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1.  A novel pathogenic role of the ER chaperone GRP78/BiP in rheumatoid arthritis 
The ER chaperone GRP78/BiP is crucial for the development of rheumatoid arthritis.
An accumulation of misfolded proteins can trigger a cellular survival response in the endoplasmic reticulum (ER). In this study, we found that ER stress–associated gene signatures were highly expressed in rheumatoid arthritis (RA) synoviums and synovial cells. Proinflammatory cytokines, such as TNF and IL-1β, increased the expression of GRP78/BiP, a representative ER chaperone, in RA synoviocytes. RA synoviocytes expressed higher levels of GRP78 than osteoarthritis (OA) synoviocytes when stimulated by thapsigargin or proinflammatory cytokines. Down-regulation of Grp78 transcripts increased the apoptosis of RA synoviocytes while abolishing TNF- or TGF-β–induced synoviocyte proliferation and cyclin D1 up-regulation. Conversely, overexpression of the Grp78 gene prevented synoviocyte apoptosis. Moreover, Grp78 small interfering RNA inhibited VEGF165-induced angiogenesis in vitro and also significantly impeded synoviocyte proliferation and angiogenesis in Matrigel implants engrafted into immunodeficient mice. Additionally, repeated intraarticular injections of BiP-inducible factor X, a selective GRP78 inducer, increased synoviocyte proliferation and angiogenesis in the joints of mice with experimental OA. In contrast, mice with Grp78 haploinsufficiency exhibited the suppression of experimentally induced arthritis and developed a limited degree of synovial proliferation and angiogenesis. In summary, this study shows that the ER chaperone GRP78 is crucial for synoviocyte proliferation and angiogenesis, the pathological hallmark of RA.
doi:10.1084/jem.20111783
PMCID: PMC3328363  PMID: 22430489
2.  CD147 overexpression on synoviocytes in rheumatoid arthritis enhances matrix metalloproteinase production and invasiveness of synoviocytes 
Macrophage-like synoviocytes and fibroblast-like synoviocytes (FLS) are known as the most active cells of rheumatoid arthritis (RA) and are close to the articular cartilage in a position enabling them to invade the cartilage. Macrophage-like synoviocytes and FLS expression of matrix metalloproteinases (MMPs) and their interaction has aroused great interest. The present article studied the expression of CD147, also called extracellular matrix metalloproteinase inducer, on monocytes/macrophages and FLS from RA patients and its potential role in enhancing MMPs and the invasiveness of synoviocytes. Expression of CD147 on FLS derived from RA patients and from osteoarthritis patients, and expression of CD147 on monocytes/macrophages from rheumatic synovial fluid and healthy peripheral blood were analyzed by flow cytometry. The levels of CD147, MMP-2 and MMP-9 mRNA in FLS were detected by RT-PCR. The role of CD147 in MMP production and the cells' invasiveness in vitro were studied by the co-culture of FLS with the human THP-1 cell line or monocytes/macrophages, by gel zymography and by invasion assay. The results showed that the expression of CD147 was higher on RA FLS than on osteoarthritis FLS and was higher on monocytes/macrophages from rheumatic synovial fluid than on monocytes/macrophages from healthy peripheral blood. RT-PCR showed that the expressions of CD147, MMP-2 and MMP-9 mRNA was higher in RA FLS than in osteoarthritis FLS. A significantly elevated secretion and activation of MMP-2 and MMP-9 were observed in RA FLS co-cultured with differentiated THP-1 cells or RA synovial monocytes/macrophages, compared with those co-cultured with undifferentiated THP-1 cells or healthy control peripheral blood monocytes. Invasion assays showed an increased number of invading cells in the co-cultured RA FLS with differentiated THP-1 cells or RA synovial monocytes/macrophages. CD147 antagonistic peptide inhibited the MMP production and the invasive potential. Our studies demonstrated that the CD147 overexpression on monocytes/macrophages and FLS in RA patients may be responsible for the enhanced MMP secretion and activation and for the invasiveness of synoviocytes. These findings suggest that CD147 may be one of the important factors in progressive joint destruction of RA and that CD147 may be a potential therapeutic target in RA treatment.
doi:10.1186/ar1899
PMCID: PMC1526600  PMID: 16507143
3.  NFAT5 is a critical regulator of inflammatory arthritis 
Arthritis and rheumatism  2011;63(7):10.1002/art.30229.
Objective
To investigate the role of nuclear factor of activated T cells 5 (NFAT5), which is known as an osmoprotective transcription factor, in synovial hyperplasia and angiogenesis in rheumatoid arthritis (RA)
Methods
Expression of NFAT5 was examined in the synovial tissues and synoviocytes of RA patients using immunohistochemistry and Western blot analysis, respectively. The mRNAs of RA synoviocytes and human umbilical vein endothelial cells (HUVEC) transfected with dummy siRNA or NFAT5 siRNA were profiled using microarray technology. Assays to determine synoviocyte apoptosis and proliferation were performed in the presence of NFAT5 siRNA.VEGF165-induced angiogenesis was assessed by measuring the proliferation, tube formation, and wounding migration of HUVEC. Experimental arthritis was induced in mice by injection of anti-type II collagen antibody.
Results
NFAT5 was highly expressed in the rheumatoid synovium and its activity was increased by proinflammatory cytokines, such as IL-1β and TNF-α. The mRNA profiling of synoviocytes and HUVEC transfected with NFAT5-targeted siRNA revealed three major changes in cellular processes associated with the pathogenesis of RA: cell cycle and survival, angiogenesis, and cell migration. Consistent with these results, NFAT5 knock-down in RA synoviocytes and HUVEC inhibited their proliferation/survival and impeded angiogenic processes in HUVEC. Mice with NFAT5 haplo-insufficiency (NFAT5+/-) developed very limited degree of synovial proliferation in histological analysis, decreased angiogenesis, and exhibited a nearly complete suppression of experimentally induced arthritis.
Conclusion
NFAT5 regulates synovial proliferation and angiogenesis in chronic arthritis.
doi:10.1002/art.30229
PMCID: PMC3084342  PMID: 21717420
NFAT5; synoviocytes proliferation; angiogenesis; rheumatoid arthritis
4.  Slug suppression induces apoptosis via Puma transactivation in rheumatoid arthritis fibroblast-like synoviocytes treated with hydrogen peroxide 
Experimental & Molecular Medicine  2010;42(6):428-436.
Inadequate apoptosis contributes to synovial hyperplasia in rheumatoid arthritis (RA). Recent study shows that low expression of Puma might be partially responsible for the decreased apoptosis of fibroblast-like synoviocytes (FLS). Slug, a highly conserved zinc finger transcriptional repressor, is known to antagonize apoptosis of hematopoietic progenitor cells by repressing Puma transactivation. In this study, we examined the expression and function of Slug in RA FLS. Slug mRNA expression was measured in the synovial tissue (ST) and FLS obtained from RA and osteoarthritis patients. Slug and Puma mRNA expression in FLS by apoptotic stimuli were measured by real-time PCR analysis. FLS were transfected with control siRNA or Slug siRNA. Apoptosis was quantified by trypan blue exclusion, DNA fragmentation and caspase-3 assay. RA ST expressed higher level of Slug mRNA compared with osteoarthritis ST. Slug was significantly induced by hydrogen peroxide (H2O2) but not by exogenous p53 in RA FLS. Puma induction by H2O2 stimulation was significantly higher in Slug siRNA-transfected FLS compared with control siRNA-transfected FLS. After H2O2 stimulation, viable cell number was significantly lower in Slug siRNA-transfected FLS compared with control siRNA-transfected FLS. Apoptosis enhancing effect of Slug siRNA was further confirmed by ELISA that detects cytoplasmic histone-associated DNA fragments and caspase-3 assay. These data demonstrate that Slug is overexpressed in RA ST and that suppression of Slug gene facilitates apoptosis of FLS by increasing Puma transactivation. Slug may therefore represent a potential therapeutic target in RA.
doi:10.3858/emm.2010.42.6.044
PMCID: PMC2892596  PMID: 20418652
apoptosis; Puma; rheumatoid arthritis; snail family transcription factors; synovial membrane
5.  Engagement of Toll-Like Receptor 3 Induces Vascular Endothelial Growth Factor and Interleukin-8 in Human Rheumatoid Synovial Fibroblasts 
Background/Aims
Angiogenesis, which is a critical step in the initiation and progression of rheumatoid arthritis (RA), involves pro-angiogenic factors, including interleukin (IL)-8 and vascular endothelial growth factor (VEGF). We investigated the role of Toll-like receptor 3 (TLR3) in the regulation of pro-angiogenic factors in RA fibroblast-like synoviocytes (FLS).
Methods
FLS were isolated from RA synovial tissues and stimulated with the TLR3 ligand, poly (I:C). The levels of VEGF and IL-8 in the culture supernatants were measured using enzyme-linked immunosorbent assays, and the mRNA levels were assessed by semiquantitative reverse transcription-polymerase chain reaction. The expression patterns of VEGF and IL-8 in the RA synovium and osteoarthritis (OA) synovium were compared using immunohistochemistry.
Results
The expression levels of TLR3, VEGF, and IL-8 were significantly higher in the RA synovium than in the OA synovium. VEGF and IL-8 production were increased in the culture supernatants of RA FLS stimulated with poly (I:C), and the genes for these proteins were up-regulated at the transcriptional level after poly (I:C) treatment. Treatment with inhibitors of nuclear factor-kappaB (NF-κB), i.e., pyrrolidine dithiocarbamate and parthenolide, abrogated the stimulatory effect of poly (I:C) on the production of VEGF and IL-8 in RA FLS.
Conclusions
Our results suggest that the activation of TLR3 in RA FLS promotes the production of proangiogenic factors, in a process that is mediated by the NF-κB signaling pathway. Therefore, targeting the TLR3 pathway may be a promising approach to preventing pathologic angiogenesis in RA.
doi:10.3904/kjim.2010.25.4.429
PMCID: PMC2997973  PMID: 21179282
Toll-like receptor 3; Arthritis, rheumatoid; Vascular endothelial growth factor; Interleukin-8; Synovial fibroblast
6.  Acute-phase serum amyloid A production by rheumatoid arthritis synovial tissue 
Arthritis Research  2000;2(2):142-144.
Acute-phase serum amyloid A (A-SAA) is a major component of the acute-phase response. A sustained acute-phase response in rheumatoid arthritis (RA) is associated with increased joint damage. A-SAA mRNA expression was confirmed in all samples obtained from patients with RA, but not in normal synovium. A-SAA mRNA expression was also demonstrated in cultured RA synoviocytes. A-SAA protein was identified in the supernatants of primary synoviocyte cultures, and its expression colocalized with sites of macrophage accumulation and with some vascular endothelial cells. It is concluded that A-SAA is produced by inflamed RA synovial tissue. The known association between the acute-phase response and progressive joint damage may be the direct result of synovial A-SAA-induced effects on cartilage degradation.
Introduction:
Serum amyloid A (SAA) is the circulating precursor of amyloid A protein, the fibrillar component of amyloid deposits. In humans, four SAA genes have been described. Two genes (SAA1 and SAA2) encode A-SAA and are coordinately induced in response to inflammation. SAA1 and SAA2 are 95% homologous in both coding and noncoding regions. SAA3 is a pseudogene. SAA4 encodes constitutive SAA and is minimally inducible. A-SAA increases dramatically during acute inflammation and may reach levels that are 1000-fold greater than normal. A-SAA is mainly synthesized in the liver, but extrahepatic production has been demonstrated in many species, including humans. A-SAA mRNA is expressed in RA synoviocytes and in monocyte/macrophage cell lines such as THP-1 cells, in endothelial cells and in smooth muscle cells of atherosclerotic lesions. A-SAA has also been localized to a wide range of histologically normal tissues, including breast, stomach, intestine, pancreas, kidney, lung, tonsil, thyroid, pituitary, placenta, skin and brain.
Aims:
To identify the cell types that produce A-SAA mRNA and protein, and their location in RA synovium.
Materials and methods:
Rheumatoid synovial tissue was obtained from eight patients undergoing arthroscopic biopsy and at joint replacement surgery. Total RNA was analyzed by reverse transcription (RT) polymerase chain reaction (PCR) for A-SAA mRNA. PCR products generated were confirmed by Southern blot analysis using human A-SAA cDNA. Localization of A-SAA production was examined by immunohistochemistry using a rabbit antihuman A-SAA polyclonal antibody. PrimaryRA synoviocytes were cultured to examine endogenous A-SAA mRNA expression and protein production.
Results:
A-SAA mRNA expression was detected using RT-PCR in all eight synovial tissue samples studied. Figure 1 demonstrates RT-PCR products generated using synovial tissue from three representative RA patients. Analysis of RA synovial tissue revealed differences in A-SAA mRNA levels between individual RA patients.
In order to identify the cells that expressed A-SAA mRNA in RA synovial tissue, we analyzed primary human synoviocytes (n = 2). RT-PCR analysis revealed A-SAA mRNA expression in primary RA synoviocytes (n = 2; Fig. 2). The endogenous A-SAA mRNA levels detected in individual primary RA synoviocytes varied between patients. These findings are consistent with A-SAA expression in RA synovial tissue (Fig. 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were relatively similar in the RA synoviocytes examined (Fig. 2). A-SAA protein in the supernatants of primary synoviocyte cultures from four RA patients was measured using ELISA. Mean values of a control and four RA samples were 77.85, 162.5, 249.8, 321.5 and 339.04 μg/l A-SAA, respectively, confirming the production of A-SAA protein by the primary RA synoviocytes. Immunohistochemical analysis was performed to localize sites of A-SAA production in RA synovial tissue. Positive staining was present in both the lining and sublining layers of all eight RA tissues examined (Fig. 3a). Staining was intense and most prominent in the cells closest to the surface of the synovial lining layer. Positively stained cells were evident in the perivascular areas of the sublining layer. In serial sections stained with anti-CD68 monoclonal antibody, positive staining of macrophages appeared to colocalize with A-SAA-positive cells (Fig. 3b). Immunohistochemical studies of cultured primary RA synoviocytes confirmed specific cytoplasmic A-SAA expression in these cells. The specificity of the staining was confirmed by the absence of staining found on serial sections and synoviocyte cells treated with IgG (Fig. 3c).
Discussion:
This study demonstrates that A-SAA mRNA is expressed in several cell populations infiltrating RA synovial tissue. A-SAA mRNA expression was observed in all eight unseparated RA tissue samples studied. A-SAA mRNA expression and protein production was demonstrated in primary cultures of purified RA synoviocytes. Using immunohistochemical techniques, A-SAA protein appeared to colocalize with both lining layer and sublining layer synoviocytes, macrophages and some endothelial cells. The detection of A-SAA protein in culture media supernatants harvested from unstimulated synoviocytes confirms endogenous A-SAA production, and is consistent with A-SAA mRNA expression and translation by the same cells. Moreover, the demonstration of A-SAA protein in RA synovial tissue, RA cultured synoviocytes, macrophages and endothelial cells is consistent with previous studies that demonstrated A-SAA production by a variety of human cell populations.
The RA synovial lining layer is composed of activated macrophages and fibroblast-like synoviocytes. The macrophage is the predominant cell type and it has been shown to accumulate preferentially in the surface of the lining layer and in the perivascular areas of the sublining layer. Nevertheless, our observations strongly suggest that A-SAA is produced not only by synoviocytes, but also by synovial tissue macrophage populations. Local A-SAA protein production by vascular endothelial cells was detected in some, but not all, of the tissues examined. The reason for the variability in vascular A-SAA staining is unknown, but may be due to differences in endothelial cell activation, events related to angiogenesis or the intensity of local inflammation.
The value of measuring serum A-SAA levels as a reliable surrogate marker of inflammation has been demonstrated for several diseases including RA, juvenile chronic arthritis, psoriatic arthropathy, ankylosing spondylitis, Behçet's disease, reactive arthritis and Crohn's disease. It has been suggested that serum A-SAA levels may represent the most sensitive measurement of the acute-phase reaction. In RA, A-SAA levels provide the strongest correlations with clinical measurements of disease activity, and changes in serum levels best reflect the clinical course.
A number of biologic activities have been described for A-SAA, including several that are relevant to the understanding of inflammatory and tissue-degrading mechanisms in human arthritis. A-SAA induces migration, adhesion and tissue infiltration of circulating monocytes and polymorphonuclear leukocytes. In addition, human A-SAA can induce interleukin-1β, interleukin-1 receptor antagonist and soluble type II tumour necrosis factor receptor production by a monocyte cell line. Moreover, A-SAA can stimulate the production of cartilage-degrading proteases by both human and rabbit synoviocytes. The effects of A-SAA on protease production are interesting, because in RA a sustained acute-phase reaction has been strongly associated with progressive joint damage. The known association between the acute-phase response and progressive joint damage may be the direct result of synovial A-SAA-induced effects on cartilage degradation.
Conclusion:
In contrast to noninflamed synovium, A-SAA mRNA expression was identified in all RA tissues examined. A-SAA appeared to be produced by synovial tissue synoviocytes, macrophages and endothelial cells. The observation of A-SAA mRNA expression in cultured RA synoviocytes and human RA synovial tissue confirms and extends recently published findings that demonstrated A-SAA mRNA expression in stimulated RA synoviocytes, but not in unstimulated RA synoviocytes.
PMCID: PMC17807  PMID: 11062604
acute-phase response; rheumatoid arthritis; serum amyloid A; synovial tissue
7.  IL-17 induces the production of IL-16 in rheumatoid arthritis 
Experimental & Molecular Medicine  2008;40(2):237-245.
The purpose of this study was to investigate the expression of IL-16 in the rheumatoid synovium and the role of inflammatory cytokines and Toll-like receptor (TLR) ligands in IL-16 production by fibroblastlike synoviocytes (FLS) of rheumatoid arthritis (RA) patients. Immunohistochemical staining was performed with a monoclonal antibody to IL-16 in synovial tissues from patients with RA and likewise in patients with osteoarthritis (OA). FLS were isolated from RA synovial tissues and stimulated with IL-15, IL-1β, IFN-γ, and IL-17. The IL-16 mRNA level was assessed by semiquantitative RT-PCR and real time (RT) PCR and a comparison was made between IL-16 mRNA levels produced by RA-FLS and OA-FLS. Production of IL-16 was identified by a western blot assay, and IL-16 production after stimulation by specific ligands of TLR2 and TLR4 was assessed by RT-PCR. While immunohistochemical staining demonstrated strong expression of IL-16 mRNA in synovial tissues from patients with RA, similar findings were not present in the OA group. Moreover, mRNA expression of IL-16 by RA-FLS increased after treatment with IL-17 but not with IL-15, IL-1β, and IFN-γ. Specifically, IL-17 increased IL-16 mRNA level by RA-FLS and peripheral blood mononuclear cells in a dose-dependent manner. However, IL-17 did not stimulate IL-16 production in OA-FLS. Peptidoglycan, a selective TLR2 ligand, also increased production of IL-16 by RA-FLS dosedependently, whereas LPS, a selective TLR4 ligand, had no such stimulatory effect. The results from our data demonstrate that IL-17 and TLR2 ligands stimulate the production of IL-16 by RA-FLS.
doi:10.3858/emm.2008.40.2.237
PMCID: PMC2679298  PMID: 18446062
interleukin-16; interleukin-17; rheumatoid arthritis; synovial membrane; Toll-like receptors
8.  Expression and regulation of cryopyrin and related proteins in rheumatoid arthritis synovium 
Annals of the Rheumatic Diseases  2004;64(5):708-714.
Background: Rheumatoid arthritis (RA) synovium is characterised by enhanced NF-κB activity and proinflammatory cytokines. Cryopyrin (CIAS-1, NALP-3, PYPAF-1) has been shown to regulate NF-κB and caspase-1 activation.
Objective: To study the expression of cryopyrin, its effector molecule ASC, and its putative antagonist pyrin in RA and osteoarthritis (OA) synovium, and the main two cellular constituents of synovial lining, cultured fibroblast-like synoviocytes (FLS) and macrophages.
Methods: FLS and macrophages were cultured in the presence of inflammatory mediators. Real time polymerase chain reaction was used to quantify message levels in synovial biopsy specimens and cells. In situ hybridisation was employed to localise expression of cryopyrin mRNA.
Results: Cryopyrin mRNA was raised in RA synovium and detected in both lining and sublining regions. FLS from RA and OA tissue expressed low baseline levels of cryopyrin transcripts that were induced by tumour necrosis factor α (TNFα). In contrast, macrophages differentiated in vitro expressed relatively high cryopyrin levels, which were further induced by TNFα, but not by interleukin 1ß. ASC mRNA levels were comparable in RA and OA tissue, FLS, and macrophages, and were depressed by TNFα in macrophages. Pyrin expression was higher in RA synovium than in OA tissue, and virtually undetectable in FLS but high in macrophages where it was unchanged by TNFα treatment.
Conclusion: These results suggest that enhanced cryopyrin levels in RA synovium are due to a greater numbers of tissue macrophages, and demonstrate transcriptional regulation of cryopyrin in a chronic inflammatory disease.
doi:10.1136/ard.2004.025577
PMCID: PMC1755498  PMID: 15498798
9.  ATP Induced Brain-Derived Neurotrophic Factor Expression and Release from Osteoarthritis Synovial Fibroblasts Is Mediated by Purinergic Receptor P2X4 
PLoS ONE  2012;7(5):e36693.
Brain-derived neurotrophic factor (BDNF), a neuromodulator involved in nociceptive hypersensitivity in the central nervous system, is also expressed in synoviocytes of osteoarthritis (OA) and rheumatoid arthritis (RA) patients. We investigated the role of P2 purinoreceptors in the induction of BDNF expression in synovial fibroblasts (SF) of OA and RA patients. Cultured SF from patients with symptomatic knee OA and RA were stimulated with purinoreceptor agonists ATP, ADP, or UTP. The expression of BDNF mRNA was measured by quantitative TaqMan PCR. BDNF release into cell culture supernatants was monitored by ELISA. P2X4 expression in synovial tissue was detected by immunohistochemistry. Endogenous P2X4 expression was decreased by siRNA transfection before ATP stimulation. Kinase pathways were blocked before ATP stimulation. BDNF mRNA expression levels in OASF were increased 2 h and 5 h after ATP stimulation. Mean BDNF levels in cell culture supernatants of unstimulated OASF and RASF were 19 (±9) and 67 (±49) pg/ml, respectively. BDNF levels in SF supernatants were only elevated 5 h after ATP stimulation. BDNF mRNA expression in OASF was induced both by P2X receptor agonists ATP and ADP, but not by UTP, an agonist of P2Y purinergic receptors. The ATP-induced BDNF mRNA expression in OASF was decreased by siRNA-mediated reduction of endogenous P2X4 levels compared to scrambled controls. Inhibition of p38, but not p44/42 signalling reduced the ATP-mediated BDNF mRNA induction. Here we show a functional role of the purinergic receptor P2X4 and p38 kinase in the ATP-induced expression and release of the neurotrophin BDNF in SF.
doi:10.1371/journal.pone.0036693
PMCID: PMC3360754  PMID: 22715356
10.  Soluble Fas ligand inhibits angiogenesis in rheumatoid arthritis 
The characteristics of rheumatoid arthritis (RA) pathology include the infiltration of inflammatory leukocytes, the proliferation of synovial cells, and the presence of extensive angiogenesis, referred to as rheumatoid pannus. Fas ligand is critical to the homeostatic regulation of the immune response, but its role in the angiogenic process of RA remains to be defined. In this study, we investigated whether soluble Fas ligand (sFasL) induces synoviocyte apoptosis and regulates angiogenesis of endothelial cells in RA. The levels of sFasL were elevated in the synovial fluids of RA patients when compared to those of osteoarthritis (OA) patients, and they correlated inversely with vascular endothelial growth factor165 (VEGF165) concentrations. sFasL, ranging from 10 to 100 ng/ml, induced the apoptosis of RA fibroblast-like synoviocytes (FLS) in vitro, and thereby decreased VEGF165 production. In addition, sFasL inhibited VEGF165-induced migration and chemotaxis of endothelial cells to basal levels in a manner independent of the Fas-mediated cell death. sFasL dose-dependently suppressed the VEGF165-stimulated increase in pAkt expression in endothelial cells, which might be associated with its anti-migratory effect on endothelial cells. Moreover, sFasL strongly inhibited neovascularization in the Matrigel plug in vivo. Our data suggest that sFasL shows anti-angiogenic activity within RA joints not only by inducing apoptosis of VEGF165-producing cells but also by blocking VEGF165-induced migration of endothelial cells, independent of Fas-mediated apoptosis.
doi:10.1186/ar2181
PMCID: PMC1906820  PMID: 17459170
11.  TNFα induces sustained signaling and a prolonged and unremitting inflammatory response in synovial fibroblasts 
Arthritis and rheumatism  2013;65(4):928-938.
Objective
The non resolving character of synovial inflammation in rheumatoid arthritis (RA) is a conundrum. To identify the contribution of fibroblast-like synoviocytes (FLS) to the perpetuation of synovitis, we investigated the molecular mechanisms that govern the TNFα-driven inflammatory program in human FLS.
Methods
FLS obtained from synovial tissues of patients with RA or osteoarthritis were stimulated with TNFα and assayed for gene expression and cytokine production by qPCR and ELISA. NF-κB signaling was evaluated using Western blotting. Histone acetylation, chromatin accessibility, and NF-κB p65 and RNA polymerase II (Pol II) occupancy at the IL6 promoter were measured by chromatin immunoprecipitation and restriction enzyme accessibility assays.
Results
In FLS, TNFα induced prolonged transcription of IL6 and progressive accumulation of IL-6 protein over four days. Similarly, induction of CXCL8/IL-8, CCL5/RANTES, MMP1 and MMP3 mRNA after TNFα stimulation was sustained for several days. This contrasted with the macrophage response to TNFα, which characteristically involved a transient increase in the expression of pro-inflammatory genes. In FLS, TNFα induced prolonged activation of NF-κB signaling and sustained transcriptional activity indicated by increased histone acetylation, chromatin accessibility, and p65 and Pol II occupancy at the IL6 promoter. Furthermore, FLS expressed low levels of the feedback inhibitors ABIN3, IRAK-M, SOCS3 and ATF3 that terminate inflammatory responses in macrophages.
Conclusions
TNFα signaling is not effectively terminated in FLS, leading to an uncontrolled inflammatory response. The results suggest that prolonged and sustained inflammatory responses by FLS, in response to synovial TNFα, contribute to the persistence of synovial inflammation in RA.
doi:10.1002/art.37853
PMCID: PMC3618592  PMID: 23335080
fibroblast-like synoviocytes; rheumatoid arthritis; signal transduction; TNFα; chromatin
12.  Cyclooxygenase-1 and -2 expression in rheumatoid synovial tissues. Effects of interleukin-1 beta, phorbol ester, and corticosteroids. 
Journal of Clinical Investigation  1994;93(3):1095-1101.
High levels of immunoreactive cyclooxygenase (Cox; prostaglandin H synthase) are present in synovia from patients with rheumatoid arthritis (RA). We now show that the recently identified inducible isoform of Cox, Cox-2, is expressed in synovia from patients with RA. To further explore modulation of the Cox isoforms in RA synovial tissues, we examined the expression and modulation of Cox-1 and -2 in rheumatoid synovial explant cultures and cultured rheumatoid synovial fibroblast-like cells (synoviocytes). Immunoprecipitation of in vitro labeled proteins and Western blot analysis demonstrated the presence of both Cox-1 and -2 under basal conditions in freshly explanted rheumatoid synovial tissues. De novo synthesis of Cox-2 polypeptide was enhanced by IL-1 beta or PMA, and dramatically suppressed by dexamethasone (dex). Cox-1 expression, under the same conditions, showed only minor variation. Since mRNA for Cox-2 is highly unstable, we examined the regulation of Cox-2 transcripts in cultured rheumatoid synoviocytes. Under basal conditions both Cox-1 and -2 mRNAs were present at low levels, but Cox-2 mRNA was markedly increased by treatment with IL-1 beta or PMA. dex markedly suppressed the induction of Cox-2 mRNA. In sharp contrast, Cox-1 transcripts were not modulated by IL-1 beta or dex. These data suggest that modulation of Cox-2 expression by IL-1 beta and corticosteroids may be an important component of the inflammatory process in synovial tissues from patients with RA.
Images
PMCID: PMC294048  PMID: 8132748
13.  Leukemia inhibitory factor is expressed in cartilage and synovium and can contribute to the pathogenesis of arthritis. 
Journal of Clinical Investigation  1992;90(3):888-896.
This study reports on leukemia inhibitory factor (LIF) in human articular connective tissues. Biologically active LIF is present in synovial fluids from patients with osteoarthritis and at higher titers in samples from patients with rheumatoid arthritis. Cultured human synoviocytes and articular chondrocytes produced biologically active LIF and synthesized and secreted LIF proteins that migrated in SDS PAGE at approximately 43 kD. This was increased after stimulation with IL-1 beta. Chondrocytes in serum-containing cultures expressed the 4.2-kb LIF mRNA. IL-1 beta, LPS, and to a lesser extent tumor necrosis factor-alpha induced LIF gene expression. LIF autoinduced its mRNA and this provides evidence for an effect of this cytokine on function of joint tissue cells. Among a series of growth factors tested, transforming growth factor (TGF beta), including the isoforms TGF-beta1, TGF-beta 2, and TGF-beta 3, platelet-derived growth factor, basic fibroblast growth factor, and insulin-like growth factor induced this cytokine gene but differed with respect to the duration of their effects. Cultured synoviocytes expressed the LIF gene in response to the same set of peptide regulatory factors. Analysis of signal transduction pathways showed that PMA increased LIF mRNA, whereas calcium ionophore and cAMP had no detectable effects. Cycloheximide was a potent LIF mRNA inducer and dexamethasone inhibited LIF induced by PMA or IL-1 beta. Cartilage organ cultures and synovial tissues stimulated with IL-1 expressed high levels of LIF mRNA as demonstrated by in situ hybridization. These results identify LIF as a new cytokine that is produced by joint tissue cells and is overexpressed in arthritis. The induction of this cytokine by factors that are present during joint inflammation and the effects of LIF on connective tissue cells suggest that LIF is a mediator that can contribute to the pathogenesis of arthritis.
Images
PMCID: PMC329943  PMID: 1522240
14.  Somatic mutations in mitochondria: the chicken or the egg? 
Arthritis Research & Therapy  2005;7(5):179-180.
Somatic mutations of mitochondrial DNA have been detected in various pathologies such as cancer, neurodegenerative diseases, cardiac disorders and aging in general. Now it has been found that patients with rheumatoid arthritis also have a higher incidence of mitochondrial mutations in synoviocytes and synovial tissue compared with patients with osteoarthritis. Furthermore, it has been shown that these mutations possibly result in changed peptides that are presented by major histocompatibility complex II and thus might be recognized as non-self by the immune system. Further studies will show whether these mutations are actually able to trigger autoimmune inflammation in rheumatoid arthritis or whether they must be considered epiphenomena of cellular damage in chronic inflammation.
doi:10.1186/ar1809
PMCID: PMC1257449  PMID: 16207343
15.  Synoviocyte innate responses: II. Pivotal role of interferon regulatory factor 3 
Introduction
Innate immune responses likely contribute to synovial inflammation in rheumatoid arthritis (RA). Of the innate receptors implicated in RA, TLR3 activates several signaling cascades, including -interferon regulatory factors 3 and 7 (IRF), resulting in production of viral-stress IFN-inducible genes. The present study was designed to investigate the contributions of IRF3 and IRF7 to the type I IFN response as well as the expression of other cytokines, chemokines, and degradative enzymes in synoviocytes.
Methods
Fibroblast-like synoviocytes (FLS) were stimulated with poly (I-C) after transfection with IRF3 or IRF7 siRNA to knockdown transcription factor expression. Western blots, luciferase assay after transfection with reporter constructs, Q-PCR, and AP-1 DNA binding ELISA was performed to evaluate the role of IRF3 and IRF7 in poly (I-C)-induced signaling and synoviocyte gene expression.
Results
IRF3 and IRF7 knockdown showed that IRF3 regulates IFN-stimulated response element (ISRE) promoter activity as well as IFNβ, IRF5, IRF7, RANTES, IP-10, MCP-1, and MIP1α gene expression in response to poly (I-C). IRF7 knockdown modestly decreased a subset of genes and ISRE activity, although the results were not significant. Surprisingly, IRF3 knockdown almost completely blocked expression of additional genes in which the ISRE is not traditionally considered a dominant promoter site in FLS, including MMP3, MMP9, IL-6 and IL-8. We then investigated a possible role for IRF3 in c-Jun activation and AP-1 binding because its promoter site is present in all four of the non-IFN regulated genes. IRF3 deficiency significantly decreased AP-1 binding of activated c-Jun compared with control.
Conclusions
In contrast to immune cells, IRF3 rather than IRF7 regulates TLR3-mediated type I IFN responses in human synoviocytes. IRF3 activates IFN-response gene expression by increasing ISRE promoter activity. In addition, IRF3 regulates other cytokines, chemokines, and MMPs through a novel mechanism that involves c-Jun and the AP-1 promoter site. Because the signaling pathway modulated by IRF3 plays a crucial role in synoviocytes, targeting IRF3 represents a potential approach to suppress diverse mediators while limiting suppression of IRF7-mediated immune responses.
doi:10.4049/jimmunol.0903944
PMCID: PMC2913682  PMID: 20483755
Rheumatoid arthritis; signal transduction; transcription factors; interferon
16.  High mobility group box 1 potentiates the pro-inflammatory effects of interleukin-1β in osteoarthritic synoviocytes 
Arthritis Research & Therapy  2010;12(4):R165.
Introduction
High mobility group box 1 (HMGB1) is released by necrotic cells or secreted in response to inflammatory stimuli. Extracellular HMGB1 may act as a pro-inflammatory cytokine in rheumatoid arthritis. We have recently reported that HMGB1 is released by osteoarthritic synoviocytes after activation with interleukin-1beta (IL-1β) The present study investigated the role of HMGB1 in synovial inflammation in osteoarthritis (OA).
Methods
HMGB1 was determined in human synovium using immunohistochemistry, comparing normal to OA. OA synoviocytes were incubated with HMGB1 at 15 or 25 ng/ml in the absence or presence of IL-1β (10 ng/ml). Gene expression was analyzed by quantitative PCR and protein expression by Western Blot and ELISA. Matrix metalloproteinase (MMP) activity was studied by fluorometric procedures and nuclear factor (NF)-κB activation by transient transfection with a NF-κB-luciferase plasmid.
Results
In the normal synovium, HMGB1 was found in the synovial lining cells, sublining cells, and in the vascular wall cells. The distribution of HMGB1 in OA synovium was similar but the number of HMGB1 positive cells was higher and HMGB1 was also present in infiltrated cells. In normal synovial membrane cells, HMGB1 was found mostly in the nuclei, whereas in OA, HMGB1 was generally found mostly in the cytoplasm. In OA synoviocytes, HMGB1 alone at concentrations of 15 or 25 ng/ml did not affect the production of IL-6, IL-8, CCL2, CCL20, MMP-1 or MMP-3, but in the presence of IL-1β, a significant potentiation of protein and mRNA expression, as well as MMP activity was observed. HMGB1 also enhanced the phosphorylated ERK1/2 and p38 levels, with a lower effect on phosphorylated Akt. In contrast, JNK1/2 phosphorylation was not affected. In addition, HMGB1 at 25 ng/ml significantly potentiated NF-κB activation in the presence of IL-1β.
Conclusions
Our results indicate that HMGB1 is overexpressed in OA synovium and mostly present in extracellular form. In OA synoviocytes, HMGB1 cooperates with IL-1β to amplify the inflammatory response leading to the production of a number of cytokines, chemokines and MMPs. Our data support a pro-inflammatory role for this protein contributing to synovitis and articular destruction in OA.
doi:10.1186/ar3124
PMCID: PMC2945068  PMID: 20799933
17.  Differential expression and functional behaviour of the αv and β3 integrin subunits in cytokine stimulated fibroblast-like cells derived from synovial tissue of rheumatoid arthritis and osteoarthritis in vitro 
Annals of the Rheumatic Diseases  1997;56(12):729-736.
OBJECTIVE—The aim of this study was to investigate in situ the expression of the classic vitronectin (VN) receptor consisting of the αv and β3 subunits in synovial lining cells (SLC) of chronic synovitis occurring in osteoarthritis (OA) and in rheumatoid arthritis (RA). The expression and function of αv and β3 as VN receptor in cultured fibroblast-like synoviocytes (FBS) derived from patients with OA and RA was also compared.
METHODS—Expression of αv and β3 was examined immunohistochemically in normal synovial tissue and in synovial tisssue from patients with OA and RA. The effect of proinflammatory cytokines and of a synovial fluid of a patient with RA on the expression of the αv and β3 subunits of cultured FBS was determined by flow cytometry. Binding of OA and RA-FBS to VN was quantified using adhesion assays and the effect of interleukin 1β (IL1β) and tumour necrosis factor α (TNFα) on adhesion was measured. The specifity of the adhesion was tested by inhibition studies using monoclonal antibodies to integrin subunits.
RESULTS—In in situ studies normal SLC showed a parallel distribution of αv and β3 subunits. OA-SLC strongly and uniformly expressed αv whereas RA-SLC showed heterogeneous expression of αv. In situ both OA-SLC and RA-SLC lacked the expression of the integrin subunit β3. In in vitro studies, OA-FBS and RA-FBS did not differ as regards expression of αv and β3, and VN attachment. Binding of RA-FBS to VN was partially blocked by antibodies against αv, β1, and β3 subunits, whereas only antibodies against αv and β3 inhibited the binding of OA-FBS to VN. The proinflammatory cytokines TNFα and IL1β increased the expression of αv and β3, and the VN binding of OA-FBS, whereas αv and β3 expression, and VN binding were downregulated in RA-FBS. Similar effects were found when the synovial fluid of an RA patient was used.
CONCLUSION—The integrin subunit β3 seems to be one partner but not the major one with which the subunit αv forms functional vitronectin receptors in OA-FBS and RA-FBS. The interaction between synovial cells and inflammatory cytokines seems to be different for OA and RA; the basis for this difference, however, remains to be established.


PMCID: PMC1752301  PMID: 9496152
18.  Early and long-standing rheumatoid arthritis: distinct molecular signatures identified by gene-expression profiling in synovia 
Introduction
Rheumatoid arthritis (RA) is a heterogeneous disease and its underlying molecular mechanisms are still poorly understood. Because previous microarray studies have only focused on long-standing (LS) RA compared to osteoarthritis, we aimed to compare the molecular profiles of early and LS RA versus control synovia.
Methods
Synovial biopsies were obtained by arthroscopy from 15 patients (4 early untreated RA, 4 treated LS RA and 7 controls, who had traumatic or mechanical lesions). Extracted mRNAs were used for large-scale gene-expression profiling. The different gene-expression combinations identified by comparison of profiles of early, LS RA and healthy synovia were linked to the biological processes involved in each situation.
Results
Three combinations of 719, 116 and 52 transcripts discriminated, respectively, early from LS RA, and early or LS RA from healthy synovia. We identified several gene clusters and distinct molecular signatures specifically expressed during early or LS RA, thereby suggesting the involvement of different pathophysiological mechanisms during the course of RA.
Conclusions
Early and LS RA have distinct molecular signatures with different biological processes participating at different times during the course of the disease. These results suggest that better knowledge of the main biological processes involved at a given RA stage might help to choose the most appropriate treatment.
doi:10.1186/ar2744
PMCID: PMC2714155  PMID: 19563633
19.  The tumour-associated glycoprotein podoplanin is expressed in fibroblast-like synoviocytes of the hyperplastic synovial lining layer in rheumatoid arthritis 
Introduction
Activated fibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA) share many characteristics with tumour cells and are key mediators of synovial tissue transformation and joint destruction. The glycoprotein podoplanin is upregulated in the invasive front of several human cancers and has been associated with epithelial-mesenchymal transition, increased cell migration and tissue invasion. The aim of this study was to investigate whether podoplanin is expressed in areas of synovial transformation in RA and especially in promigratory RA-FLS.
Methods
Podoplanin expression in human synovial tissue from 18 RA patients and nine osteoarthritis (OA) patients was assessed by immunohistochemistry and confirmed by Western blot analysis. The expression was related to markers of synoviocytes and myofibroblasts detected by using confocal immunofluoresence microscopy. Expression of podoplanin, with or without the addition of proinflammatory cytokines and growth factors, in primary human FLS was evaluated by using flow cytometry.
Results
Podoplanin was highly expressed in cadherin-11-positive cells throughout the synovial lining layer in RA. The expression was most pronounced in areas with lining layer hyperplasia and high matrix metalloproteinase 9 expression, where it coincided with upregulation of α-smooth muscle actin (α-sma). The synovium in OA was predominantly podoplanin-negative. Podoplanin was expressed in 50% of cultured primary FLSs, and the expression was increased by interleukin 1β, tumour necrosis factor α and transforming growth factor β receptor 1.
Conclusions
Here we show that podoplanin is highly expressed in FLSs of the invading synovial tissue in RA. The concomitant upregulation of α-sma and podoplanin in a subpopulation of FLSs indicates a myofibroblast phenotype. Proinflammatory mediators increased the podoplanin expression in cultured RA-FLS. We conclude that podoplanin might be involved in the synovial tissue transformation and increased migratory potential of activated FLSs in RA.
doi:10.1186/ar3274
PMCID: PMC3132020  PMID: 21385358
20.  Fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes 
Fibroblast activation protein (FAP), as described so far, is a type II cell surface serine protease expressed by fibroblastic cells in areas of active tissue remodelling such as tumour stroma or healing wounds. We investigated the expression of FAP by fibroblast-like synoviocytes (FLSs) and compared the synovial expression pattern in rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Synovial tissue from diseased joints of 20 patients, 10 patients with refractory RA and 10 patients with end-stage OA, was collected during routine surgery. As a result, FLSs from intensively inflamed synovial tissues of refractory RA expressed FAP at high density. Moreover, FAP expression was co-localised with matrix metalloproteinases (MMP-1 and MMP-13) and CD44 splice variants v3 and v7/8 known to play a major role in the concert of extracellular matrix degradation. The pattern of signals appeared to constitute a characteristic feature of FLSs involved in rheumatoid arthritic joint-destructive processes. These FAP-expressing FLSs with a phenotype of smooth muscle actin-positive myofibroblasts were located in the lining layer of the synovium and differ distinctly from Thy-1-expressing and non-proliferating fibroblasts of the articular matrix. The intensity of FAP-specific staining in synovial tissue from patients with RA was found to be different when compared with end-stage OA. Because expression of FAP by RA FLSs has not been described before, the findings of this study highlight a novel element in cartilage and bone destruction of arthritic joints. Moreover, the specific expression pattern qualifies FAP as a therapeutic target for inhibiting the destructive potential of fibroblast-like synovial cells.
doi:10.1186/ar2080
PMCID: PMC1794515  PMID: 17105646
21.  IL-26 Is Overexpressed in Rheumatoid Arthritis and Induces Proinflammatory Cytokine Production and Th17 Cell Generation 
PLoS Biology  2012;10(9):e1001395.
Human interleukin-26 induces Th17 cells, is over-expressed in rheumatoid arthritis, and is thus a promising therapeutic target in chronic inflammatory disease.
Interleukin-26 (IL-26), a member of the IL-10 cytokine family, induces the production of proinflammatory cytokines by epithelial cells. IL-26 has been also reported overexpressed in Crohn's disease, suggesting that it may be involved in the physiopathology of chronic inflammatory disorders. Here, we have analyzed the expression and role of IL-26 in rheumatoid arthritis (RA), a chronic inflammatory disorder characterized by joint synovial inflammation. We report that the concentrations of IL-26 are higher in the serums of RA patients than of healthy subjects and dramatically elevated in RA synovial fluids compared to RA serums. Immunohistochemistry reveals that synoviolin+ fibroblast-like synoviocytes and CD68+ macrophage-like synoviocytes are the main IL-26-producing cells in RA joints. Fibroblast-like synoviocytes from RA patients constitutively produce IL-26 and this production is upregulated by IL-1-beta and IL-17A. We have therefore investigated the role of IL-26 in the inflammatory process. Results show that IL-26 induces the production of the proinflammatory cytokines IL-1-beta, IL-6, and tumor necrosis factor (TNF)-alpha by human monocytes and also upregulates the expression of numerous chemokines (mainly CCL20). Interestingly, IL-26-stimulated monocytes selectively promote the generation of RORgamma t+ Th17 cells, through IL-1-beta secretion by monocytes. More precisely, IL-26-stimulated monocytes switch non-Th17 committed (IL-23R− or CCR6− CD161−) CD4+ memory T cells into Th17 cells. Finally, synovial fluids from RA patients also induce Th17 cell generation and this effect is reduced after IL-26 depletion. These findings show that IL-26 is constitutively produced by RA synoviocytes, induces proinflammatory cytokine secretion by myeloid cells, and favors Th17 cell generation. IL-26 thereby appears as a novel proinflammatory cytokine, located upstream of the proinflammatory cascade, that may constitute a promising target to treat RA and chronic inflammatory disorders.
doi:10.1371/journal.pbio.1001395
PMCID: PMC3463509  PMID: 23055831
22.  Role of Interferon Regulatory Factor 7 in Serum Transfer Arthritis: Regulation of Interferon β production 
Arthritis and Rheumatism  2011;64(4):1046-1056.
Objective
Innate immune responses activate synoviocytes and recruit inflammatory cells into the rheumatoid joint. Type I interferons (IFN) play a role in autoimmunity and IFN gene transcription is activated by IFN-regulatory factors (IRF) in response to innate sensor recognition. This study examined the effect of genetic deficiency of IRF7 in a passive K/BxN serum transfer model of arthritis.
Methods
Passive transfer arthritis was induced in Irf7−/− mice and additional groups were treated with IFNβ or poly (I-C). Clinical arthritis scores, histology, microcomputed tomography, and synovial tissue Q-PCR were performed. Mouse serum was analyzed by ELISA.
Results
In the K/BxN passive model, arthritis severity was significantly increased in Irf7−/− mice compared with wild type (WT). In addition, synovial and serum IFNβ expression was decreased, potentially contributing to increased arthritis. Irf7−/− mice injected with replacement IFNβ had a decrease in arthritis. Poly (I-C) treatment diminished arthritis in Irf7−/− mice, restored synovial IFNβ gene expression, and increased serum IFNβ. In vitro studies demonstrated that poly (I-C) stimulation of Irf7−/− fibroblast-like synoviocytes (FLS) resulted in increased induction of pro-inflammatory gene expression compared with WT FLS; however, IFNβ expression was not significantly different. In contrast, Irf7−/− macrophages showed significantly less induction of IFNβ in response to poly (I-C) stimulation.
Conclusions
IRF7 deficiency exacerbated arthritis and replacement treatment with IFNβ or poly (I-C) decreased arthritis severity. Both macrophage and synoviocyte specific roles for IRF7 likely contribute to increased arthritis. IRF7 might play an anti-inflammatory role in passive transfer arthritis through regulation of macrophage IFNβ production.
doi:10.1002/art.33454
PMCID: PMC3290705  PMID: 22076939
transcription factors; signal transduction; inflammation; rheumatoid arthritis
23.  Increased AP-1 and NF-κB activation and recruitment with the combination of the proinflammatory cytokines IL-1β, tumor necrosis factor alpha and IL-17 in rheumatoid synoviocytes 
Arthritis Research & Therapy  2004;6(3):R190-R198.
To determine the contribution of IL-1β, tumor necrosis factor alpha (TNF-α) and IL-17 to AP-1, NF-κB and Egr-1 activation in rheumatoid arthritis, the effect of the cytokines used alone or in combination was measured on TF expression in rheumatoid synoviocytes. Effects on mRNA expression were measured by RT-PCR and effects on nuclear translocation were measured by immunocytochemistry. To assess the functional consequences of cytokine induction, osteoprotegerin levels were measured in synoviocyte supernatants.
IL-1β and TNF-α alone at optimal concentration (100 pg/ml) induced the nuclear translocation of NF-κB and almost all AP-1 members, except JunB and Egr-1 for IL-1β and except Fra-2 and Egr-1 for TNF-α. IL-17 was clearly less potent since no nuclear translocation was observed, except for a weak activation of Fra-1 and NF-κB. More importantly, when these cytokines were used at low concentrations, their combination showed a synergistic effect on almost all the TFs, except for Egr-1, with a particular effect on Fra-1 and NF-κB. Increased recruitment of additional factors was induced when the three cytokines were combined. IL-1 and TNF-α induced mRNA expression of c-jun while IL-17 had no effect. A synergistic effect was seen with their combination. A similar synergistic effect was observed for osteoprotegerin production when these three cytokines were combined at low concentrations.
AP-1 and NF-κB pathways were highly sensitive to the combination through synergistic mechanisms. These effects observed in rheumatoid arthritis synoviocytes may reflect the conditions found in the rheumatoid arthritis joint and may contribute to the mode of action of cytokine inhibitors.
doi:10.1186/ar1159
PMCID: PMC416439  PMID: 15142264
proinflammatory cytokines; rheumatoid arthritis; synoviocytes; transcription factors
24.  Histone deacetylase inhibitors suppress rheumatoid arthritis fibroblast-like synoviocyte and macrophage IL-6 production by accelerating mRNA decay 
Annals of the Rheumatic Diseases  2011;71(3):424-431.
Background
Histone deacetylase inhibitors (HDACi) display potent therapeutic efficacy in animal models of arthritis and suppress inflammatory cytokine production in rheumatoid arthritis (RA) synovial macrophages and tissue.
Objectives
To determine the molecular mechanisms contributing to the suppressive effects of HDACi on RA synovial cell activation, using interleukin 6 (IL-6) regulation as a model.
Methods
RA fibroblast-like synoviocytes (FLS) and healthy donor macrophages were treated with IL-1β, tumour necrosis factor (TNF)α, lipopolysaccharide or polyinosinic:polycytidylic acid (poly(I:C)) in the absence or presence of the HDACi trichostatin A (TSA) or ITF2357 (givinostat). IL-6 production and mRNA expression was measured by ELISA and quantitative PCR (qPCR), respectively. Protein acetylation and the activation of intracellular signalling pathways were assessed by immunoblotting. The DNA-binding activity of nuclear factor κB (NFκB) and activator protein 1 (AP-1) components was measured by ELISA-based assays.
Results
HDACi (0.25–1.0 μM) suppressed RA FLS IL-6 production induced by IL-1β, TNFα and Toll-like receptor ligands. Phosphorylation of mitogen-activated protein kinases and inhibitor of κBα (IκBα) following IL-1β stimulation were unaffected by HDACi, as were AP-1 composition and binding activity, and c-Jun induction. TSA induced a significant reduction in nuclear retention of NFκB in FLS 24 h after IL-1β stimulation, but this did not reduce NFκB transcriptional activity or correlate temporally with reductions in IL-6 mRNA accumulation. HDACi significantly reduced the stability of IL-6 mRNA in FLS and macrophages.
Conclusions
Our study identifies a novel, shared molecular mechanism by which HDACi can disrupt inflammatory cytokine production in RA synovial cells, namely the promotion of mRNA decay, and suggests that targeting HDAC activity may be clinically useful in suppressing inflammation in RA.
doi:10.1136/ard.2011.154211
PMCID: PMC3277722  PMID: 21953341
25.  A cell-cycle independent role for p21 in regulating synovial fibroblast migration in rheumatoid arthritis 
Rheumatoid arthritis (RA) is characterized by synovial hyperplasia and destruction of cartilage and bone. The fibroblast-like synoviocyte (FLS) population is central to the development of pannus by migrating into cartilage and bone. We demonstrated previously that expression of the cell cycle inhibitor p21 is significantly reduced in RA synovial lining, particularly in the FLS. The aim of this study was to determine whether reduced expression of p21 in FLS could alter the migratory behavior of these cells. FLS were isolated from mice deficient in p21 (p21(-/-)) and were examined with respect to growth and migration. p21(-/-) and wild-type (WT) FLS were compared with respect to migration towards chemoattractants found in RA synovial fluid in the presence and absence of cell cycle inhibitors. Restoration of p21 expression was accomplished using adenoviral infection. As anticipated from the loss of a cell cycle inhibitor, p21(-/-) FLS grow more rapidly than WT FLS. In examining migration towards biologically relevant RA synovial fluid, p21(-/-) FLS display a marked increase (3.1-fold; p < 0.05) in migration compared to WT cells. Moreover, this effect is independent of the cell cycle since chemical inhibitors that block the cell cycle have no effect on migration. In contrast, p21 is required to repress migration as restoration of p21 expression in p21(-/-) FLS reverses this effect. Taken together, these data suggest that p21 plays a novel role in normal FLS, namely to repress migration. Loss of p21 expression that occurs in RA FLS may contribute to excessive invasion and subsequent joint destruction.
doi:10.1186/ar1999
PMCID: PMC1779389  PMID: 16846525

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