Synovial fibroblasts from rheumatoid arthritis show resistance to apoptotic stimuli, indicating they may be difficult to treat. To clearly understand these mechanisms of resistance, rheumatoid and osteoarthritis synovial fibroblasts (RASF and OASF) were exposed to endoplasmic reticulum (ER) stress such as thapsigargin, Ca2+-ATPase inhibitor.
Fibroblasts were assessed microscopically for cell viability by trypan blue exclusion and for autophagic cells by LC-3II formation. Caspase-3 activity was measured as aminomethyl-coumarin (AMC) liberated from AC-DEVD-AMC. Immunoblotting was performed to compare protein expression in OASF and RASF.
ER stress caused cell death in OASF but not in RASF. Thapsigargin, a Ca2+-ATPase inhibitor, did not change the expression of GRP78, an ER chaperone in OASF and RASF, but induced another ER stress protein, CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) differently, showing high levels in OASF and low levels in RASF. Thapsigargin increased the autophagy response in RASF, with autophagosome formation, beclin expression, and LC3-II conversion. Transfection with beclin siRNA inhibited autophagy and increased the susceptibility to ER stress-induced cell death. On the other hand, CHOP siRNA increased autophagy and improved cell survival, especially in RASF, indicating that CHOP is involved in regulation of autophagy and cell death, but that low expression of CHOP protects RASF from apoptosis.
Autophagy induction and CHOP under-expression increases cell resistance against ER stress-induced cell death in fibroblasts from rheumatoid arthritis patients.
Several microRNA, which are ~22-nucleotide noncoding RNAs, exhibit tissue-specific or developmental stage–specific expression patterns and are associated with human diseases. The objective of this study was to identify the expression pattern of microRNA-146 (miR-146) in synovial tissue from patients with rheumatoid arthritis (RA).
The expression of miR-146 in synovial tissue from 5 patients with RA, 5 patients with osteoarthritis (OA), and 1 normal subject was analyzed by quantitative reverse transcription–polymerase chain reaction (RT-PCR) and by in situ hybridization and immunohistochemistry of tissue sections. Induction of miR-146 following stimulation with tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) of cultures of human rheumatoid arthritis synovial fibroblasts (RASFs) was examined by quantitative PCR and RT-PCR.
Mature miR-146a and primary miR-146a/b were highly expressed in RA synovial tissue, which also expressed TNFα, but the 2 microRNA were less highly expressed in OA and normal synovial tissue. In situ hybridization showed primary miR-146a expression in cells of the superficial and sublining layers in synovial tissue from RA patients. Cells positive for miR-146a were primarily CD68+ macrophages, but included several CD3+ T cell subsets and CD79a+ B cells. Expression of miR-146a/b was markedly up-regulated in RASFs after stimulation with TNFα and IL-1β.
This study shows that miR-146 is expressed in RA synovial tissue and that its expression is induced by stimulation with TNFα and IL-1β. Further studies are required to elucidate the function of miR-146 in these tissues.
Rheumatoid synovial fibroblasts (RASFs) mediate joint inflammation and destruction in rheumatoid arthritis (RA). Endothelial protein C receptor (EPCR) is a specific receptor for the natural anticoagulant activated protein C (APC). It mediates the cytoprotective properties of APC and is expressed in rheumatoid synovial tissue. A recent report shows that group V secretory phospholipase A2 (sPLA2V) prevents APC from binding to EPCR in endothelium and inhibits EPCR/APC function. The aim of this study was to investigate the expression and function of EPCR on RASFs.
Human synovial fibroblasts (SFs) were isolated from RA or osteoarthritis (OA) synovial tissues and treated with control, EPCR, or sPLA2V small interfering RNA (siRNA); recombinant human APC, tumor necrosis factor-alpha (TNF-α), or sPLA2V. RASF viability and migration/invasion were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and collagen gel migration/invasion assays, respectively, and cartilage degradation by 1,9-dimethylmethylene blue (DMMB) assay in the presence of human OA articular cartilage explants. The expression or activation of cytokines, EPCR, cadherin-11, mitogen-activated protein (MAP) kinases, and nuclear factor-kappa-B (NF-κB) or both were detected by enzyme-linked immunosorbent assay, Western blotting, or immunostaining.
EPCR was expressed by both OASFs and RASFs but was markedly increased in RASFs. When EPCR was suppressed by siRNA or blocking antibody cell viability, cell invasion and cartilage degradation were reduced by more than 30%. Inflammatory mediators interleukin-1-beta (IL-1β), cadherin-11, and NF-κB were significantly reduced by EPCR suppression under control or TNF-α-stimulated conditions. The expression or activation (or both) of MAP kinases ERK, p38, and JNK were also markedly decreased in cells transfected with EPCR siRNA. Further analysis revealed that sPLA2V co-localized with EPCR on RASFs. Suppression of sPLA2V reduced cell viability and cartilage degradation and increased APC binding to RASFs. Conversely, recombinant sPLA2V increased cartilage degradation, blocked APC binding to RASFs, and could not rescue the effects induced by EPCR suppression.
Our results demonstrate that EPCR is overexpressed by RASFs and mediates the aggressive behavior of RASFs. This function of EPCR is contrary to its cytoprotective role in other settings and is likely driven by sPLA2V.
The purpose of this study was to investigate the profile of histone deacetylase (HDAC) expression in the synovial tissue of rheumatoid arthritis (RA) compared with that of normal control and osteoarthritis (OA), and to examine whether there is a link between HDAC activity and synovial inflammation.
HDAC activity and histone acetyltransferase (HAT) activity were determined in nuclear extracts of total synovial tissue surgically obtained from normal, OA and RA joints. The level of cytoplasmic tumor necrosis factor a (TNFα) fraction was measured by ELISA. Total RNA of synovial tissue was used for RT-PCR of HDAC1-8. In synovial fibroblasts from RA (RASFs), the effects of TNFα on nuclear HDAC activity and class I HDACs (1, 2, 3, 8) mRNA expressions were examined by quantitative real-time PCR. The protein expression and distribution of class I HDACs were examined by Western blotting.
Nuclear HDAC activity was significantly higher in RA than in OA and normal controls and correlated with the amount of cytoplasmic TNFα. The mRNA expression of HDAC1 in RA synovial tissue was higher than in OA and normal controls, and showed positive correlation with TNFα mRNA expression. The protein level of nuclear HDAC1 was higher in RA synovial tissue compared with OA synovial tissue. Stimulation with TNFα significantly increased the nuclear HDAC activity and HDAC1 mRNA expression at 24 hours and HDAC1 protein expression at 48 hours in RASFs.
Our results showed nuclear HDAC activity and expression of HDAC1 were significantly higher in RA than in OA synovial tissues, and they were upregulated by TNFα stimulation in RASFs. These data might provide important clues for the development of specific small molecule HDAC inhibitors.
The rheumatoid arthritis (RA) synovium is characterised by the presence of an aggressive population of activated synovial fibroblasts (RASFs) that are prominently involved in the destruction of articular cartilage and bone. Accumulating evidence suggests that RASFs are relatively resistant to Fas-ligand (FasL)-induced apoptosis, but the data concerning tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) have been conflicting. Here, we hypothesise that the susceptibility of RASFs to receptor-mediated apoptosis depends on the proliferation status of these cells and therefore analysed the cell cycle dependency of FasL- and TRAIL-induced programmed cell death of RASFs in vitro.
Synovial fibroblasts were isolated from patients with RA by enzymatic digestion and cultured under standard conditions. Cell cycle analysis was performed using flow cytometry and staining with propidium iodide. RASFs were synchronised or arrested in various phases of the cell cycle with 0.5 mM hydroxyurea or 2.5 μg/ml nocodazol and with foetal calf serum-free insulin-transferrin-sodium selenite supplemented medium. Apoptosis was induced by stimulation with 100 ng/ml FasL or 100 ng/ml TRAIL over 18 hours. The apoptotic response was measured using the Apo-ONE® Homogenous Caspase-3/7 Assay (Promega GmbH, Mannheim, Germany) and the Cell Death Detection (ELISAPlus) (enzyme-linked immunosorbent assay) (Roche Diagnostics GmbH, Mannheim, Germany). Staurosporin-treated cells (1 μg/ml) served as a positive control. Expression of Fas and TRAIL receptors (TRAILR1-4) was determined by fluorescence-activated cell sorting analysis.
Freshly isolated RASFs showed only low proliferation in vitro, and the rate decreased further over time, particularly when RASFs became confluent. RASFs expressed Fas, TRAIL receptor-1, and TRAIL receptor-2, and the expression levels were independent of the cell cycle. However, the proliferation rate significantly influenced the susceptibility to FasL- and TRAIL-induced apoptosis. Specifically, proliferating RASFs were less sensitive to FasL- and TRAIL-induced apoptosis than RASFs with a decreased proliferation rate. Furthermore, RASFs that were synchronised in S phase or G2/M phase were less sensitive to TRAIL-induced apoptosis than synchronised RASFs in G0/G1 phase.
Our data indicate that the susceptibility of RASFs to FasL- and TRAIL-induced apoptosis depends on the cell cycle. These results may explain some conflicting data on the ability of RASFs to undergo FasL- and TRAIL-mediated cell death and suggest that strategies to sensitise RASFs to apoptosis may include the targeting of cell cycle-regulating genes.
The repellent factor family of Slit molecules has been described to have repulsive function in the developing nervous system on growing axons expressing the Robo receptors. However, until today no data are available on whether these repellent factors are involved in the regulation of synovial fibroblast (SF) activity in rheumatoid arthritis (RA).
mRNA expression in primary synovial fibroblasts was quantified by quantitative reverse transcription PCR and protein expression was measured by fluorescence activated cell sorting (FACS) analysis. Different functional assays were performed with rheumatoid arthritis synovial fibroblasts (RASF): proliferation, migration and a novel in-vitro cartilage destruction assay.
First, we found increased expression of Robo3 expression in RASF compared to normal SF. Interestingly, analysis of data from a recently published genome-wide association study suggests a contribution of ROBO3 gene polymorphisms to susceptibility of RA. Functional assays performed with RASF revealed induction of migration and cartilage destruction by Robo3 and increased matrix metalloproteinase (MMP)1 and MMP3 expression. Treatment of RASF in early passages with Slit3 led to inhibition of migration whereas RASF in later passages, having reduced Robo3 expression in cell culture, were not inhibited by Slit3 treatment. Here, reduction of Robo3 expression from passage 3 to 10 might reflect an important step in losing repulsive activity of Slit3.
Taken together, our data showed that deregulation of the Robo3 receptor in synovial fibroblasts in RA correlates with aggressiveness of the fibroblasts. Slit3 reduces the migratory activity of synovial cells from patients with RA, potentially by repulsion of the cells in analogy to the neuronal system. Further studies will be necessary to prove Slit activity in vivo.
MicroRNAs (miRNAs), endogenous small noncoding RNAs regulating the activities of target mRNAs and cellular processes, are present in human plasma in a stable form. In this study, we investigated whether miRNAs are also stably present in synovial fluids and whether plasma and synovial fluid miRNAs could be biomarkers of rheumatoid arthritis (RA) and osteoarthritis (OA).
We measured concentrations of miR-16, miR-132, miR-146a, miR-155 and miR-223 in synovial fluid from patients with RA and OA, and those in plasma from RA, OA and healthy controls (HCs) by quantitative reverse transcription-polymerase chain reaction. Furthermore, miRNAs in the conditioned medium of synovial tissues, monolayer fibroblast-like synoviocytes, and mononuclear cells were examined. Correlations between miRNAs and biomarkers or disease activities of RA were statistically examined.
Synovial fluid miRNAs were present and as stable as plasma miRNAs for storage at -20°C and freeze-thawing from -20°C to 4°C. In RA and OA, synovial fluid concentrations of miR-16, miR-132, miR-146a, and miR-223 were significantly lower than their plasma concentrations, and there were no correlation between plasma and synovial fluid miRNAs. Interestingly, synovial tissues, fibroblast-like synoviocytes, and mononuclear cells secreted miRNAs in distinct patterns. The expression patterns of miRNAs in synovial fluid of OA were similar to miRNAs secreted by synovial tissues. Synovial fluid miRNAs of RA were likely to originate from synovial tissues and infiltrating cells. Plasma miR-132 of HC was significantly higher than that of RA or OA with high diagnosability. Synovial fluid concentrations of miR-16, miR-146a miR-155 and miR-223 of RA were significantly higher than those of OA. Plasma miRNAs or ratio of synovial fluid miRNAs to plasma miRNAs, including miR-16 and miR-146a, significantly correlated with tender joint counts and 28-joint Disease Activity Score.
Plasma miRNAs had distinct patterns from synovial fluid miRNAs, which appeared to originate from synovial tissue. Plasma miR-132 well differentiated HCs from patients with RA or OA, while synovial fluid miRNAs differentiated RA and OA. Furthermore, plasma miRNAs correlated with the disease activities of RA. Thus, synovial fluid and plasma miRNAs have potential as diagnostic biomarkers for RA and OA and as a tool for the analysis of their pathogenesis.
Circadian rhythms play an important role in the body and in single cells. Rhythms of molecular clocks have not been investigated in synovial fibroblasts (SF) of patients with osteoarthritis (OA) and rheumatoid arthritis (RA). The study was initiated to fill this gap and to study effects of interleukin (IL)-1β/tumor necrosis factor (TNF) on rhythmicity in synovial fibroblasts of RA and OA patients.
The presence of BMAL-1, CLOCK, Period 1 and Period 2 proteins in synovial tissue was investigated by immunofluorescence. The presence of mRNA of molecular clocks was studied during 72 h by qPCR. Characteristics of rhythms were studied with time series analysis.
BMAL-1, CLOCK, Period 1 and Period 2 proteins were abundantly present in synovial tissue of OA, RA and controls. Receiving synovial tissue at different operation time points during the day (8:00 am to 4:00 pm) did not reveal a rhythm of BMAL-1 or Period 1 protein. In OASF and RASF, no typical rhythm curve of molecular clock mRNA was observed. Time series analysis identified a first peak between 2 and 18 hours after synchronization but a period was not detectable due to loss of rhythm. TNF inhibited mRNA of CLOCK, Period 1 and Period 2 in OASF, while IL-1β and TNF increased these factors in RASF. This was supported by dose-dependently increased levels in MH7A RA fibroblasts. In RASF, IL-1β and TNF shifted the first peak of BMAL-1 mRNA to later time points (8 h to 14 h).
Rhythmicity is not present in primary OASF and RASF, which is unexpected because fibroblasts usually demonstrate perfect rhythms during several days. This might lead to uncoupling of important cellular pathways.
Glioblastoma (GBM) is the most common and malignant primary adult brain cancer. Allelic deletion on chromosome 14q plays an important role in the pathogenesis of GBM, and this site was thought to harbor multiple tumor suppressor genes associated with GBM, a region that also encodes microRNA-203 (miR-203). In this study, we sought to identify the role of miR-203 as a tumor suppressor in the pathogenesis of GBM. We analyzed the miR-203 expression data of GBM patients in 10 normal and 495 tumor tissue samples derived from The Cancer Genome Atlas data set. Quantitative real-time PCR and in situ hybridization in 10 high-grade GBM and 10 low-grade anaplastic astrocytoma tumor samples showed decreased levels of miR-203 expression in anaplastic astrocytoma and GBM tissues and cell lines. Exogenous expression of miR-203 using a plasmid expressing miR-203 precursor (pmiR-203) suppressed glioma cell proliferation, migration, and invasion. We determined that one relevant target of miR-203 was Robo1, given that miR-203 expression decreased mRNA and protein levels as determined by RT-PCR and Western blot analysis. Moreover, cotransfection experiments using a luciferase-based transcription reporter assay have shown direct regulation of Robo1 by miR-203. We also show that Robo1 mediates miR-203 mediated antimigratory functions as up-regulation of Robo1 abrogates miR-203 mediated antimigratory effects. We also show that miR-203 expression suppressed ERK phosphorylation and MMP-9 expression in glioma cells. Furthermore, we demonstrate that miR-203 inhibits migration of the glioma cells by disrupting the Robo1/ERK/MMP-9 signaling axis. Taken together, these studies demonstrate that up-regulation of Robo1 in response to the decrease in miR-203 in glioma cells is responsible for glioma tumor cell migration and invasion.
glioma; miR-203; Robo1; cell migration; invasion
MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression via degradation or translational repression of their targeted mRNAs. It is known that aberrant microRNA expression can play important roles in cancer, but the role of microRNAs in autoimmune diseases is only beginning to emerge. In this study, the expression of selected microRNAs is examined in rheumatoid arthritis.
Total RNA was isolated from peripheral blood mononuclear cells obtained from patients with rheumatoid arthritis, and healthy and disease control individuals, and the expression of miR-146a, miR-155, miR-132, miR-16, and microRNA let-7a was analyzed using quantitative real-time PCR.
Rheumatoid arthritis peripheral blood mononuclear cells exhibited between 1.8-fold and 2.6-fold increases in miR-146a, miR-155, miR-132, and miR-16 expression, whereas let-7a expression was not significantly different compared with healthy control individuals. In addition, two targets of miR-146a, namely tumor necrosis factor receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK-1), were similarly expressed between rheumatoid arthritis patients and control individuals, despite increased expression of miR-146a in patients with rheumatoid arthritis. Repression of TRAF6 and/or IRAK-1 in THP-1 cells resulted in up to an 86% reduction in tumor necrosis factor-α production, implicating that normal miR-146a function is critical for the regulation of tumor necrosis factor-α production.
Recent studies have shown that synovial tissue and synovial fibroblasts from patients with rheumatoid arthritis exhibit increased expression of certain microRNAs. Our data thus demonstrate that microRNA expression in rheumatoid arthritis peripheral blood mononuclear cells mimics that of synovial tissue/fibroblasts. The increased microRNA expression in rheumatoid arthritis patients is potentially useful as a marker for disease diagnosis, progression, or treatment efficacy, but this will require confirmation using a large and well defined cohort. Our data also suggest a possible mechanism contributing to rheumatoid arthritis pathogenesis, whereby miR-146a expression is increased but unable to properly function, leading to prolonged tumor necrosis factor-α production in patients with rheumatoid arthritis.
It is generally believed that some inflammatory antigens can recognize Toll-like receptors on synovial fibroblasts (SFs) and then activate downstream signals, leading to the formation of RASFs and inducing rheumatoid arthritis (RA). The objective of the current work was to study on the hypothesis that outer PAMP (LPS) binds to the inner DAMP (HMGB1) and becomes a complex that recognizes TLRs/RAGE on SFs, thus initiating a signaling cascade that leads to the secretion of inflammatory cytokines and chemokines, production of tissue-destructive enzymes, and formation of RASFs, finally resulting in RA. Osteoarthritis synovial fibroblasts (OASFs) were co-cultured with HMGB1–LPS complex in vitro for five generations to induce the transformation of human SFs to RA-like SFs (tOASFs). Then, changes of tOASFs in cell cycle and apoptosis–autophagy balance were investigated in vitro, and the pathogenicity of tOASFs was evaluated in a SCID mouse model in vivo. In vitro cell cycle analysis showed more tOASFs passing through the G1/S checkpoint and moving to S or G2 phase. Flow cytometry and confocal microscopy showed that apoptosis was reduced and autophagy was enhanced significantly in tOASFs as compared with those in OASFs. The expression of certain receptors and adhesion molecules in tOASFs was upregulated. In vivo experiments showed that tOASFs attached to, invaded, and degraded the co-implanted cartilage. In addition, histochemistry showed excessive proliferation of tOASFs and the expression of matrix metalloproteinases (MMPs). Based on the above findings, we conclude that HMGB1–LPS complex could promote the formation of RASFs.
rheumatoid arthritis synovial fibroblast; HMGB1; autophagy
Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease of unknown origin, which exhibits a complex heterogeneity in its pathophysiological background, resulting in differential responses to a range of therapies and poor long-term prognosis. RA synovial fibroblasts (RASFs) are key player cells in RA pathogenesis. Identification of DNA methylation biomarkers is a field that provides potential for improving the process of diagnosis and prognosis of various human diseases. We utilized a genome-wide technique, methylated DNA isolation assay (MeDIA), in combination with a high resolution CpG microarray for discovery of novel hypermethylated genes in RASFs. Thirteen genes (APEX1, EBF3, EGR2, EN1, IRX1, IRX6, KIF12, LHX2, MIPOL1, SGTA, SIN3A, TOLLIP, and ZHX2) with three consecutive hypermethylated probes were isolated as candidate genes through two CpG microarrays. Pyrosequencing assay was performed to validate the methylation status of TGF-β signaling components, EBF3 and IRX1 genes in RASFs and osteoarthritis (OA) SFs. Hypermethylation at CpG sites in the EBF3 and IRX1 genes was observed with a high methylation index (MI) in RASFs (52.5% and 41.4%, respectively), while a lower MI was observed in OASFs and healthy SFs (13.2% for EBF3 and 4.3% for IRX1). In addition, RT-PCR analysis showed a remarkable decrease in their mRNA expression in the RA group, compared with the OA or healthy control, and their reduction levels correlated with MI. The current findings suggest that methylation-associated down-regulation of EBF3 and IRX1 genes may play an important role in a pathogenic effect of TGF-β on RASFs. However, further clinical validation with large numbers of patients is needed in order to confirm our findings.
EBF3; hypermethylation; IRX1; rheumatoid arthritis; synovial fibroblast
Identification of parameters for early diagnosis and treatment response would be beneficial for patients with early rheumatoid arthritis (ERA) to prevent ongoing joint damage. miRNAs have features of potential biomarkers, and an altered expression of miRNAs was shown in established rheumatoid arthritis (RA).
To analyse RA associated miRNAs in the sera of patients with ERA to find markers of early disease, clinical activity or predictors of disease outcome.
Total RNA was isolated from whole sera in ERA patients (prior to and after 3 and 12 months of therapy with disease modifying antirheumatic drugs), in patients with established RA and in healthy controls (HC) using phenol–chloroform extraction. Expression of miR-146a, miR-155, miR-223, miR-16, miR-203, miR-132 and miR-124a was analysed by TaqMan Real Time PCR.
From all analysed miRNAs, levels of miR-146a, miR-155 and miR-16 were decreased in the sera of ERA patients in comparison with established RA. A change in circulating miR-16 in the first 3 months of therapy was associated with a decrease in DAS28 in long term follow-up in ERA (p=0.002). Levels of circulating miR-223 in treatment naïve ERA correlated with C reactive protein (p=0.008), DAS28 (p=0.031) and change in DAS28 after 3 months (p=0.003) and 12 months (p=0.011) of follow-up. However, neither miR-16 nor miR-223 could distinguish ERA from HC.
Differential expression of circulating miR-146a, miR-155 and miR-16 in the sera of ERA patients may characterise an early stage of the disease. We suggest miR-223 as a marker of disease activity and miR-16 and miR-223 as possible predictors for disease outcome in ERA.
Rheumatoid Arthritis; DAS28; Early Rheumatoid Arthritis
The ING family of type II tumour suppressors serve as both epigenetic ‘readers' and target histone acetyl transferase (HAT) and histone deacetylase (HDAC) ‘writers' of the epigenetic histone code. The ING1 protein has also been implicated in regulating microRNA (miRNA) levels. In this study, we identify a link between ING1b and the miRNA epigenetic network.
Primary fibroblasts infected with adenoviruses expressing GFP control or GFP plus ING1b were examined for alterations in miRNA profiles using a miRNA PCR array. Additional experiments confirmed specificity and consequences of altered miRNA expression.
MicroRNAs miR-203, miR-375, miR-449b and miR-200c were increased by ING1b overexpression. Ectopic expression of miR-203 inhibited U2OS and MDA-MB-231 cancer cell growth, and induced G1 cell cycle arrest in U2OS cells as estimated by flow cytometry. Transfection with miR-203 inhibitor reversed the proliferation inhibition induced by ING1b in U2OS cells. CHIP assays showed that ING1b bound to the promoter of miR-203. Western blot analyses showed that CDK6, c-Abl and Src were downregulated by the transfection of miR-203.
These results indicate that ING1b epigenetically regulates several miRNAs including miR-203. The several-fold increase in miR-203 by ING1b might inhibit cancer cell proliferation through coordinate downregulation of CDK6, c-Abl and Src.
ING1; transcriptional regulation; mir-203; CDK6; c-Abl; Src
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial fibroblast hyperplasia and bone and cartilage erosion. Synovial fibroblast- and T cell-mediated inflammation plays crucial roles in the pathogenesis of RA. However how this inflammation is initiated, propagated, and maintained remains controversial. Here, we systemically examined the contribution of toll-like receptors (TLRs) to the inflammatory mediator production as well as Th1 and Th17 cell hyperactivity in RA. Our results show that rheumatoid arthritis synovial fibroblasts (RASF) express a series of TLRs, including TLR2, TLR3, TLR4, and TLR9, with the predominant expression of TLR3. Moreover, the expression levels of these TLRs were higher than those in osteoarthritis synovial fibroblasts (OASF). Ligation of TLR3, as well as TLR2 and TLR4, resulted in vigorous production of inflammatory cytokines, matrix metalloproteinases (MMPs), and vascular endothelial growth factor (VEGF) in RASF, with activation of the NF-κB, MAPK, and IRF3 pathways. More important, activation of these TLRs expressed by RASF exacerbated inflammatory Th1 and Th17 cell expansion both in cell-cell contact-dependent and inflammatory cytokine-dependent manners, which induced more IFN-γ and IL-17 accumulation. Targeting TLRs may modulate the inflammation in RA and provide new therapeutic strategies for overcoming this persistent disease.
In addition to its direct proinflammatory activity, extracellular high mobility group box protein 1 (HMGB1) can strongly enhance the cytokine response evoked by other proinflammatory molecules, such as lipopolysaccharide (LPS), CpG-DNA and IL-1β, through the formation of complexes. Extracellular HMGB1 is abundant in arthritic joint tissue where it is suggested to promote inflammation as intra-articular injections of HMGB1 induce synovitis in mice and HMGB1 neutralizing therapy suppresses development of experimental arthritis. The aim of this study was to determine whether HMGB1 in complex with LPS, interleukin (IL)-1α or IL-1β has enhancing effects on the production of proinflammatory mediators by rheumatoid arthritis synovial fibroblasts (RASF) and osteoarthritis synovial fibroblasts (OASF). Furthermore, we examined the toll-like receptor (TLR) 4 and IL-1RI requirement for the cytokine-enhancing effects of the investigated HMGB1-ligand complexes.
Synovial fibroblasts obtained from rheumatoid arthritis (RA) and osteoarthritis (OA) patients were stimulated with HMGB1 alone or in complex with LPS, IL-1α or IL-1β. Tumour necrosis factor (TNF) production was determined by enzyme-linked immunospot assay (ELISPOT) assessment. Levels of IL-10, IL-1-β, IL-6 and IL-8 were measured using Cytokine Bead Array and matrix metalloproteinase (MMP) 3 production was determined by ELISA.
Stimulation with HMGB1 in complex with LPS, IL-1α or IL-1β enhanced production of TNF, IL-6 and IL-8. HMGB1 in complex with IL-1β increased MMP production from both RASF and OASF. The cytokine production was inhibited by specific receptor blockade using detoxified LPS or IL-1 receptor antagonist, indicating that the synergistic effects were mediated through the partner ligand-reciprocal receptors TLR4 and IL-1RI, respectively.
HMGB1 in complex with LPS, IL-1α or IL-1β boosted proinflammatory cytokine- and MMP production in synovial fibroblasts from RA and OA patients. A mechanism for the pathogenic role of HMGB1 in arthritis could thus be through enhancement of inflammatory and destructive mechanisms induced by other proinflammatory mediators present in the arthritic joint.
This study was performed to investigate the effect of microRNA-203 (miR-203) on cell proliferation and migration in triple-negative breast cancer (TNBC).
Real-time PCR was performed to detect the expression of miR-203 in TNBC cell lines. miR-203 precursor and control microRNA (miRNA) were transfected into triple-negative breast cancer (TNBC) cell lines and the effects of miR-203 up-regulation on the proliferation and migration of cells were investigated. Meanwhile, the mRNA and protein levels of baculoviral IAP repeat-containing protein 5 (BIRC5) and Lim and SH3 domain protein 1 (LASP1) were measured. Luciferase assays were also performed to validate BIRC5 and LASP1 as miR-203 targets.
Both miR-203 and BIRC5 siRNA signicantly inhibited cell proliferation in TNBC cells. Both miR-203 and LASP1 siRNA signicantly inhibited cell migration in TNBC cells, also. Moreover, up-regulated of BIRC5 and LASP1 was able to abrogate the effects induced by transfection with the miR-203 precursor.
These data suggest that miR-203 may function as a tumor suppressor in TNBC cells. Thus, miR-203 could be a potential therapeutic target for this disease.
Triple-negative breast cancer, MiR-203; baculoviral IAP repeat-containing protein 5, Lim and SH3 domain protein 1, Proliferation, Migration
Tyrosine kinase inhibitors (TKIs) are effective in treating malignant disorders and were lately suggested to have an impact on non-malignant diseases. However, in some inflammatory conditions like rheumatoid arthritis (RA) the in vivo effect seemed to be moderate. As most TKIs are taken up actively into cells by cell membrane transporters, this study aimed to evaluate the role of such transporters for the accumulation of the TKI Imatinib mesylates in RA synovial fibroblasts as well as their regulation under inflammatory conditions.
The transport and accumulation of Imatinib was investigated in transporter-transfected HEK293 cells and human RA synovial fibroblasts (hRASF). Transporter expression was quantified by qRT-PCR. In transfection experiments, hMATE1 showed the highest apparent affinity for Imatinib among all known Imatinib transporters. Experiments quantifying the Imatinib uptake in the presence of specific transporter inhibitors and after siRNA knockdown of hMATE1 indeed identified hMATE1 to mediate Imatinib transport in hRASF. The anti-proliferative effect of Imatinib on PDGF stimulated hRASF was quantified by cell counting and directly correlated with the uptake activity of hMATE1. Expression of hMATE1 was investigated by Western blot and immuno-fluorescence. Imatinib transport under disease-relevant conditions, such as an altered pH and following stimulation with different cytokines, was also investigated by HPLC. The uptake was significantly reduced by an acidic extracellular pH as well as by the cytokines TNFα, IL-1β and IL-6, which all decreased the expression of hMATE1-mRNA and protein.
The regulation of Imatinib uptake via hMATE1 in hRASF and resulting effects on their proliferation may explain moderate in vivo effects on RA. Moreover, our results suggest that investigating transporter mediated drug processing under normal and pathological conditions is important for developing intracellular acting drugs used in inflammatory diseases.
Ulceration of the oesophageal squamous mucosa (ulcerative oesophagitis) is a pathological manifestation of gastro-oesophageal reflux disease, and is a major risk factor for the development of Barrett’s oesophagus. Barrett’s oesophagus is characterised by replacement of reflux-damaged oesophageal squamous epithelium with a columnar intestinal-like epithelium. We previously reported discovery of microRNAs that are differentially expressed between oesophageal squamous mucosa and Barrett’s oesophagus mucosa. Now, to better understand early steps in the initiation of Barrett’s oesophagus, we assessed the expression, location and function of these microRNAs in oesophageal squamous mucosa from individuals with ulcerative oesophagitis.
Quantitative real-time PCR was used to compare miR-21, 143, 145, 194, 203, 205 and 215 expression levels in oesophageal mucosa from individuals without pathological gastro-oesophageal reflux to individuals with ulcerative oesophagitis. Correlations between microRNA expression and messenger RNA differentiation markers BMP-4, CK8 and CK14 were analyzed. The cellular localisation of microRNAs within the oesophageal mucosa was determined using in-situ hybridisation. microRNA involvement in proliferation and apoptosis was assessed following transfection of a human squamous oesophageal mucosal cell line (Het-1A).
miR-143, miR-145 and miR-205 levels were significantly higher in gastro-oesophageal reflux compared with controls. Elevated miR-143 expression correlated with BMP-4 and CK8 expression, and elevated miR-205 expression correlated negatively with CK14 expression. Endogenous miR-143, miR-145 and miR-205 expression was localised to the basal layer of the oesophageal epithelium. Transfection of miR-143, 145 and 205 mimics into Het-1A cells resulted in increased apoptosis and decreased proliferation.
Elevated miR-143, miR-145 and miR-205 expression was observed in oesophageal squamous mucosa of individuals with ulcerative oesophagitis. These miRNAs localised to the basal layer of the oesophageal epithelium. They reduced proliferation and increased apoptosis, and may play roles in regulating epithelial restoration in response to injury caused by gastro-oesophageal reflux.
microRNA; Gastro-oesophageal reflux disease; Ulcerative oesophagitis; Apoptosis; Proliferation; Barrett’s oesophagus
MicroRNA alterations are likely to contribute to the development of pancreatic cancer and may serve as markers for the early detection of pancreatic neoplasia.
To identify the microRNA alterations that arise during the development of pancreatic cancer we determined the levels of 735 miRNAs in 34 pancreatic intraepithelial neoplasias (PanINs) and 15 normal pancreatic duct samples isolated by laser capture microdissection using TaqMan miRNA microarrays. Differential expression of selected miRNAs was confirmed by fluorescent in-situ hybridization analysis and by qRT-PCR analysis of selected candidate microRNAs in an independent set of PanIN and normal duct samples.
We identified 107 aberrantly expressed miRNAs in different PanIN grades compared with normal pancreatic duct samples, and 35 aberrantly expressed miRNAs in PanIN-3 lesions compared with normal pancreatic duct samples. These differentially expressed miRNAs included those that have been previously identified as differentially expressed in pancreatic ductal adenocarcinomas (including miR-21, miR-200a/b/c, miR-216a/b, miR-217, miR-146a, miR-155, miR-182, miR-196b, miR-203, miR-222, miR-338-3p, miR-486-3p and others) as well as miRNAs not previously described as differentially expressed in these lesions (miR-125b, miR-296-5p, miR-183*, miR-603, miR-625/*, miR-708 and others). MiR-196b was the most selectively differentially expressed miRNA in Panin-3 lesions.
Many miRNAs undergo aberrant expression in PanIN lesions and are likely to be important in the development of pancreatic ductal adenocarcinoma. MicroRNAs such as miR-196b whose expression is limited to PanIN-3 lesions or pancreatic cancers could be useful as diagnostic markers.
Gene expression is potently regulated through the action of microRNAs (miRNAs). Here, we present evidence of a miRNA regulating Hakai protein. Hakai was discovered as an E3 ubiquitin-ligase that mediates the posttranslational downregulation of E-cadherin, a major component of adherens junctions in epithelial cells and a potent tumour suppressor. Recent data have provided evidence that Hakai affects cell proliferation in an E-cadherin-independent manner, thus revealing a role for Hakai in the early stages of tumour progression. Furthermore, Hakai is highly up-regulated in human colon adenocarcinomas compared to normal tissues. However, the molecular mechanisms that regulate Hakai abundance are unknown. We identified two putative sites of miR-203 interaction on the Hakai mRNA, in its 3′-untranslated region (UTR). In several human carcinoma cell lines tested, overexpression of a miR-203 precursor (Pre-miR-203) reduced Hakai abundance, while inhibiting miR-203 by using an antisense RNA (Anti-miR-203) elevated Hakai levels. The repressive influence of miR-203 on the Hakai 3′-UTR was confirmed using heterologous reporter constructs. In keeping with Hakai's proliferative influence, Anti-miR-203 significantly increased cell number and BrdU incorporation, while Pre-miR-203 reduced these parameters. Importantly, the growth-promoting effects of anti-miR-203 required the presence of Hakai, because downregulation of Hakai by siRNA suppressed its proliferative action. Finally, in situ hybridization showed that miR-203 expression is attenuated in colon tumour tissues compared to normal colon tissues, suggesting that miR-203 could be a potential new prognostic marker and therapeutic target to explore in colon cancer. In conclusion, our findings reveal, for the first time, a post-transcriptional regulator of Hakai expression. Furthermore, by lowering Hakai abundance, miR-203 also reduces Hakai-regulated-cell division.
Objective. This study was to screen for the miRNAs differently expressed in peripheral blood mononuclear cells (PBMC) of RA, to further identify the expression of miR-155 in RA PBMC and fibroblast-like synoviocytes (FLS), and to evaluate the function of miR-155 in RA-FLS. Methods. Microarray was used to screen for differentially expressed miRNAs in RA PBMC. miR-155 expression in PBMC and FLS of RA were identified by real-time PCR. Enforced overexpression and downexpression of miR-155 were used to investigate the function of miR-155 in RA-FLS. Expression of IKBKE which was previously identified as the actual target of miR-155 was examined by Western blot and real-time PCR in RA-FLS. Results. miR-155 levels were increased in both PBMC and FLS of RA and could be induced by TNF-α. Upregulation of miR-155 decreased MMP-3 levels and suppressed proliferation and invasion of RA-FLS. Inverse relationship between the expressions of miR-155 and the MMPs production-related protein IKBKE was found. Conclusion. An inflammatory milieu may alter miRNA expression profiles in rheumatoid arthritis. miR-155 is upregulated in RA-FLS, and it may be a protective factor against the inflammatory effect in part by attenuating expression of IKBKE.
The Epstein-Barr virus (EBV) is highly associated with nasopharyngeal carcinoma (NPC), and it regulates some microRNAs (miRNAs) that are involved in the development of cancer. The role of EBV in the deregulation of cellular miRNAs and how this affects the progression of NPC remain to be investigated. An analysis of the miRNA profile in an EBV-infected cell line revealed that miRNA 203 (miR-203) was downregulated. miR-203 is expressed specifically in epithelial cells. This downregulation of miR-203 was further verified and functionally analyzed. miR-203 was downregulated substantially in epithelial cells and NPC tissues that were latently infected with EBV. Downregulation of miR-203 also occurred during the early stage of EBV infection. Furthermore, the viral oncoprotein, latent membrane protein 1 (LMP1), was responsible for downregulation of miR-203. Removal of the latent EBV genome or suppression of LMP1 resulted in restoration of miR-203 expression. EBV-LMP1 mediated the downregulation of miR-203 at the primary transcript level. E2F3 and CCNG1 were identified as target genes of miR-203. Ectopic expression of miR-203 inhibited EBV-induced S-phase entry and transformation in vivo. Overexpression of the targets overcame the effects of miR-203 mimics on the cell cycle, and the expression of target genes in tumor models was inhibited by miR-203. Inhibitors of Jun N-terminal protein kinase (JNK) and NF-κB blocked miR-203 downregulation. These results imply that EBV promotes malignancy by downregulating cellular miR-203, which contributes to the etiology of NPC.
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.
A surprising feature of the inflammatory infiltrate in rheumatoid arthritis is the accumulation of neutrophils within synovial fluid and at the pannus cartilage boundary. Recent findings suggest that a distinct subset of IL-17-secreting T-helper cells (TH17 cells) plays a key role in connecting the adaptive and innate arms of the immune response and in regulating neutrophil homeostasis. We therefore tested the hypothesis that synovial fibroblasts bridge the biological responses that connect TH17 cells to neutrophils by producing neutrophil survival factors following their activation with IL-17.
IL-17-expressing cells in the rheumatoid synovium, and IL-17-expressing cells in the peripheral blood, and synovial fluid were examined by confocal microscopy and flow cytometry, respectively. Peripheral blood neutrophils were cocultured either with rheumatoid arthritis synovial fibroblasts (RASF) or with conditioned medium from RASF that had been pre-exposed to recombinant human IL-17, TNFα or a combination of the two cytokines. Neutrophils were harvested and stained with the vital mitochondrial dye 3,3'-dihexyloxacarbocyanine iodide before being enumerated by flow cytometry.
TH17-expressing CD4+ cells were found to accumulate within rheumatoid synovial tissue and in rheumatoid arthritis synovial fluid. RASF treated with IL-17 and TNFα (RASFIL-17/TNF) effectively doubled the functional lifespan of neutrophils in coculture. This was entirely due to soluble factors secreted from the fibroblasts. Specific depletion of granulocyte–macrophage colony-stimulating factor from RASFIL-17/TNF-conditioned medium demonstrated that this cytokine accounted for approximately one-half of the neutrophil survival activity. Inhibition of phosphatidylinositol-3-kinase and NF-κB pathways showed a requirement for both signalling pathways in RASFIL-17/TNF-mediated neutrophil rescue.
The increased number of neutrophils with an extended lifespan found in the rheumatoid synovial microenvironment is partly accounted for by IL-17 and TNFα activation of synovial fibroblasts. TH17-expressing T cells within the rheumatoid synovium are likely to contribute significantly to this effect.