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.

     

Inhibition of inflammatory bone erosion by constitutively active STAT‐6 through blockade of JNK and NF‐κB activation

Objective

NF‐κB and JNK signaling pathways play key roles in the pathogenesis of inflammatory arthritis. Both factors are also activated in response to osteoclastogenic factors, such as RANKL and tumor necrosis factor α. Inflammatory arthritis and bone erosion subside in the presence of antiinflammatory cytokines such as interleukin‐4 (IL‐4). We have previously shown that IL‐4 inhibits osteoclastogenesis in vitro through inhibition of NF‐κB and JNK activation in a STAT‐6–dependent manner. This study was undertaken to investigate the potential of constitutively active STAT‐6 to arrest the activation of NF‐κB and JNK and to subsequently ameliorate the bone erosion associated with inflammatory arthritis in mice.

Methods

Inflammatory arthritis was induced in wild‐type and STAT‐6–null mice by intraperitoneal injection of arthritis‐eliciting serum derived from K/B×N mice. Bone erosion was assessed in the joints by histologic and immunostaining techniques. Cell‐permeable Tat‐STAT‐6 fusion proteins were administered intraperitoneally. Cells were isolated from bone marrow and from joints for the JNK assay, the DNA‐binding assays (electrophoretic mobility shift assays), and for in vitro osteoclastogenesis.

Results

Activation of NF‐κB and JNK in vivo was increased in extracts of cells retrieved from the joints of arthritic mice. Cell‐permeable, constitutively active STAT‐6 (i.e., STAT‐6‐VT) was effective in blocking NF‐κB and JNK activation in RANKL‐treated osteoclast progenitors. More importantly, STAT‐6‐VT protein significantly inhibited the in vivo activation of NF‐κB and JNK, attenuated osteoclast recruitment in the inflamed joints, and decreased bone destruction.

Conclusion

Our findings indicate that the administration of STAT‐6‐VT presents a novel approach to the alleviation of bone erosion in inflammatory arthritis.

     

NF‐κB protects Behçet's disease T cells against CD95‐induced apoptosis up‐regulating antiapoptotic proteins

Objective

To determine whether prolongation of the inflammatory reaction in patients with Behçet's disease (BD) is related to apoptosis resistance and is associated with the up‐regulation of antiapoptotic factors.

Methods

The percentage of cell death was evaluated by flow cytometry in peripheral blood mononuclear cells from 35 patients with BD and 30 healthy volunteers. The expression levels of antiapoptotic factors and NF‐κB regulatory proteins were measured using Western blotting and immunohistochemical analyses. To down‐regulate NF‐κB nuclear translocation, BD T lymphocytes were exposed in vitro to thalidomide and subjected to transfection with NF‐κB small interfering RNA.

Results

Although CD95 is highly expressed in BD T cells, the absence of sensitivity to CD95‐induced apoptosis observed may be attributable to the inhibitory action of antiapoptotic genes. Immunoblot analysis for major antiapoptotic proteins showed considerable up‐regulation of the short form of cellular FLIP (cFLIP) and Bcl‐x_L in BD activated T cells, while levels of Bcl‐2, caspase 3, and caspase 8 in activated T cells from patients with BD were comparable with those in activated T cells from normal donors. Moreover, expression of IKK and IκB was up‐regulated, whereas NF‐κB translocated to the nucleus in BD T cells, suggesting that NF‐κB activation may modulate the expression of antiapoptotic genes. Interestingly, thalidomide and NF‐κB small interfering RNA down‐regulated cFLIP and Bcl‐x_L expression levels and sensitized BD activated T cells to CD95‐induced apoptosis.

Conclusion

Taken together, these results indicate that NF‐κB contributes to the regulation of the apoptosis‐related factors and death receptors leading to apoptosis resistance in BD T cell subsets. Our results suggest that NF‐κB plays a crucial role in the pathogenesis of BD, and that its pharmacologic control could represent a key strategy in modulating specific immune‐mediated disease.

     

Altered expression of microRNA‐203 in rheumatoid arthritis synovial fibroblasts and its role in fibroblast activation

Objective

MicroRNA (miRNA) are recognized as important regulators of a variety of fundamental biologic processes. Previously, we described increased expression of miR‐155 and miR‐146a in rheumatoid arthritis (RA) and showed a repressive effect of miR‐155 on matrix metalloproteinase (MMP) expression in RA synovial fibroblasts (RASFs). The present study was undertaken to examine alterations in expression of miR‐203 in RASFs and analyze its role in fibroblast activation.

Methods

Differentially expressed miRNA in RASFs versus osteoarthritis synovial fibroblasts (OASFs) were identified by real‐time polymerase chain reaction (PCR)–based screening of 260 individual miRNA. Transfection of miR‐203 precursor was used to analyze the function of miR‐203 in RASFs. Levels of interleukin‐6 (IL‐6) and MMPs were measured by real‐time PCR and enzyme‐linked immunosorbent assay. RASFs were stimulated with IL‐1β, tumor necrosis factor α (TNFα), lipopolysaccharide (LPS), and 5‐azacytidine (5‐azaC). Activity of IκB kinase 2 was inhibited with SC‐514.

Results

Expression of miR‐203 was higher in RASFs than in OASFs or fibroblasts from healthy donors. Levels of miR‐203 did not change upon stimulation with IL‐1β, TNFα, or LPS; however, DNA demethylation with 5‐azaC increased the expression of miR‐203. Enforced expression of miR‐203 led to significantly increased levels of MMP‐1 and IL‐6. Induction of IL‐6 by miR‐203 overexpression was inhibited by blocking of the NF‐κB pathway. Basal expression levels of IL‐6 correlated with basal expression levels of miR‐203.

Conclusion

The current results demonstrate methylation‐dependent regulation of miR‐203 expression in RASFs. Importantly, they also show that elevated levels of miR‐203 lead to increased secretion of MMP‐1 and IL‐6 via the NF‐κB pathway and thereby contribute to the activated phenotype of synovial fibroblasts in RA.

     

Up‐regulation of stromal cell–derived factor 1 (CXCL12) production in rheumatoid synovial fibroblasts through interactions with T lymphocytes: Role of interleukin‐17 and CD40L–CD40 interaction

Objective

Stromal cell–derived factor 1 (SDF‐1) is a potent chemoattractant for memory T cells in inflamed rheumatoid arthritis (RA) synovium. This study was undertaken to investigate the effect of interleukin‐17 (IL‐17) and CD40–CD40L interaction on SDF‐1 production in RA fibroblast‐like synoviocytes (FLS).

Methods

Synovial fluid (SF) and serum levels of SDF‐1 in RA patients were measured by enzyme‐linked immunosorbent assay (ELISA). The SDF‐1 produced by cultured RA FLS was evaluated by real‐time polymerase chain reaction and ELISA after FLS were treated with IL‐17 and inhibitors of intracellular signal molecules. The SDF‐1 level was also determined after FLS were cocultured with T cells in the presence and absence of IL‐17.

Results

Concentrations of SDF‐1 in the sera and SF were higher in RA patients than in osteoarthritis patients, although the increase in the serum levels did not reach statistical significance. The production of SDF‐1 in RA FLS was enhanced by IL‐17 stimulation. This effect of IL‐17 was blocked by inhibitors of phosphatidylinositol 3‐kinase (PI 3‐kinase), NF‐κB, and activator protein 1 (AP‐1). When FLS were cocultured with T cells, SDF‐1 production was up‐regulated, especially in the presence of IL‐17, but FLS were inhibited by neutralizing anti–IL‐17 and anti‐CD40L antibodies. Addition of RA SF to cultured RA FLS significantly up‐regulated SDF‐1 messenger RNA expression, which was hampered by pretreatment with anti–IL‐17 antibody.

Conclusion

SDF‐1 is overproduced in RA FLS, and IL‐17 could up‐regulate the expression of SDF‐1 in RA FLS via pathways mediated by PI 3‐kinase, NF‐κB, and AP‐1. Our findings suggest that inhibition of the interaction between IL‐17 from T cells and SDF‐1 in FLS may provide a new therapeutic approach in RA.

     

Antiphospholipid antibodies from patients with the antiphospholipid syndrome induce monocyte tissue factor expression through the simultaneous activation of NF‐κB/Rel proteins via the p38 mitogen‐activated protein kinase pathway,and of the MEK‐1/ERK pathway

Objective

Antiphospholipid syndrome (APS) is characterized by thrombosis and the presence of antiphospholipid antibodies (aPL). In patients with primary APS, expression of tissue factor (TF) on the surface of monocytes is increased, which may contribute to thrombosis in these patients. However, the intracellular mechanisms involved in aPL‐mediated up‐regulation of TF on monocytic cells are not understood. This study was undertaken to investigate the intracellular signals induced by aPL that mediate TF activation in monocytes from APS patients.

Methods

We analyzed, both in vivo and in vitro, aPL interactions with proteins that have signaling functions, including mitogen‐activated protein kinases (MAP kinases) and NF‐κB/Rel proteins.

Results

In vivo studies demonstrated significantly higher levels of both TF messenger RNA and TF protein in monocytes from APS patients compared with controls. At the molecular level, increased proteolysis of IκBα and activation of NF‐κB were observed. Constitutive activation of both p38 and ERK‐1 MAP kinases was also found. Treatment of normal monocytes with aPL activated ERK‐1 and p38 MAP kinases, as well as the IκB/NF‐κB pathway, in a dose‐dependent manner. NF‐κB activation and IκBα degradation induced by aPL were inhibited by the NF‐κB inhibitor SN50 and the p38 MAP kinase inhibitor SB203580, thus suggesting crosstalk between these pathways. However, the MEK‐1/ERK inhibitor PD98059 did not affect aPL‐induced NF‐κB binding activity. TF expression induced by aPL was significantly inhibited by combined treatment with the 3 inhibitors.

Conclusion

Our results suggest that aPL induces TF expression in monocytes from APS patients by activating, simultaneously and independently, the phosphorylation of MEK‐1/ERK proteins, and the p38 MAP kinase–dependent nuclear translocation and activation of NF‐κB/Rel proteins.

     

RhoA‐mediated,tumor necrosis factor α–induced activation of NF‐κB in rheumatoid synoviocytes: Inhibitory effect of simvastatin

Objective

Increasing evidence indicates that RhoA may play a central role in the inflammatory response. This study was conducted to examine the role of RhoA in mediating the activation of NF‐κB in tumor necrosis factor α (TNFα)–stimulated rheumatoid synoviocytes, and to evaluate the modulatory effects of statins on the TNFα‐induced activation of RhoA and NF‐κB and the secretion of proinflammatory cytokines by rheumatoid synoviocytes.

Methods

Rheumatoid synoviocytes obtained from patients with active rheumatoid arthritis were stimulated with TNFα and incubated with simvastatin (SMV) (1 μM). RhoA activity was assessed by a pull‐down assay. NF‐κB DNA binding activity and nuclear translocation of NF‐κB were measured by a sensitive multiwell colorimetric assay and confocal fluorescence microscopy, respectively.

Results

TNFα stimulation elicited a robust increase in RhoA activity in a dose‐dependent manner, and SMV mitigated this increase. TNFα also hastened NF‐κB nuclear translocation of subunit p65 and increased DNA binding activity, luciferase reporter gene expression, degradation of IκB, and secretion of interleukin‐1β (IL‐1β) and IL‐6. SMV prevented the increase in NF‐κB activation and rise in IL‐1β and IL‐6 levels induced by TNFα, whereas mevalonate and geranylgeranyl pyrophosphate reversed the inhibitory effects of SMV on activation of NF‐κB and RhoA. Furthermore, cotransfection with a dominant‐negative mutant of RhoA demonstrated that the TNFα‐induced signaling pathway involved sequential activation of RhoA, leading to NF‐κB activation and, ultimately, to secretion of cytokines.

Conclusion

This study identifies RhoA as the key regulator of TNFα‐induced NF‐κB activation, which ultimately results in the secretion of proinflammatory cytokines in rheumatoid synoviocytes. The findings provide a new rationale for the antiinflammatory effects of statins in inflammatory arthritis.

     

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.

     

Expression of toll‐like receptor 2 on CD16+ blood monocytes and synovial tissue macrophages in rheumatoid arthritis

Objective

CD16 (IgG Fcγ receptor type IIIA [FcγRIIIA])–expressing CD14+ monocytes express high levels of Toll‐like receptor 2 (TLR‐2) and are able to efficiently produce proinflammatory cytokines such as tumor necrosis factor α (TNFα). To understand the role of CD16 and TLR‐2 in monocyte and macrophage activation in rheumatoid arthritis (RA), we investigated the expression of TLR‐2 on CD16+ blood monocytes and synovial tissue macrophages and the effect of CD16 and TLR‐2 activation on cytokine production.

Methods

The expression of CD14, CD16, TLR‐2, and TLR‐4 on blood monocytes was measured by flow cytometric analysis. CD16 and TLR‐2 expression in RA synovial tissue was detected by 2‐color immunofluorescence labeling. CD16+ mature monocytes were prepared by incubating blood monocytes in plastic plates for 24 hours. These adhered monocytes were stimulated with lipoteichoic acid (LTA), anti‐FcγRIII antibody, and Hsp60 for 5 hours, and culture supernatants were measured for various cytokines by immunoassay. The activation of NF‐κB was detected by electrophoretic mobility shift assay.

Results

The frequency of CD16+ cells in all blood monocytes was significantly increased in patients with RA compared with healthy controls. TLR‐2 was expressed at higher levels on CD16+ monocytes than on CD16− monocytes, while TLR‐4 was expressed similarly on both monocytes. In RA synovial tissue, CD16+/TLR‐2+ cells were distributed mainly in the lining layer. TLR‐2 expression on monocytes was enhanced by macrophage colony‐stimulating factor (M‐CSF) and interleukin‐10 (IL‐10), but was reduced by transforming growth factor β1, while CD16 expression was inducible by these cytokines. Adhered monocytes (∼50% CD16+) produced TNFα, IL‐1β, IL‐6, IL‐8, IL‐12 p40, IL‐1 receptor antagonist, and IL‐10 after LTA stimulation. This cytokine response was inhibited significantly by anti–TLR‐2 antibody and partly by anti–TLR‐4 antibody. Anti‐FcγRIII antibody stimulation markedly enhanced the LTA‐induced TNFα response. Hsp60 could stimulate TNFα production by adhered monocytes, which was inhibited similarly by anti–TLR‐2 antibody and anti–TLR‐4 antibody. NF‐κB activation in adhered monocytes was induced by LTA, but this NF‐κB activity was not augmented by anti‐FcγRIII antibody stimulation.

Conclusion

These results suggest that CD16+ monocytes and synovial tissue macrophages with high TLR‐2 expression may be induced by M‐CSF and IL‐10, and their production of TNFα could be simulated by endogenous TLR ligands such as Hsp60 and FcγRIIIA ligation by small immune complexes in RA joints.

     

Adiponectin stimulates prostaglandin E2 production in rheumatoid arthritis synovial fibroblasts

Objective

Adipokines may influence inflammatory and/or immune responses. This study was undertaken to examine whether adiponectin affects the production of prostaglandin E₂ (PGE₂) by rheumatoid arthritis synovial fibroblasts (RASFs).

Methods

Synovial tissue was obtained from patients with RA who were undergoing joint replacement surgery. Fibroblast‐like cells from the third or fourth passage were used as RASFs. Expression of adiponectin receptor messenger RNA (mRNA) and protein was detected. PGE₂ (converted from arachidonic acid) was measured by enzyme‐linked immunosorbent assay (ELISA). Expression of mRNA and protein for cyclooxygenase 2 (COX‐2) and membrane‐associated PGE synthase 1 (mPGES‐1), key enzymes involved in PGE₂ synthesis, was detected in RASFs. The effects of RNA interference (RNAi) targeting the adiponectin receptor genes and the receptor signal inhibitors were examined. The influence of adiponectin on NF‐κB activation in RASFs was measured with an ELISA kit.

Results

Adiponectin receptors were detected in RASFs. Adiponectin increased both COX‐2 and mPGES‐1 mRNA and protein expression by RASFs in a time‐ and concentration‐dependent manner. PGE₂ production by RASFs was also increased by the addition of adiponectin, and this increase was inhibited by RNAi for the adiponectin receptor gene, or coincubation with the receptor signal inhibitors. Enhancement of NF‐κB activation by adiponectin as well as by interleukin‐1β was observed in RASFs.

Conclusion

Our findings indicate that adiponectin induces COX‐2 and mPGES‐1 expression, resulting in the enhancement of PGE₂ production by RASFs. Thus, adiponectin may play a role in the pathogenesis of synovitis in RA patients.