Trex‐1 deficiency in rheumatoid arthritis synovial fibroblasts

Objective

To explore whether the increased expression of long interspersed nuclear element 1 (LINE‐1; L1) messenger RNA (mRNA) and protein in rheumatoid arthritis synovial fibroblasts (RASFs) is associated with decreased expression of Trex‐1, an exonuclease involved in the metabolization of L1 DNA:RNA hybrids.

Methods

Chromatin immunoprecipitation was used to detect L1‐related p40 protein (L1‐ORF1p) binding sequences in RASFs. Luciferase activity was measured in the synovial fibroblasts following cotransfection of the episomal plasmid with pJM105 expressing L1‐ORF1p and pGL3‐TS3 carrying the target sequence for L1‐ORF1p. This luciferase reporter assay was used to compare the activity between RASFs and osteoarthritis synovial fibroblasts (OASFs) and to assess correlations of luciferase activity with the expression of Trex‐1 measured by flow cytometry. The expression of Trex‐1 mRNA and protein was also compared using real‐time polymerase chain reaction, immunohistochemistry, and Western blot analyses. The role of Trex‐1 in the L1‐ORF1p–mediated luciferase activity assay was studied using interfering RNAs (iRNA) and a Trex‐1 expression vector.

Results

Increased luciferase activity occurred after cotransfection of synovial fibroblasts with pJM105 and pGL3‐TS3. L1‐ORF1p activity was increased in RASFs as compared with OASFs, and this was correlated inversely with the expression of Trex‐1. Levels of Trex‐1 mRNA and protein were lower in RASFs than in OASFs. After transfection of the L1 expression plasmid, Trex‐1 mRNA levels increased in OASFs, but not in RASFs. The addition of iRNA against Trex‐1, however, resulted in an enhancement of L1‐ORF1p activity in OASFs to the levels measured in RASFs. Overexpression of Trex‐1 inhibited 5‐azacytidine–induced expression of p38δ MAPK, a gene carrying the TS3 sequence.

Conclusion

The deficiency of Trex‐1 in RASFs allows a longer half‐life of gene products encoded by active endogenous L1 retrotransposons. This pathway may play a role in diseases in which the cells exhibit a “spontaneous” aggressive behavior.

     

Tumor Necrosis Factor α–Induced MicroRNA‐18a Activates Rheumatoid Arthritis Synovial Fibroblasts Through a Feedback Loop in NF‐κB Signaling

Objective

To elucidate whether the microRNA (miRNA) cluster miR‐17–92 contributes to the activated phenotype of rheumatoid arthritis synovial fibroblasts (RASFs).

Methods

RASFs were stimulated with tumor necrosis factor α (TNFα), and the expression and regulation of the miR‐17–92 cluster were studied using real‐time quantitative PCR (PCR) and promoter activity assays. RASFs were transfected with single precursor molecules of miRNAs from miR‐17–92 and the expression of matrix‐degrading enzymes and cytokines was measured by quantitative PCR and enzyme‐linked immunosorbent assay. Potential miRNA targets were identified by computational prediction and were validated using reporter gene assays and Western blotting. The activity of NF‐κB signaling was determined by reporter gene assays.

Results

We found that TNFα induces the expression of miR‐17–92 in RASFs in an NF‐κB–dependent manner. Transfection of RASFs with precursor molecules of single members of miR‐17–92 revealed significantly increased expression levels of matrix‐degrading enzymes, proinflammatory cytokines, and chemokines in precursor miR‐18a (pre‐miR‐18a)–transfected RASFs. Using reporter gene assays, we identified the NF‐κB pathway inhibitor TNFα‐induced protein 3 as a new target of miR‐18a. In addition, pre‐miR‐18a–transfected RASFs showed stronger activation of NF‐κB signaling, both constitutively and in response to TNFα stimulation.

Conclusion

Our data suggest that the miR‐17–92–derived miR‐18a contributes to cartilage destruction and chronic inflammation in the joint through a positive feedback loop in NF‐κB signaling, with concomitant up‐regulation of matrix‐degrading enzymes and mediators of inflammation in RASFs.

     

DNA hypomethylation in rheumatoid arthritis synovial fibroblasts

Objective

Rheumatoid arthritis synovial fibroblasts (RASFs) are phenotypically activated and aggressive. We undertook this study to investigate whether the intrinsic activation of RASFs is due to global genomic hypomethylation, an epigenetic modification.

Methods

Global genomic hypomethylation was assessed by immunohistochemistry, flow cytometry, and L1 promoter bisulfite sequencing. The levels of Dnmt1 were determined in synovial tissue and cultured SFs by Western blotting before and after treatment with cytokines and growth factors. Normal SFs were treated for 3 months with a nontoxic dose of the DNA hypomethylation drug 5‐azacytidine (5‐azaC), and changes in gene expression were revealed using complementary DNA arrays. The phenotypic changes were confirmed by flow cytometry.

Results

In situ and in vitro, RASF DNA had fewer 5‐methylcytosine and methylated CG sites upstream of an L1 open‐reading frame than did DNA of osteoarthritis SFs, and proliferating RASFs were deficient in Dnmt1. Using 5‐azaC, we reproduced the activated phenotype of RASFs in normal SFs. One hundred eighty‐six genes were up‐regulated >2‐fold by hypomethylation, with enhanced protein expression. These included growth factors and receptors, extracellular matrix proteins, adhesion molecules, and matrix‐degrading enzymes. The hypomethylating milieu induced irreversible phenotypic changes in normal SFs, which resembled those of the activated phenotype of RASFs.

Conclusion

DNA hypomethylation contributes to the chronicity of RA and could be responsible for the limitation of current therapies.

     

RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via toll‐like receptor 3

Objective

To assess the expression of Toll‐like receptor 3 (TLR‐3) protein in synovial tissues and cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to investigate the consequences of stimulation of cultured synovial fibroblasts with TLR‐3 ligands.

Methods

TLR‐3 expression in synovial tissues was determined by immunohistochemistry and immunofluorescence, and expression in cultured RA synovial fibroblasts (RASFs) was determined by fluorescence‐activated cell sorting and real‐time polymerase chain reaction techniques. TLR‐3 signaling was assessed by incubating RASFs with poly(I‐C), lipopolysaccharide, palmitoyl‐3‐cysteine‐serine‐lysine‐4, or necrotic synovial fluid cells from RA patients in the presence or absence of hydroxychloroquine or Benzonase. Subsequent determination of interferon‐β (IFNβ), CXCL10, CCL5, and interleukin‐6 (IL‐6) protein production in the culture supernatants was performed by enzyme‐linked immunosorbent assays.

Results

TLR‐3 protein expression was found to be higher in RA synovial tissues than in OA synovial tissues. TLR‐3 expression was localized predominantly in the synovial lining, with a majority of the TLR‐3–expressing cells coexpressing fibroblast markers. Stimulation of cultured RASFs with the TLR‐3 ligand poly(I‐C) resulted in the production of high levels of IFNβ, CXCL10, CCL5, and IL‐6 protein. Similarly, coincubation of RASFs with necrotic synovial fluid cells from patients with RA resulted in up‐regulation of these cytokines and chemokines in a TLR‐3–dependent manner.

Conclusion

Our findings demonstrate the expression of TLR‐3 in RA synovial tissue and the activation of RASFs in vitro by the TLR‐3 ligand poly(I‐C) as well as by necrotic RA synovial fluid cells, and indicate that RNA released from necrotic cells might act as an endogenous TLR‐3 ligand for the stimulation of proinflammatory gene expression in RASFs.

     

Protease‐activated receptor 2, rather than protease‐activated receptor 1, contributes to the aggressive properties of synovial fibroblasts in rheumatoid arthritis

Objective

To investigate whether protease‐activated receptor 1 (PAR‐1) and/or PAR‐2 promotes the invasiveness/proliferation of synovial fibroblasts (SFs) and to determine the signaling mechanisms of these pathways.

Methods

SFs were isolated from the synovial tissue of patients with rheumatoid arthritis (RA), patients with osteoarthritis (OA), and PAR‐1– or PAR‐2–knockout (KO) mice. Expression of PAR‐1 and PAR‐2 was detected by immunofluorescence and Western blotting. The invasion and proliferation of SFs were measured by invasion assay and MTT assay, respectively. Matrix metalloproteinase 2 (MMP‐2) and MMP‐9 were detected by zymography, and cytokines were measured by enzyme‐linked immunosorbent assay.

Results

PAR‐1 and PAR‐2 were colocalized with SFs in RA and OA synovium and, to a considerably lesser extent, in normal synovium. Inhibition of PAR‐2 by small interfering RNA (siRNA) inhibited RASF invasion and proliferation, whereas blocking of PAR‐1 by siRNA had the reverse effects. SFs from PAR‐2–KO mice exhibited slower rates of proliferation and invasion. SFs from PAR‐1–KO mice produced less MMP‐2 and, in response to tumor necrosis factor α (TNFα) stimulation, had increased MMP‐9 secretion when compared to SFs from wild‐type and PAR‐2–KO mice. Inhibition of PAR‐1, but not PAR‐2, stimulated the secretion of interleukin‐17 (IL‐17) and TNFα by RASFs. Furthermore, PAR‐1 and PAR‐2 had opposing effects on the activation of ERK, p38, and NF‐κB.

Conclusion

Activation of PAR‐1 stimulates MMP‐2 secretion, inhibits RASF growth and invasion, and decreases production of IL‐17 and TNFα by RASFs, whereas activation of PAR‐2 stimulates RASF growth and invasion and increases production of TNFα. Thus, although PAR‐1 and PAR‐2 are coexpressed by RASFs, PAR‐2 alone appears to be responsible for the aggressive properties of RASFs and is likely to contribute to the pathologic progression of RA.

     

Function of interleukin‐20 as a proinflammatory molecule in rheumatoid and experimental arthritis

Objective

The pathogenesis of rheumatoid arthritis (RA) reflects an ongoing imbalance between proinflammatory and antiinflammatory cytokines. Interleukin‐20 (IL‐20) has proinflammatory properties for keratinocytes. In this study, we sought to determine whether IL‐20 is involved in RA.

Methods

We analyzed IL‐20 levels in synovial fluid from RA patients. IL‐20 and its receptors were detected in RA synovial fibroblasts (RASFs), using immunohistochemical staining. The effect of IL‐20 on endothelial cells, neutrophils, and RASFs was investigated using MTT and migration assays. The expression of IL‐20 and its receptors in healthy rats and in rats with collagen‐induced arthritis (CIA) was also analyzed. Soluble IL‐20 receptor type I (sIL‐20RI) or sIL‐20RII was administered to rats with CIA by intramuscular electroporation, and the severity of arthritis was monitored.

Results

RA patients expressed significantly higher levels of synovial fluid IL‐20 than did the rheumatic disease controls. IL‐20 and its receptors were expressed in the synovial membranes and RASFs. IL‐20 induced RASFs to secrete monocyte chemoattractant protein 1, IL‐6, and IL‐8, and it promoted neutrophil chemotaxis, RASF migration, and endothelial cell proliferation. Both IL‐20 and IL‐20RI were up‐regulated in the rat CIA model. In vivo, electroporated sIL‐20RI plasmid DNA decreased the severity of arthritis in the rats with CIA.

Conclusion

IL‐20 was up‐regulated in the synovial fluid of RA patients and acted as a chemokine that attracted the migration of neutrophils and RASFs in vitro. The rat CIA model demonstrated that IL‐20 was involved in the pathogenesis of arthritis, because sIL‐20RI significantly reduced arthritis in rats with CIA. Thus, IL‐20 may modulate the incidence and severity of arthritis and play important roles at local sites of inflammation.

     

The CCL5/CCR5 axis promotes interleukin‐6 production in human synovial fibroblasts

Objective

CCL5 (RANTES) was originally identified as a product of activated T cells and plays a crucial role in the inflammatory response. This study was undertaken to investigate the intracellular signaling pathways involved in CCL5‐induced interleukin‐6 (IL‐6) production in human synovial fibroblasts.

Methods

CCL5‐mediated IL‐6 expression was assessed by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay. The mechanisms of action of CCL5 in different signaling pathways were studied using Western blotting. Knockdown of CCR5 and protein kinase Cδ (PKCδ) protein was achieved by transfection of small interfering RNA (siRNA). Chromatin immunoprecipitation assays were used to study in vivo binding of c‐Jun to the IL‐6 promoter. Transient transfection was used to examine IL‐6 and activator protein 1 (AP‐1) activity.

Results

Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of CCL5 and CCR5, and expression was higher than that in normal synovial fibroblasts. Stimulation of OASFs with CCL5 induced concentration‐ and time‐dependent increases in IL‐6 production. CCL5‐mediated IL‐6 production was attenuated by CCR5 monoclonal antibody, CCR5 inhibitor (Met‐RANTES), and CCR5 siRNA. Pretreatment with a PKCδ inhibitor (rottlerin), a c‐Src inhibitor (PP2), or an AP‐1 inhibitor (tanshinone IIA) also blocked the potentiating action of CCL5. Treatment of OASFs with CCL5 increased the accumulation of phosphorylated c‐Jun in the nucleus, AP‐1 luciferase activity, and c‐Jun binding to the AP‐1 element on the IL‐6 promoter. CCL5‐mediated AP‐1 luciferase activity and c‐Jun binding to the AP‐1 element were inhibited by Met‐RANTES, rottlerin, and PP2.

Conclusion

The present results suggest that the interaction between CCL5 and CCR5 increases IL‐6 production in human synovial fibroblasts via the PKCδ/c‐Src/c‐Jun and AP‐1 signaling pathways.

     

Inhibition of fibroblast activation protein and dipeptidylpeptidase 4 increases cartilage invasion by rheumatoid arthritis synovial fibroblasts

Objective

Since fibroblasts in the synovium of patients with rheumatoid arthritis (RA) express the serine proteases fibroblast activation protein (FAP) and dipeptidylpeptidase 4 (DPP‐4)/CD26, we undertook the current study to determine the functional role of both enzymes in the invasion of RA synovial fibroblasts (RASFs) into articular cartilage.

Methods

Expression of FAP and DPP‐4/CD26 by RASFs was analyzed using fluorescence‐activated cell sorting and immunocytochemistry. Serine protease activity was measured by cleavage of fluorogenic substrates and inhibited upon treatment with L‐glutamyl L‐boroproline. The induction and expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in RASFs were detected using real‐time polymerase chain reaction. Densitometric measurements of MMPs using immunoblotting confirmed our findings on the messenger RNA level. Stromal cell–derived factor 1 (SDF‐1 [CXCL12]), MMP‐1, and MMP‐3 protein levels were measured using enzyme‐linked immunosorbent assay. The impact of FAP and DPP‐4/CD26 inhibition on the invasiveness of RASFs was analyzed in the SCID mouse coimplantation model of RA using immunohistochemistry.

Results

Inhibition of serine protease activity of FAP and DPP‐4/CD26 in vitro led to increased levels of SDF‐1 in concert with MMP‐1 and MMP‐3, which are downstream effectors of SDF‐1 signaling. Using the SCID mouse coimplantation model, inhibition of enzymatic activity in vivo significantly promoted invasion of xenotransplanted RASFs into cotransplanted human cartilage. Zones of cartilage resorption were infiltrated by FAP‐expressing RASFs and marked by a significantly higher accumulation of MMP‐1 and MMP‐3, when compared with controls.

Conclusion

Our results indicate a central role for the serine protease activity of FAP and DPP‐4/CD26 in protecting articular cartilage against invasion by synovial fibroblasts in RA.

     

Expression,regulation, and signaling of the pattern‐recognition receptor nucleotide‐binding oligomerization domain 2 in rheumatoid arthritis synovial fibroblasts

Objective

Since pattern‐recognition receptors (PRRs), in particular Toll‐like receptors (TLRs), were found to be overexpressed in the synovium of rheumatoid arthritis (RA) patients and to play a role in the production of disease‐relevant molecules, we sought to determine the expression, regulation, and function of the PRR nucleotide‐binding oligomerization domain 2 (NOD‐2) in RA.

Methods

Expression of NOD‐2 in synovial tissues was analyzed by immunohistochemistry. Expression and induction of NOD‐2 in RA synovial fibroblasts (RASFs) were measured by conventional and real‐time polymerase chain reaction (PCR) analyses. Levels of interleukin‐6 (IL‐6) and IL‐8 were measured by enzyme‐linked immunosorbent assay (ELISA) and expression of matrix metalloproteinases (MMPs) by ELISA and/or real‐time PCR. NOD‐2 expression was silenced with small interfering RNA. Western blotting with antibodies against phosphorylated and total p38, JNK, and ERK, as well as inhibitors of p38, JNK, and ERK was performed. Activation of NF‐κB was measured by electrophoretic mobility shift assay.

Results

NOD‐2 was expressed by fibroblasts and macrophages in the synovium of RA patients, predominantly at sites of invasion into articular cartilage. In cultured RASFs, no basal expression of messenger RNA for NOD‐2 was detectable, but was induced by poly(I‐C), lipopolysaccharide, and tumor necrosis factor α. After up‐regulation of NOD‐2 by TLR ligands, its ligand muramyl dipeptide (MDP) increased the expression of IL‐6 and IL‐8 via p38 and NF‐κB. Stimulation with MDP further induced the expression of MMP‐1, MMP‐3, and MMP‐13.

Conclusion

Not only TLRs, but also the PRR NOD‐2 is expressed in the synovium of RA patients, and activation of NOD‐2 acts synergistically with TLRs in the production of proinflammatory and destructive mediators. Therefore, NOD‐2 might contribute to the initiation and perpetuation of chronic, destructive inflammation in RA.

     

Bacterial peptidoglycans but not CpG oligodeoxynucleotides activate synovial fibroblasts by toll‐like receptor signaling

Objective

To test the hypothesis that bacterial products acting as adjuvants, such as CpG oligodeoxynucleotides (ODNs) and peptidoglycans (PGs), are able to activate synoviocytes, and to determine the involvement of Toll‐like receptors (TLRs) in this activation process.

Methods

Cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) were stimulated with CpG ODNs or PGs. The expression of various integrins was determined by fluorescence‐activated cell sorting. TLR and matrix metalloproteinase (MMP) messenger RNA (mRNA) was measured by real‐time polymerase chain reaction. Additionally, levels of interleukin‐6 (IL‐6) and IL‐8 in the culture supernatants were assessed by enzyme‐linked immunosorbent assay. Blocking experiments were performed by adding anti–TLR‐2 and anti–TLR‐4 monoclonal antibodies to cultures stimulated with bacterial PGs.

Results

Incubation of synovial fibroblasts with CpG ODNs resulted in neither up‐regulation of the expression of integrins on the cell surface, up‐regulation of MMP mRNA expression, nor IL‐6 and IL‐8 production. However, incubation of RA synovial fibroblasts as well as OA synovial fibroblasts with staphylococcal PGs led to an up‐regulation of CD54 (ICAM‐1) surface expression and to increased expression of MMP‐1, MMP‐3, and MMP‐13 mRNA. Furthermore, production of the proinflammatory cytokines IL‐6 and IL‐8 was increased by treatment with PGs. We demonstrated that cultured synovial fibroblasts express low levels of TLR‐2 and TLR‐9 mRNA. TLR‐2 was up‐regulated after stimulation with PGs, whereas TLR‐9 mRNA remained at baseline levels after stimulation with CpG ODNs. Anti–TLR‐2 monoclonal antibodies significantly inhibited production of IL‐6 and IL‐8 induced by stimulation with PGs.

Conclusion

We demonstrate that bacterial PGs activate synovial fibroblasts, at least partially via TLR‐2, to express integrins, MMPs, and proinflammatory cytokines. Inhibition of TLR signaling pathways might therefore have a beneficial effect on both joint inflammation and joint destruction.

     

Stimulation of collagenase 3 expression in synovial fibroblasts of patients with rheumatoid arthritis by contact with a three‐dimensional collagen matrix or with normal cartilage when coimplanted in NOD/SCID mice

Objective

To study the expression of collagenase 3 (matrix metalloproteinase 13 [MMP‐13]) and collagenase 1 (MMP‐1) in synovial fibroblasts from patients with rheumatoid arthritis (RA) when cultured within 3‐dimensional collagen gels or coimplanted with normal cartilage in immunodeficient NOD/SCID mice.

Methods

Messenger RNA (mRNA) and protein expression of collagenase 3 and collagenase 1 were characterized in synovial and skin fibroblasts by Northern blot and Western blot analysis. The mRNA expression of both collagenases in cell–cartilage implants in NOD/SCID mice was investigated by in situ hybridization in combination with immunohistochemistry of human fibroblasts.

Results

Synovial fibroblasts coimplanted with normal cartilage in NOD/SCID mice deeply invaded adjacent cartilage tissue. In this in vivo system of cartilage destruction, collagenase 3 mRNA was induced in synovial fibroblasts at sites of cartilage erosion, while the expression of collagenase 1 mRNA could not be detected. Culture of synovial fibroblasts within 3‐dimensional collagen gels was a ssociated with a marked increase in collagenase 3 mRNA expression and proenzyme production. This stimulatory effect was 1 order of magnitude higher in comparison with a 2–4‐fold increase upon treatment with interleukin‐1 β or tumor necrosis factor α. In contrast, mRNA expression and proenzyme production of collagenase 1 were increased strongly, and to a similar extent, either by contact with 3‐dimensional collagen or by proinflammatory cytokines.

Conclusion

The expression of collagenase 3, in contrast to that of collagenase 1, is preferentially stimulated in synovial fibroblasts by 3‐dimensional collagen rather than by proinflammatory cytokines. The induction of collagenase 3 by cell–matrix interactions represents a potential mechanism contributing to the invasive phenotype of synovial fibroblasts at sites of synovial invasion into cartilage in RA.

     

MicroRNA‐140 is expressed in differentiated human articular chondrocytes and modulates interleukin‐1 responses

Objective

MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue‐specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA‐140 (miR‐140).

Methods

To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR‐140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin‐1β (IL‐1β) on miR‐140 expression. Double‐stranded miR‐140 (ds–miR‐140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA.

Results

Microarray analysis showed that miR‐140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR‐140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR‐140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL‐1β suppressed miR‐140 expression. Transfection of chondrocytes with ds–miR‐140 down‐regulated IL‐1β–induced ADAMTS5 expression and rescued the IL‐1β–dependent repression of AGGRECAN gene expression.

Conclusion

This study shows that miR‐140 has a chondrocyte differentiation–related expression pattern. The reduction in miR‐140 expression in OA cartilage and in response to IL‐1β may contribute to the abnormal gene expression pattern characteristic of OA.