Rheumatoid Arthritis Synovial Fibroblasts Research Articles

Ursolic acid facilitates apoptosis in rheumatoid arthritis synovial fibroblasts by inducing SP1-mediated Noxa expression and proteasomal degradation of Mcl-1.

Rheumatoid arthritis (RA) is characterized by hyperplastic pannus formation mediated by activated synovial fibroblasts (RASFs) that cause joint destruction. We have shown earlier that RASFs exhibit resistance to apoptosis, primarily as a result of enhanced expression of myeloid cell leukemia-1 (Mcl-1). In this study, we discovered that ursolic acid (UA), a plant-derived pentacyclic triterpenoid, selectively induces B-cell lymphoma 2 homology 3-only protein Noxa in human RASFs. We observed that UA-induced Noxa expression was followed by a consequent decrease in Mcl-1 expression in a dose-dependent manner. Subsequent evaluation of the signaling pathways showed that UA-induced Noxa is primarily mediated by the JNK pathway in human RASFs. Chromatin immunoprecipitation (IP) studies into the promoter region of Noxa indicated the role of transcription factor specificity protein 1 in JNK-mediated Noxa expression. Furthermore, the results from IP studies and proximity ligation assays indicated that UA-induced Noxa colocalizes and associates with Mcl-1 to prime it for proteasomal degradation through K48-linked ubiquitination by the selective recruitment of Mcl-1 ubiquitin ligase E3, a homologous to E6-associated protein C terminus domain-containing E3 ubiquitin ligase. These findings unveil a novel mechanism of inducing apoptosis in RASFs and a potential adjunct therapeutic strategy of regulating synovial hyperplasia in RA.-Kim, E. Y., Sudini, K., Singh, A. K., Haque, M., Leaman, D., Khuder, S., Ahmed, S. Ursolic acid facilitates apoptosis in rheumatoid arthritis synovial fibroblasts by inducing SP1-mediated Noxa expression and proteasomal degradation of Mcl-1.

Selective killing of proinflammatory synovial fibroblasts via activation of transient receptor potential ankyrin (TRPA1)

BackgroundStudies in rheumatoid arthritis synovial fibroblasts (RASF) demonstrated the expression of several transient receptor potential channels (TRP) such as TRPV1, TRPV2, TRPV4, TRPA1 and TRPM8. Upon ligation, these receptors increase intracellular calcium but they have also been linked to modulation of inflammation in several cell types. TNF was shown to increase the expression of TRPA1, the receptor for mustard oil and environmental poisons in SF, but the functional consequences have not been investigated yet. MethodsTRPA1 was detected by immunocytochemistry, western blot and cell-based ELISA. Calcium measurements were conducted in a multimode reader. Cell viability was assessed by quantification of lactate dehydrogenase (LDH) in culture supernatants and “RealTime-Glo” luminescent assays. IL-6 and IL-8 production by SF was quantified by ELISA. Proliferation was determined by cell titer blue incorporation. ResultsAfter 72 h, mimicking proinflammatory conditions by the innate cytokine TNF up-regulated TRPA1 protein levels in RASF which was accompanied by increased sensitivity to TRPA1 agonists AITC and polygodial. Under unstimulated conditions, polygodial elicited calcium flux only in the highest concentrations used (50 µM and 25 µM). TNF preincubation substantially lowered the activation threshold for polygodial (from 25 µM to 1 µM). In the absence of TNF pre-stimulation, only polygodial in high concentrations was able to reduce viability of synovial fibroblasts as determined by a real-time viability assay. However, following TNF preincubation, stimulation of TRPA1 led to a fast (<30 min) viability loss by necrosis of synovial fibroblasts. TRPA1 activation was also associated with decreased proliferation of RASFs, an effect that was also substantially enhanced by TNF preincubation. On the functional level, IL-6 and IL-8 production was attenuated by the TRPA1 antagonist A967079 but also polygodial, although the latter mediated this effect by reducing cell viability. ConclusionSimulating inflamed conditions by preincubation of synovial fibroblasts with TNF up-regulates and sensitizes TRPA1. Subsequent activation of TRPA1 increases calcium flux and substantially reduces cell viability by inducing necrosis. Since TRPA1 agonists in the lower concentration range only show effects in TNF-stimulated RASF, this cation channel might be an attractive therapeutic target in chronic inflammation to selectively reduce the activity of proinflammatory SF in the joint.

Analysis of early changes in DNA methylation in synovial fibroblasts of RA patients before diagnosis

DNA methylation is an important epigenetic modification that is known to be altered in rheumatoid arthritis synovial fibroblasts (RASF). Here, we compared the status of promoter DNA methylation of SF from patients with very early RA with SF from patients with resolving arthritis, fully established RA and from non-arthritic patients. DNA was hybridized to Infinium Human methylation 450k and 850k arrays and differential methylated genes and pathways were identified. We could identify a significant number of CpG sites that differed between the SF of different disease stages, showing that epigenetic changes in SF occur early in RA development. Principal component analysis confirmed that the different groups of SF were separated according to their DNA methylation state. Furthermore, pathway analysis showed that important functional pathways were altered in both very early and late RASF. By focusing our analysis on CpG sites in CpG islands within promoters, we identified genes that have significant hypermethylated promoters in very early RASF. Our data show that changes in DNA methylation differ in RASF compared to other forms of arthritis and occur at a very early, clinically yet unspecific stage of disease. The identified differential methylated genes might become valuable prognostic biomarkers for RA development.

Atomic force microscopy technique used for assessment of the anti-arthritic effect of licochalcone A via suppressing NF-κB activation

Atomic force microscopy (AFM) is appropriately applied to the examination of hard surfaces and soft samples with extremely high resolution and ultrasensitive force, which cannot be obtained by other imaging techniques, including optical and electron microscopy. In the current study, AFM was employed to evaluate the anti-arthritic effect of licochalcone A (LCA), a flavonoid isolated from the root of Chinese medicinal herb Glycyrrhiza inflate, on rheumatoid arthritis synovial fibroblasts (RASFs) at the nanoscale for the first time. The morphology, ultrastructure and stiffness of RASFs was modified by LCA as determined by AFM, suggesting that LCA most likely exerts an anti-arthritic effect based on the key role of RASFs in the progression of RA. Further studies showed that the inhibitory effect of LCA on IκBα phosphorylation and degradation as well as on p65 nuclear translocation and phosphorylation contributed to altering the morphology, ultrastructure and stiffness of the RASF membrane. Interestingly, IKKβ phosphorylation was not detectable in RASFs, indicating that LCA altered the morphology, ultrastructure and stiffness of the RASF membrane by inhibiting NF-κB activation independent of IKKβ phosphorylation. Antigen-induced arthritis (AIA) was established in Sprague Dawley (SD) rats to validate the anti-arthritic effect of LCA, and LCA significantly decreased both the arthritis scores and paw swelling in the AIA rats, suggesting that LCA inhibits the progression and development of arthritis in vivo. Collectively, AFM provides evidence at the nanoscale to predict the anti-arthritic effect of drugs on RASFs, and LCA should be further investigated as a candidate agent for the treatment of arthritis.

MiR‑152 inhibits rheumatoid arthritis synovial fibroblast proliferation and induces apoptosis by targeting ADAM10.

miR‑152 has been reported to be downregulated in rheumatoid arthritis (RA). However, the functional significance and molecular mechanisms underlying the role of miR‑152 in RA remain largely unknown. The present study aimed to explore the functional role and the underlying mechanisms of miR‑152 in RA. The expression of miR‑152 in serum, synovial tissues, and fibroblast‑like synoviocytes (FLS) from patients with RA and healthy controls was detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). Cell proliferation, cell cycle phase distribution and apoptosis of FLS were measured by Cell Counting Kit‑8 and flow cytometry assays. The effects of miR‑152 on the production of pro‑inflammatory cytokines, including tumor necrosis factor (TNF)‑α, interleukin (IL)‑1β, IL‑6 and IL‑8, were examined by ELISA. The target gene of miR‑152 was discovered by miRNA‑target prediction bioinformatics analysis, and confirmed by dual‑luciferase reporter assay, RT‑qPCR and western blotting. Spearman's correlation analysis was performed to assess the relationship between miR‑152 expression and a disintegrin and metalloproteinase domain‑containing protein10 (ADAM10). The results demonstrated that miR‑152 expression levels were significantly decreased in RA serum, synovial tissues and RA‑FLS compared with healthy controls. Overexpression of miR‑152 significantly inhibited cell proliferation, promoted cell apoptosis, and decreased TNF‑α, IL‑1β, IL‑6 and IL‑8 production in RA‑FLS cells. Additionally, ADAM10 was demonstrated to be a target of miR‑152, and expression of the two genes was significantly negatively correlated. Of note, restoration of ADAM10 expression partially reversed the effects of miR‑152 on cell proliferation and apoptosis in RA‑FLS. Thus, miR‑152 may serve as a potential target for therapeutic intervention in RA.

Isolation of endophytic fungi from Zanthoxylum simulans and screening of its active strain

This study aims at making full use of microbial resources, and screening the active endophytic fungi of anti-rheumatoid arthritis from Zanthoxylum simulans. The endophytic fungi were cultured and isolated by tissue culture and scribing method, and the active strain of inhibiting the proliferation of human rheumatoid arthritis synovial fibroblasts (HFLS-RA) was screened by MTT method. Morphological characteristics and rDNA ITS1-5.8S-ITS2 sequences were applied for the taxonomy of endophytic fungi. Strains were isolated from Z. simulans. Among them, MK-05, MK-17, MK-19, MK-23 having inhibiting activity to HFLS-RA, the IC₅₀ were 0.367, 0.775, 0.689, 0.757 g·L⁻¹, respectively. By classic morphologic classification and sequencing the PCR-amplified rDNA ITS1-5.8S-ITS2 regions, four effective strains were identified as Botryosphaeria dothidea, Phomopsis sp., P. liquidambari and Diaporthe perseae. The active endophyic fungi that inhibited the proliferation of HFLS-RA were screened from Z. simulans for the first time, and the results lay the foundation for the development and utilization of the Z. simulans resources.

Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

BackgroundEffects of methotrexate (MTX) on the proliferation of rheumatoid arthritis (RA) synovial fibroblasts are incompletely understood. We explored actions of MTX in view of circadian transcriptions of synovial fibroblasts.MethodsUnder treatment with MTX, expression of core circadian clock genes, circadian transcriptional factor proline and acidic amino acid-rich basic leucine zipper (PAR bZIP), and proapoptotic molecule Bcl-2 interacting killer (Bik) was examined by real-time polymerase chain reaction. Protein expression of circadian clock gene PERIOD2 (PER2) and CYTOCHROME C was also examined by western blotting and ELISA. Promoter activities of Per2 and Bik were measured by Luciferase assay. Expression of PER2, BIK, and CYTOCHROME C and morphological changes of the nucleus were observed by fluorescent immunostaining. Synovial fibroblasts were transfected with Per2/Bik small interfering RNA, and successively treated with MTX to determine cell viabilities. Finally, synovial fibroblasts were treated with MTX according to the oscillation of Per2/Bik expression.ResultsMTX (10 nM) significantly decreased cell viabilities, but increased messenger RNA expression of Per2, Bik, and PAR ZIP including D site of the albumin promoter binding protein (Dbp), hepatic leukemia factor (Hlf), and thyrotroph embryonic factor (Tef). MTX also increased protein expression of PER2 and CYTOCHROME C, and promoter activities of Per2 and Bik via D-box. Under fluorescent observations, expression of PER2, BIK, and CYTOCHROME C was increased in apoptotic cells. Cytotoxicity of MTX was attenuated by silencing of Per2 and/or Bik, and revealed that MTX was significantly effective in situations where Per2/Bik expression was high.ConclusionsWe present here novel unique action of MTX on synovial fibroblasts that upregulates PAR bZIP to transcribe Per2 and Bik, resulting in apoptosis induction. MTX is important in modulating circadian environments to understand a new aspect of pathogenesis of RA.

Interrupting oral infection of Porphyromonas gingivalis with anti-FimA antibody attenuates bacterial dissemination to the arthritic joint and improves experimental arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease that typically results in strong inflammation and bone destruction in the joints. It is generally known that the pathogenesis of RA is linked to cardiovascular and periodontal diseases. Though rheumatoid arthritis and periodontitis share many pathologic features such as a perpetual inflammation and bone destruction, the precise mechanism underlying a link between these two diseases has not been fully elucidated. Collagen-induced arthritis (CIA) mice were orally infected with Porphyromonas gingivalis (Pg) or Pg preincubated with an anti-FimA antibody (FimA Ab) specific for fimbriae that are flexible appendages on the cell surface. Pg-infected CIA mice showed oral microbiota disruption and increased alveolar bone loss and had synovitis and joint bone destruction. However, preincubation with FimA Ab led to a significant reduction in the severity of both oral disease and arthritis. Moreover, FimA Ab attenuated bacterial attachment and aggregation on human gingival and rheumatoid arthritis synovial fibroblasts. In addition, we discovered bacteria may utilize dendritic cells, macrophages and neutrophils to migrate into the joints of CIA mice. These results suggest that disrupting Pg fimbriae function by FimA Ab ameliorates RA.

Adiponectin aggravates bone erosion by promoting osteopontin production in synovial tissue of rheumatoid arthritis

BackgroundWe have previously reported that adiponectin (AD), an adipokine that is secreted by adipocytes, correlates well with progressive bone erosion in rheumatoid arthritis (RA). The exact mechanism of AD in promoting joint destruction remains unclear. Osteopontin (OPN) is required for osteoclast recruitment. We hypothesized that AD exacerbates bone erosion by inducing OPN expression in synovial tissue. This study aimed to evaluate a novel role for AD in RA.MethodsThe serum levels of AD and OPN were determined in 38 patients with RA, 40 patients with osteoarthritis (OA), and 20 healthy controls using enzyme-linked immunosorbent assay (ELISA). AD and OPN production were measured by double immunofluorescence in RA and OA synovial tissue. Quantitative real-time PCR and immunofluorescence were used to evaluate the mRNA and protein expression levels of OPN in RA synovial fibroblasts (RASFs) and OA synovial fibroblasts after pre-incubation with AD, respectively. Migration of the RAW264.7 osteoclast precursor cell line was assessed using the Transwell migration assay and co-culture system. Bone destruction and osteoclastogenesis were assessed by immunohistochemical staining, microcomputed tomography and tartrate-resistant acid phosphatase (TRAP) staining in AD-treated collagen-induced arthritis (CIA) mice with or without OPN silencing. The expression levels of OPN and integrin αvβ3 in the ankle joint tissues of the mice were examined by double immunofluorescence.ResultsOur results indicated that the AD and OPN expression levels increased noticeably and were associated with each other in the RA serum. The AD distribution was coincident with that of OPN in the RA synovial tissue. AD stimulation of RASFs increased OPN production in a dose-dependent manner. AD-treated RASFs promoted RAW264.7 cell migration, and the effect was blocked with a specific antibody against OPN. Silencing of OPN using lentiviral-OPN short hairpin RNA reduced the number of TRAP-positive osteoclasts and the extent of bone erosion in the AD-treated CIA mice. When bound to integrin αvβ3, OPN functions as a mediator of AD and osteoclasts.ConclusionsOur study provides new evidence of AD involvement in bone erosion. AD induces the expression of OPN, which recruits osteoclasts and initiates bone erosion. These data highlight AD as a novel target for RA treatment.

Elevated Levels of Soluble ST2 were Associated with Rheumatoid Arthritis Disease Activity and Ameliorated Inflammation in Synovial Fibroblasts.

Background:Much evidence has demonstrated that interleukin (IL)-33 plays an important role in rheumatoid arthritis (RA). However, there have been limited studies about soluble ST2, a receptor for IL-33, in RA. The aims of this study were to detect the levels of ST2 in the serum and synovial fluid of RA patients and to reveal the association of these levels with disease activity and the function of ST2 in RA.Methods:A total of 56 RA patients and 38 age-matched healthy controls were enrolled in this study. Synovial fluid samples were collected from another 30 RA patients and 20 osteoarthritis patients. Serum and synovial fluid levels of ST2 were measured by ELISA. In addition, the levels of ST2 in the serum of RA patients before and after therapy were detected. The function of ST2 in RA was revealed by the results of an in vitro cell assay, where recombinant ST2 proteins were used to treat peripheral blood mononuclear cells (PBMCs) and RA synovial fibroblasts (RASFs).Results:Serum-soluble ST2 levels were significantly higher in RA patients (127.14 ± 61.43 pg/ml) than those in healthy controls (78.37 ± 41.93 pg/ml, P < 0.01). Synovial fluid-soluble ST2 levels (41.90 ± 33.58 pg/ml) were much higher in RA patients than those in osteoarthritis patients (19.71 ± 16.72 pg/ml, P < 0.05). RA patients who received effective therapy for 6 months showed decreased serum-soluble ST2 levels (113.01 ± 53.90 pg/ml) compared to baseline (139.59 ± 68.36 pg/ml) (P = 0.01). RA patients with high disease activity had higher serum-soluble ST2 levels (162.02 ± 56.78 pg/ml) than those with low disease activity (94.67 ± 40.27 pg/ml, P = 0.001). Soluble ST2 did not affect IL-1β, IL-6, IL-8, or tumor necrosis factor-α (TNF-α) expression in PBMCs from RA patients. However, soluble ST2 ameliorated the expressions of IL-33 and IL-1β but not that of IL-6, IL-8, or TNF-α in resting RASFs. Interestingly, in the RASFs stimulated by TNF-α plus IL-1β, soluble ST2 showed extensive suppressive effects on the expression of IL-6, IL-8, and TNF-α.Conclusion:Elevated levels of ST2 in the serum and synovial fluid were associated with disease activity and ameliorated IL-33 expression and IL-33-induced inflammation in RASFs, suggesting that soluble ST2 might be a potential therapeutic candidate for RA.

MiR-522 Modulated the Expression of Proinflammatory Cytokines and Matrix Metalloproteinases Partly via Targeting Suppressor of Cytokine Signaling 3 in Rheumatoid Arthritis Synovial Fibroblasts.

microRNAs have been reported to play important roles in the pathogenesis of rheumatoid arthritis (RA). This study examined the effects of miR-522 on the biological behaviors of RA synovial fibroblasts. The expression levels of miR-522 and relevant genes were measured by quantitative real-time PCR. The protein levels of cytokines were determined by ELISA assay. The protein levels of matrix metalloproteinases (MMPs) and suppressor of cytokine signaling 3 (SOCS3) were determined by western blot assay. Luciferase reporter assay was used to confirm the potential target of miR-522. Our results showed that miR-522 was upregulated in synovial fibroblasts from RA patients, and miR-522 expression level was significantly associated with the RA-associated clinical parameters. miR-522 overexpression increased the mRNA and protein expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and MMPs (MMP-1, MMP-3, and MMP-13) in RA synovial fibroblasts. Lipopolysaccharide induced the upregulation of TNF-α, IL-1β, and MMPs in RA synovial fibroblasts, which was reversed by miR-522 knockdown. Bioinformatics analysis identified SOCS3 as a potential target of miR-522, and this target of miR-522 was confirmed by luciferase reporter assay, and miR-522 overexpression suppressed the mRNA and protein expression levels of SOCS3. The enforced expression of SOCS3 attenuated the enhanced effects of miR-522 on mRNA expression levels of TNF-α, IL-1β, and MMPs. Collectively, our results suggested that miR-522 regulated the expression of proinflammatory cytokines and MMPs partly via targeting SOCS3 in RA synovial fibroblasts, which may contribute to pathogenesis of RA.

Effects of Jinwu Jiangu recipe on IL-17/STAT3 signals in rheumatoid arthritis synoviocytes

This paper aimed to investigate the effects of Jinwu Jiangu recipe total extract on the IL-17/STAT3 signals in rheumatoid arthritis synovial fibroblasts(RASF). The primary RASFs were cultured by tissue piece method in vitro， and divided into blank control group， Jinwu Jiangu recipe low dose group， Jinwu Jiangu recipe middle dose group， Jinwu Jiangu recipe high dose group， and tripterygium glycosides control group. They were then treated with corresponding serum free medium， different doses of Jinwu Jiangu recipe total extract(0.06， 0.6， 6.0 g·L⁻¹)， and tripterygium glycosides(0.03 g·L⁻¹) respectively for 24 hours. The gene expression levels of RORα， RORγt， and STAT3 mRNA were detected by polymerase chain reaction(PCR)， and the protein activity of IL-17R and pSTAT3 were measured by Western blot assay. The results showed that as compared with blank control group， the expression levels of RORα， RORγt， IL-17R and STAT3 mRNA in RASF were significantly declined(P<0.01). As compared with tripterygium glycosides control group， Jinwu Jiangu recipe total extract middle dose group and high dose group can down-regulate the expression levels of RORα， RORγt， IL-17R and STAT3 mRNA(P<0.05)， and the effect was more obvious in high dose group(P<0.01). As compared with blank control group， the protein expression levels of IL-17R and pSTAT3 in each treatment group were obviously decreased(P<0.01). As compared with tripterygium glycosides control group， Jinwu Jiangu recipe high dose group had more obvious effect in down-regulating the protein expression of pSTAT3(P<0.01). Therefore， Miao medicine Jinwu Jiangu recipe total extract can down-regulate the expressions of RORα， RORγt， and STAT3 mRNA， and inhibit the protein activity of IL-17R and pSTAT3 in RASF.

TLR3 Ligand Poly(I:C) Exerts Distinct Actions in Synovial Fibroblasts When Delivered by Extracellular Vesicles.

Extracellular vesicles (EV) can modulate the responses of cells to toll-like receptor (TLR) ligation; conversely, TLR ligands such as double-stranded RNA (dsRNA) can enhance the release of EV and influence of the composition and functions of EV cargos. Inflamed synovial joints in rheumatoid arthritis (RA) are rich in EV and extracellular RNA; besides, RNA released from necrotic synovial fluid cells can activate the TLR3 signaling in synovial fibroblasts (SFs) from patients with RA. Since EV occur prominently in synovial joints in RA and may contribute to the pathogenesis, we questioned whether EV can interact with dsRNA, a TLR3 ligand, and modify its actions in arthritis. We have used as model the effects on RA SFs, of EV released from monocyte U937 cells and peripheral blood mononuclear cells upon stimulation with Poly(I:C), a synthetic analog of dsRNA. We show that EV released from unstimulated cells and Poly(I:C)-stimulated U937 cells [Poly(I:C) EV] differ in size but bind similar amounts of Annexin V and express comparable levels of MAC-1, the receptor for dsRNA, on the vesicular membranes. Specifically, Poly(I:C) EV contain or associate with Poly(I:C) and at least partially protect Poly(I:C) from RNAse III degradation. Poly(I:C) EV shuttle Poly(I:C) to SFs and reproduce the proinflammatory and antiviral gene responses of SFs to direct stimulation with Poly(I:C). Poly(I:C) EV, however, halt the death receptor-induced apoptosis in SFs, thereby inverting the proapoptotic nature of Poly(I:C). These prosurvival effects sharply contrast with the high toxicity of cationic liposome-delivered Poly(I:C) and may reflect the route of Poly(I:C) delivery via EV or the fine-tuning of Poly(I:C) actions by molecular cargo in EV. The demonstration that EV may safeguard extracellular dsRNA and allow dsRNA to exert antiapoptotic effects on SFs highlights the potential of EV to amplify the pathogenicity of dsRNA in arthritis beyond inflammation (by concurrently enhancing the expansion of the invasive synovial stroma).

The Calcium-Induced Regulation in the Molecular and Transcriptional Circuitry of Human Inflammatory Response and Autoimmunity

Rheumatoid arthritis synovial fibroblasts (RASFs) are fundamental effector cells in RA driving the joint inflammation and deformities. Celastrol is a natural compound that exhibits a potent anti-arthritic effect promoting endoplasmic reticulum (ER) stress mediated by intracellular calcium (Ca2+) mobilization. Ca2+ is a second messenger regulating a variety of cellular processes. We hypothesized that the compound, celastrol, affecting cytosolic Ca2+ mobilization could serve as a novel strategy to combat RA. To address this issue, celastrol was used as a molecular tool to assay the inflammatory gene expression profile regulated by Ca2+. We confirmed that celastrol treatment mobilized cytosolic Ca2+ in patient-derived RASFs. It was found that 23 genes out of 370 were manipulated by Ca2+ mobilization using an inflammatory and autoimmunity PCR array following independent quantitative PCR validation. Most of the identified genes were downregulated and categorized into five groups corresponding to their cellular responses participating in RA pathogenesis. Accordingly, a signaling network map demonstrating the possible molecular circuitry connecting the functions of the products of these genes was generated based on literature review. In addition, a bioinformatics analysis revealed that celastrol-induced Ca2+ mobilization gene expression profile showed a novel mode of action compared with three FDA-approved rheumatic drugs (methotrexate, rituximab and tocilizumab). To the best of our knowledge, this is a pioneer work charting the Ca2+ signaling network on the regulation of RA-associated inflammatory gene expression.

Functional analysis of choline transporter-like proteins in rheumatoid arthritis synovial fibroblasts: A novel molecular target for rheumatoid arthritis

Functional analysis of choline transporter-like proteins in rheumatoid arthritis synovial fibroblasts: A novel molecular target for rheumatoid arthritis

류마티스 관절염에서 광금전초 추출물의 관절염 억제 효과

Desomodii Herba (DH) has been shown to exhibit pharmacologyical activities, such as increase myocaridal contraction and secretion of hepatic bile. DH is used to reduce pain caused by rheumatoid arthritis(RA) in Korean medicine. However, the DH exact(DHE) effect and mechanism on rheumatoid arthritis are unknown. In this study, we aimed at the inhibitory effect of DHE on rheumatoid arthritis, and investigated the effect in collagen-induced mice arthritis model and TNF-αinduced MMP-1 and MMP-3 expression including the molecular basis in rheumatoid arthritis synovial fibroblasts (RASFs).The effect of DHE on RA was measured by clinical scoring system. In RASFs, expression of MMP-1 and MMP-3 was assessed by Western blotting and real-time PCR. Also, Western blotting used to evaluate the phosphorylation levels of p38, ERK and JNK and activation of NF-κB and AP-1. Our results showed that DHE reduced collagen-induced arthritis in mice. DHE inhibits TNF-αinduced MMP-1 and MMP-3 expression and mRNA levels in RASFs. The inhibitory effect of DHE was mediated by the inhibition of the AP-1/JNK signaling pathway. Taken together, our results suggest that the DHE may have preventive potential for rheumatoid arthritis.

CXCL10 and TRAIL Are Upregulated by TXNDC5 in Rheumatoid Arthritis Fibroblast-like Synoviocytes.

Thioredoxin domain containing 5 (TXNDC5) is highly expressed in synovial membranes of rheumatoid arthritis (RA). Our study aimed to investigate the pathogenic role of TXNDC5 in RA. PCR arrays, CCK-8 assays, flow cytometry, and transwell migration assays were used to analyze cultured rheumatoid arthritis synovial fibroblasts (RASF). Increased CXCL10 and tumor necrosis factor-related apoptosis-inducing ligand levels were detected in RASF transfected with anti-TXNDC5 small interfering RNA (siRNA), and decreased expression was detected in RASF transfected with TXNDC5-expressing plasmids. Significantly attenuated RASF proliferation and migration, and increased RASF apoptosis, were observed in the siRNA-transfected RASF. Downregulation of TXNDC5 could contribute to RASF antiangiogenic and proapoptotic features through the suppression of CXCL10 and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand).

Licochalcone A activates Keap1-Nrf2 signaling to suppress arthritis via phosphorylation of p62 at serine 349.

Licochalcone A (LCA) is derived from glycyrrhizae radix with antimicrobial, antitumor and anti-inflammatory activities. However, the anti-arthritic function of LCA and underlying mechanism has not been yet explored. The current study investigated the anti-arthritic effect of LCA and elucidated the underlying mechanism. The results showed that LCA significantly suppressed arthritis via the activation of SQSTM1 (p62)/nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling in the collagen-induced arthritis (CIA) model of DBA mice. In coincided with the results, this anti-arthritic effect of LCA was remarkably diminished in the collagen antibody-induced arthritis (CAIA) model of Nrf2-/- mice. These findings indicate that p62/Nrf2 signaling is a crucial pathway for the induction and treatment of arthritis. To further validate the effect of LCA on the arthritis, rheumatoid arthritis synovial fibroblasts (RASFs) isolated from the synovium of RA patients were employed in the study. In coincided with in vivo results, LCA inhibited the cell proliferation and arrested the cell cycle, induced apoptosis, suppressed pro-inflammatory cytokine secretion and increased expression of antioxidant enzymes via the activation of Keap1-Nrf2 signaling by enhancing p62 phosphorylation and expression, Nrf2 accumulation and Nrf2 nucleus translocation. Findings in the current study provide evidence that p62-Keap1-Nrf2 axis is a pivotal signaling pathway in development of arthritis and therapeutic efficacy of drugs, and LCA activates of Keap1-Nrf2 signaling to suppress arthritis by phosphorylation of p62 at Ser349. Collectively, LCA is valuable to be further investigated as a lead compound for application in anti-arthritis, and interference with the interaction between Nrf2 and Keap1 by phosphorylation of p62 may be a promising strategy for the discovery of anti-arthritic agents.

Myostatin Promotes Interleukin-1β Expression in Rheumatoid Arthritis Synovial Fibroblasts through Inhibition of miR-21-5p.

Rheumatoid arthritis (RA) is characterized by the infiltration of a number of pro-inflammatory cytokines into synovial fluid and patients with RA often develop joint destruction and deficits in muscle mass. The growth factor myostatin is a key regulator linking muscle mass and bone structure. We sought to determine whether myostatin regulates rheumatoid synovial fibroblast activity and inflammation in RA. We found that levels of myostatin and interleukin (IL)-1β (a key pro-inflammatory cytokine in RA) in synovial fluid from RA patients were overexpressed and positively correlated. In in vitro investigations, we found that myostatin dose-dependently regulated IL-1β expression through the ERK, JNK, and AP-1 signal-transduction pathways. Computational analysis confirmed that miR-21-5p directly targets the expression of the 3′ untranslated region (3′ UTR) of IL-1β. Treatment of cells with myostatin inhibited miR-21-5p expression and miR-21-5p mimic prevented myostatin-induced enhancement of IL-1β expression, showing an inverse correlation between miR-21-5p and IL-1β expression during myostatin treatment. We also found significantly increased paw swelling in an animal model of collagen-induced arthritis (CIA), compared with controls; immunohistochemistry staining revealed substantially higher levels of myostatin and IL-1β expression in CIA tissue. Our evidence indicates that myostatin regulates IL-1β production. Thus, targeting myostatin may represent a potential therapeutic target for RA.

Differential Effects of Activated Protein C on Synovial Fibroblasts in Response to Hypoxia and Normoxia

Rheumatoid arthritis (RA) is a chronic disease characterized by inflammation of the joints and their lining or synovium. Previous studies showed that the synovium in RA patients is more hypoxic than normal synovium. Activated protein C (APC) has anticoagulant and anti-inflammatory effects and is highly expressed in the joints of RA patients. We examined the effect of APC on RA and normal synovial fibroblasts under hypoxic conditions. Human synovial fibroblasts were isolated from the synovial tissues of RA patients and normal controls and cells were exposed to recombinant APC under normoxic (21% oxygen) or hypoxic (1% oxygen) conditions. Cell proliferation was measured using MTT assays. Cell lysates and conditioned media were collected and assayed for matrix metalloproteinase (MMP)-2, MMP-9 and p38 using zymography and western blots. Proliferation of both normal and RA synovial fibroblasts dose-dependently increased after APC treatment in normoxic conditions. Under hypoxia, APC enhanced RA cell proliferation but had no effect on normal fibroblasts. MMP-2 production and activation were significantly augmented by APC in both cell types under normoxia and hypoxia conditions. However, activated MMP-2 was more reduced in cells under hypoxia than normoxia. APC substantially reduced the phosphorylation of p38 in normal and RA synovial fibroblasts under hypoxia. No difference in p38 phosphorylation was observed under normoxia. The receptor for APC, endothelial protein C receptor (EPCR), was elevated in normal fibroblasts under hypoxic conditions whereas in RA cells, EPCR was highly expressed under both normoxic and hypoxic conditions. We found that hypoxia enhanced the effect of APC on RA synovial fibroblasts through activation of MMP2 and inhibition of p38 phosphorylation. Our results suggested that APC may suppress joint destruction and progression of inflammation in a hypoxic RA environment.