STAT3 suppression, together with up-regulation of SOCS3.

IL-17-mediated Bcl-2 expression regulates survival of fibroblast-like synoviocytes in rheumatoid arthritis through STAT3 activation

IntroductionFibroblast-like synoviocytes (FLSs) are a major cell population of the pannus that invades adjacent cartilage and bone in rheumatoid arthritis (RA). The study was undertaken to determine the effect of interleukin-17 (IL-17) on the survival and/or proliferation of FLSs from RA patients and to investigate whether signal tranducer and activator of transcription 3 (STAT3) is implicated in this process.MethodsBcl-2 and Bax expression in FLSs was determined using the real-time PCR and western blot analysis. The expression of Bcl-2 and phosphoSTAT3 in synovial tissues was investigated by confocal microscope. Apoptosis of FLSs was detected by Annexin V/propidium iodide staining and/or phase contrast microscopy. The proliferation of FLSs was determined by CCK-8 ELISA assay.ResultsThe pro-apoptotic Bax is decreased and anti-apoptotic Bcl-2 is increased in FLSs from RA patients compared with those from patients with osteoarthritis (OA). IL-17 upregulated the expression of Bcl-2 in FLSs from RA patients, but not in FLSs from OA patients. STAT3 was found to mediate IL-17-induced Bcl-2 upregulation in FLSs from RA patients. Additionally, IL-17 promoted the survival and proliferation of FLSs from RA patients. Most importantly, treatment with STAT3 inhibitor reversed the protective effect of IL-17 on FLSs apoptosis induced by sodium nitroprusside (SNP).ConclusionsOur data demonstrate that STAT3 is critical in IL-17-induced survival of FLS from RA patients. Therefore, therapeutic strategies that target the IL-17/STAT3 pathway might be strong candidates for RA treatment modalities.

Characterisation of fibroblast-like synoviocytes from a murine model of joint inflammation

IntroductionFibroblast-like synoviocytes (FLS) play a central role in defining the stromal environment in inflammatory joint diseases. Despite a growing use of FLS isolated from murine inflammatory models, a detailed characterisation of these cells has not been performed.MethodsIn this study, FLS were isolated from inflamed joints of mice expressing both the T cell receptor transgene KRN and the MHC class II molecule Ag7 (K/BxN mice) and their purity in culture determined by immunofluorescence and real-time reverse transcription polymerase chain reaction (real-time RT-PCR). Basal expression of proinflammatory genes was determined by real-time RT-PCR. Secreted interleukin 6 (IL-6) was measured by enzyme-linked immunosorbent assay (ELISA), and its regulation by tumor necrosis factor-alpha (TNF-α and corticosterone (the major glucocorticoid in rodents) measured relative to other mesenchymal cell populations.ResultsPurity of FLS culture was identified by positive expression of fibronectin, prolyl 4-hydroxylase, cluster of differentiation 90.2 (CD90.2) and 248 (CD248) in greater than 98% of the population. Cultured FLS were able to migrate and invade through matrigel, a process enhanced in the presence of TNF-α. FLS isolated from K/BxN mice possessed significantly greater basal expression of the inflammatory markers IL-6, chemokine ligand 2 (CCL-2) and vascular cell adhesion molecule 1 (VCAM-1) when compared to FLS isolated from non-inflamed tissue (IL-6, 3.6 fold; CCL-2, 11.2 fold; VCAM-1, 9 fold; P < 0.05). This elevated expression was abrogated in the presence of corticosterone at 100 nmol/l. TNF-α significantly increased expression of all inflammatory markers to a much greater degree in K/BxN FLS relative to other mesenchymal cell lines (K/BxN; IL-6, 40.8 fold; CCL-2, 1343.2 fold; VCAM-1, 17.8 fold; ICAM-1, 13.8 fold; P < 0.05), with secreted IL-6 mirroring these results (K/BxN; con, 169 ± 29.7 versus TNF-α, 923 ± 378.8 pg/ml/1 × 105 cells; P < 0.05). Dose response experiments confirmed effective concentrations between 10 and 100 nmol/l for corticosterone and 1 and 10 ng/ml for TNF-α, whilst inflammatory gene expression in FLS was shown to be stable between passages four and seven.ConclusionsThis study has established a well characterised set of key inflammatory genes for in vitro FLS culture, isolated from K/BxN mice and non-inflamed wild-type controls. Their response to both pro- and anti-inflammatory signalling has been assessed and shown to strongly resemble that which is seen in human FLS culture. Additionally, this study provides guidelines for the effective characterisation, duration and treatment of murine FLS culture.

Epirubicin potentiates recombinant adeno‐associated virus type 2/5–mediated TRAIL expression in fibroblast‐like synoviocytes and augments the antiarthritic effects of rAAV2/5‐TRAIL

Synovial cells in rheumatoid synovium display abnormal proliferation, which leads to joint destruction. TRAIL has been described as a proapoptotic factor in fibroblast-like synoviocytes (FLS). This study was undertaken to investigate the functions of rAAV2/5-TRAIL in human FLS and in arthritic mice. Primary human FLS were infected with rAAV2/5-TRAIL in the presence or absence of epirubicin. Transgene expression was monitored by both enzyme-linked immunosorbent assay and flow cytometry. The disease-modulating activity of epirubicin plus rAAV2/5-TRAIL was investigated in mice with collagen-induced arthritis (CIA). Subtoxic doses of epirubicin potentiated rAAV2/5-mediated TRAIL expression in FLS and simultaneously enhanced the sensitivity of FLS to TRAIL. Epirubicin treatment up-regulated death receptor 4 (DR-4) and DR-5 expression and down-regulated FLIP expression, thereby enhancing the activation of procaspase 3, procaspase 8, and procaspase 9. An in vivo study showed that the combination of rAAV2/5-TRAIL gene therapy and epirubicin chemotherapy provided augmented antiarthritic effects in a mouse model of CIA. The intraarticular injection of rAAV2/5-TRAIL combined with epirubicin treatment significantly reduced the severity and incidence of CIA and joint swelling in the animals. Histologic evaluations revealed that inflammatory cell infiltration, cartilage destruction, and bone erosion were significantly reduced in the joints of the mice receiving the synthetic treatment. Results of a viral genome copy number assay indicated that epirubicin dramatically augmented the expression of rAAV2/5-TRAIL without altering its tissue distribution. These results suggest that epirubicin enhances the antiarthritic effect of rAAV2/5-TRAIL and that combination treatment might be an important therapeutic alternative, with practical significance for rheumatoid arthritis.

Disorders in angiogenesis and redox pathways are main factors contributing to the progression of rheumatoid arthritis: A comparative proteomics study

To clarify the pathogenesis of rheumatoid arthritis (RA) by comparing protein expression in fibroblast-like synoviocytes (FLS) from patients with RA with that in FLS from normal subjects, using proteomics analysis. Proteins extracted from primary cultures of FLS obtained from 50 patients with RA and 10 normal subjects were analyzed by automated 2-dimensional nano-electrospray ionization liquid chromatography tandem mass spectometry. Differentially expressed proteins were screened by 2-sample t-test (P < 0.05) and fold change (>1.5), based on the bioinformatics analysis. The expression of vasculature development-related proteins (Thy-1, connective tissue growth factor [CTGF], and thrombospondin 1 [TSP-1]) and redox-related proteins (superoxide dismutase 2 [SOD2]) in synovial tissue was confirmed by real-time polymerase chain reaction and Western blotting. The effect of Thy-1 and CTGF knockdown on Thy-1, CTGF, TSP-1, and vascular endothelial growth factor (VEGF) was analyzed by RNA interference experiments. According to the criteria of having >1 unique peptide per protein present and a false discovery rate of ≤5%, 1,060 proteins were identified from patients with RA, and 1,292 proteins were identified from normal subjects, from which 100 differentially expressed proteins were screened out from the RA proteins. Of these, 46 proteins were up-regulated, and the remaining 54 proteins were down-regulated. Gene ontology and pathway analyses showed that 6 vasculature development-related proteins were up-regulated, including Thy-1, CTGF, and TSP-1, while 11 redox-related proteins were down-regulated, including SOD2. The results were consistent with those obtained using mass spectrometry. Thy-1, VEGF, CTGF, and TSP-1 were down-regulated after Thy-1 knockdown, and VEGF and CTGF were down-regulated after CTGF knockdown. Recombinant human CTGF could enhance proliferation and Transwell migration of human umbilical vein endothelial cells. Up-regulation of vasculature development-related proteins and down-regulation of redox-related proteins in FLS are predominant factors that may contribute to the pathogenesis of RA.

Paeoniflorin Inhibits Proliferation of Fibroblast-Like Synoviocytes through Suppressing G-Protein-Coupled Receptor Kinase 2

Paeoniflorin (Pae) is a monoterpene glucoside and the main component of the total glucosides of paeony (TGP) extracted from the roots of Paeonia lactiflora. Its anti-inflammatory effect is associated with regulating G-protein-coupled receptors (GPCRs) signaling. The aim of this study was to explore the expression change of G-protein-coupled receptor kinase 2 (GRK2) in fibroblast-like synoviocytes (FLS) and the effect of Pae. Pae was obtained and purified from the roots of Paeonia lactiflora. We investigated the expression of GRK2 in synovium during the inflammatory process and assessed the effects of a specific GRK2 inhibitor and Pae on proliferation, cAMP level, and protein kinase A (PKA) activity of FLS in vitro. Additionally, the effect of Pae on GRK2 expression in FLS was detected in vitro. Expression of GRK2 in synovium from CIA rats increased during the inflammatory process. The specific GRK2 inhibitor suppressed proliferation and increased the cAMP level as well as PKA activity of FLS, and Pae had the same effects. Furthermore, Pae decreased GRK2 expression in FLS in vitro. Our results indicate that a chronic inflammatory process in CIA induces upregulation of GRK2 expression in FLS, and Pae can reverse this change, which might be one of the important mechanisms for Pae regulating GPCRs signaling and suppressing the proliferation of FLS in CIA.

The Sp1 transcription factor is essential for the expression of gliostatin/thymidine phosphorylase in rheumatoid fibroblast-like synoviocytes

IntroductionGliostatin/thymidine phosphorylase (GLS/TP) has angiogenic and arthritogenic activities, and aberrant GLS production has been observed in the active synovial membranes of rheumatoid arthritis (RA) patients. The human GLS gene promoter contains at least seven consensus binding sites for the DNA binding protein Sp1. Here we examined whether Sp1 is necessary for GLS production in RA. We also studied the effects of the Sp1 inhibitor mithramycin on GLS production in RA fibroblast-like synoviocytes (FLSs).MethodsFLSs from RA patients were treated with specific inhibitors. The gene and protein expression of GLS were studied using the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and an enzyme immunoassay. Intracellular signalling pathway activation was determined by western blotting analysis, a luciferase assay, a chromatin immunoprecipitation (ChIP) assay and a small interfering RNA (siRNA) transfection.ResultsThe luciferase and ChIP assays showed that Sp1 binding sites in the GLS promoter were essential for GLS messenger RNA (mRNA) expression. GLS production was suppressed in FLSs by siRNA against Sp1 transfection. Mithramycin decreased GLS promoter activity, mRNA and protein expression in FLSs. Tumour necrosis factor-α (TNF-α) significantly increased GLS expression in RA FLSs; this effect was reduced by pre-treatment with cycloheximide and mithramycin.ConclusionsPretreatment of mithramycin and Sp1 silencing resulted in a significant suppression of GLS production in TNF-α-stimulated FLSs compared to controls. GLS gene expression enhanced by TNF-α was partly mediated through Sp1. As physiological concentrations of mithramycin can regulate GLS production in RA, mithramycin is a promising candidate for anti-rheumatic therapy.

Inhibitory effect of paeoniflorin on rat fibroblast-like synoviocytes hyperplasia induced by PGE2 via β-arrestin 2 regulating EP2 receptor

Aim To investigate the potential molecular mechanism of paeoniflorin(Pae) in inhibiting PGE2 induced rat fibroblast-like synoviocytes(FLSs) hyperplasy.Methods Rat FLSs were cultured in the presence of prostaglandin E2(PGE2) with or without different concentrations of Pae.FLSs proliferations were determined by 3H-TdR incorporation assay.Cyclic adenosine monophosphate(cAMP) levels in synoviocytes were assessed by radioimmunoassay(RIA).The expressions of EP2 and β-arrestin 2 in whole FLSs or on membrane were measured by Western blot or flow cytometry.Results Pae significantly inhibited rat FLS proliferation induced by PGE2 via increasing cAMP levels.High levels of total EP2,β-arrestin 2 and membrane β-arrestin 2 expression in FLSs induced by PGE2 were decreased by different concentrations of Pae administration.EP2 was downregulated on FLSs membrane after PGE2 stimulation;however,was restored by Pae in various degrees.Conclusions Our findings suggest that Pae inhibits rat FLSs proliferation induced by PGE2 via arresting EP2 receptor internalization by inhibiting β-arrestin 2 recruit to cell membrane.