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.

     

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.

     

Down‐regulation of myeloid cell leukemia 1 by epigallocatechin‐3‐gallate sensitizes rheumatoid arthritis synovial fibroblasts to tumor necrosis factor α–induced apoptosis

Objective

Overexpression of the antiapoptotic protein myeloid cell leukemia 1 (Mcl‐1) in rheumatoid arthritis (RA) synovial fibroblasts is a major cause of their resistance to tumor necrosis factor α (TNFα)–induced apoptosis. This study was undertaken to evaluate the efficacy of epigallocatechin‐3‐gallate (EGCG) in down‐regulating Mcl‐1 expression and its mechanism of RA synovial fibroblast sensitization to TNFα‐induced apoptosis.

Methods

EGCG effects on cultured RA synovial fibroblast cell morphology, proliferation, and viability over 72 hours were determined by microscopy and a fluorescent cell enumeration assay. Caspase 3 activity was determined by a colorimetric assay. Western blotting was used to evaluate the apoptosis mediators poly(ADP‐ribose) polymerase (PARP), Mcl‐1, Bcl‐2, Akt, and nuclear translocation of NF‐κB.

Results

In RA synovial fibroblasts, EGCG (5–50 μM) inhibited constitutive and TNFα‐induced Mcl‐1 protein expression in a concentration‐ and time‐dependent manner (P < 0.05). Importantly, EGCG specifically abrogated Mcl‐1 expression in RA synovial fibroblasts and affected Mcl‐1 expression to a lesser extent in osteoarthritis and normal synovial fibroblasts or endothelial cells. Inhibition of Mcl‐1 by EGCG triggered caspase 3 activity in RA synovial fibroblasts, which was mediated via down‐regulation of the TNFα‐induced Akt and NF‐κB pathways. Caspase 3 activation by EGCG also suppressed RA synovial fibroblast growth, and this effect was mimicked by Akt and NF‐κB inhibitors. Interestingly, Mcl‐1 degradation by EGCG sensitized RA synovial fibroblasts to TNFα‐induced PARP cleavage and apoptotic cell death.

Conclusion

Our findings indicate that EGCG itself induces apoptosis and further sensitizes RA synovial fibroblasts to TNFα‐induced apoptosis by specifically blocking Mcl‐1 expression and, hence, may be of promising adjunct therapeutic value in regulating the invasive growth of synovial fibroblasts in RA.

     

Interleukin‐18 enhances monocyte tumor necrosis factor α and interleukin‐1β production induced by direct contact with T lymphocytes: Implications in rheumatoid arthritis

Objective

At sites of inflammation, T cells exert pathologic effects through direct contact with monocyte/macrophages, inducing massive up‐regulation of interleukin‐1 (IL‐1) and tumor necrosis factor α (TNFα). We examined the regulatory effects of IL‐18 on monocyte activation by direct contact with T lymphocytes in rheumatoid arthritis (RA).

Methods

Activated T cells were isolated from RA synovial fluid. Resting T cells and monocytes were isolated from peripheral blood mononuclear cells. RA synovial T cells or phytohemagglutinin (PHA)–stimulated T cells were fixed by paraformaldehyde and then cocultured with monocytes at a ratio of 4:1. Levels of TNFα, IL‐1β, IL‐10, and IL‐18 were measured by enzyme‐linked immunosorbent assay. Expression of adhesion molecules, IL‐18 receptor, and TNF receptors was analyzed by flow cytometry. Expression of NF‐κB p65, phosphorylated IκBα, and phosphatidylinositol 3‐kinase (PI 3‐kinase) p110 was analyzed by Western blotting.

Results

IL‐18 dose‐dependently enhanced the production of IL‐1β and TNFα, but not IL‐10, by monocytes following contact with RA synovial T cells or PHA‐prestimulated T cells. NF‐κB inhibitors N‐acetyl‐L ‐cysteine and Bay 11‐7085 and PI 3‐kinase inhibitor LY294002 inhibited the enhancing effects of IL‐18, but MAPK p38 inhibitor SB203580, ERK inhibitor PD98059, and JNK inhibitor SP600125 did not. Increased levels of NF‐κB in the nucleus, phosphorylated IκB, and PI 3‐kinase were confirmed in monocytes cocultured with PHA‐prestimulated T cells, and the levels were further increased by stimulation with IL‐18. Neutralizing antibody to IL‐18 inhibited monocyte activation induced by direct contact with PHA‐prestimulated T cells. Via cell–cell contact, PHA‐prestimulated T cells increased autocrine production of IL‐18 by monocytes, which was mediated by activation of the NF‐κB and PI 3‐kinase pathways, and up‐regulated the expression of the IL‐18 receptor in monocytes. IL‐18 up‐regulated the expression of the TNF receptors vascular cell adhesion molecule 1 (VCAM‐1) and intercellular adhesion molecule 1 (ICAM‐1) on monocytes. Blocking the binding of the TNF receptors VCAM‐1 or ICAM‐1 on monocytes to their ligands on stimulated T cells suppressed the IL‐18–enhanced production of TNFα and IL‐1β in monocytes induced by contact with PHA‐prestimulated T cells.

Conclusion

IL‐18 augments monocyte activation induced by contact with activated T cells in RA synovitis, which is dependent on activation of the NF‐κB and PI 3‐kinase pathways. IL‐18 up‐regulates the expression of the TNF receptors VCAM‐1 and ICAM‐1 on monocytes, which mediate the enhancing effects of IL‐18 on T cell–monocyte contact.

     

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–regulated expression of cellular FLIP protects rheumatoid arthritis synovial fibroblasts from tumor necrosis factor α–mediated apoptosis

Objective

Little apoptosis has been observed in rheumatoid arthritis (RA) synovial tissues. Tumor necrosis factor α (TNFα) is expressed in the joints of patients with RA, yet RA synovial fibroblasts are relatively resistant to apoptosis induced by TNFα. Recently, we demonstrated that FLIP is highly expressed in the RA joint. These studies were performed to determine if TNFα‐induced NF‐κB controls the expression of FLIP long (FLIP_L) and FLIP short (FLIP_S) in RA synovial fibroblasts and to determine the role of FLIP in the control of TNFα‐induced apoptosis.

Methods

RA synovial fibroblasts were isolated from RA synovial tissues and used between passages 3 and 9. RA synovial or control fibroblasts were sham infected or infected with a control adenovirus vector or one expressing the super‐repressor IκBα (srIκBα). The cells were stimulated with TNFα or a control vehicle, and expression of FLIP_L and FLIP_S was determined by isoform‐specific real‐time polymerase chain reaction and Western blot analysis. Cell viability was determined by XTT cleavage, and apoptosis was determined by annexin V staining, DNA fragmentation, and activation of caspases 8 and 3.

Results

TNFα induced the expression of both isoforms of FLIP messenger RNA (mRNA) in RA synovial fibroblasts; however, FLIP_L was the dominant isoform detected by Western blot analysis. In control fibroblasts, TNFα induced the expression of FLIP_L and FLIP_S mRNA and protein. The TNFα‐induced, but not the basal, expression of FLIP was regulated by NF‐κB. When NF‐κB activation was suppressed by the expression of srIκBα, TNFα‐mediated apoptosis was induced. TNFα‐induced apoptotic cell death was mediated by caspase 8 activation and was prevented by the ectopic expression of FLIP_L or the caspase 8 inhibitor CrmA.

Conclusion

The TNFα‐induced, but not the basal, expression of FLIP is regulated by NF‐κB in RA synovial fibroblasts. The resistance of RA synovial fibroblasts to TNFα‐induced apoptosis is mediated by the NF‐κB–regulated expression of FLIP. These observations support the role of NF‐κB and FLIP as attractive therapeutic targets in RA.

     

Normalization of A2A and A3 adenosine receptor up‐regulation in rheumatoid arthritis patients by treatment with anti–tumor necrosis factor α but not methotrexate

Objective

To investigate A₁, A_2A, A_2B, and A₃ adenosine receptors in lymphocytes and neutrophils from patients with early rheumatoid arthritis (ERA) as well as from RA patients treated with methotrexate (MTX) or anti–tumor necrosis factor α (anti‐TNFα), as compared with those in age‐matched healthy controls, and to examine correlations between the status and functionality of adenosine receptors and TNFα release and NF‐κB activation.

Methods

Adenosine receptors were analyzed by saturation binding assays and Western blot analyses. We investigated the potency of typical A_2A and A₃ agonists in the production of cAMP in control subjects, ERA patients, and RA patients treated with MTX or anti‐TNFα. In a separate cohort of RA patients, TNFα release and NF‐κB activation were evaluated in plasma and nuclear extracts, respectively.

Results

In ERA patients, we found a high density and altered functionality of A_2A and A₃ receptors. The binding and functional parameters of A_2A and A₃ receptors normalized after anti‐TNFα, but not MTX, treatment. TNFα release was increased in ERA patients and in MTX‐treated RA patients, whereas in anti‐TNFα–treated RA patients, release was comparable to that in the controls. NF‐κB activation was elevated in ERA patients and in MTX‐treated RA patients. Anti‐TNFα treatment mediated decreased levels of NF‐κB activation.

Conclusion

A_2A and A₃ receptor up‐regulation in ERA patients and in MTX‐treated RA patients was associated with high levels of TNFα and NF‐κB activation. Treatment with anti‐TNFα normalized A_2A and A₃ receptor expression and functionality. This new evidence of A_2A and A₃ receptor involvement opens the possibility of exploiting their potential role in human diseases characterized by a marked inflammatory component.

     

Anti–citrullinated protein antibodies bind surface‐expressed citrullinated Grp78 on monocyte/macrophages and stimulate tumor necrosis factor α production

Objective

Anti–citrullinated protein antibodies (ACPAs), which are the most specific autoantibody marker in patients with rheumatoid arthritis (RA), correlate with disease activity; however, the role of ACPAs in RA pathogenesis has not been elucidated. We hypothesized that ACPAs may directly stimulate mononuclear cells to produce inflammatory cytokines. Thus, we identified cognate antigens of ACPAs on monocyte/macrophages and examined their immunopathologic roles in the pathogenesis of RA.

Methods

ACPAs were purified from pooled ACPA‐positive RA sera by cyclic citrullinated peptide–conjugated affinity column. After coculture of U937 cells with ACPAs, the tumor necrosis factor α (TNFα) production and NF‐κB DNA binding activity of the cells were measured by enzyme‐linked immunosorbent assay. The cognate antigens of ACPAs on the U937 cell surface were probed by ACPAs, and the reactive bands were examined via proteomic analysis.

Results

ACPAs specifically enhanced TNFα production and increased the DNA‐binding activity of NF‐κB in U937 cells. Proteomic analysis revealed that Grp78 protein (72 kd) was one of the cognate antigens of ACPAs. The truncated form of cell surface–expressed Grp78 (55 kd) on U937 cells contained citrulline capable of binding with ACPAs. After citrullination, glutathione S‐transferase–tagged recombinant Grp78 (97.52 kd) became a 72‐kd fragment and bound with ACPAs. ACPAs also bound to human monocytes and lymphocytes to promote TNFα production.

Conclusion

We clearly demonstrated that ACPAs enhance NF‐κB activity and TNFα production in monocyte/macrophages via binding to surface‐expressed citrullinated Grp78.