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.

     

Cytokine Combinations Increase p75 Tumor Necrosis Factor Receptor Binding and Stimulate Receptor Shedding in Rheumatoid Synovial Fibroblasts

Objective. To identify cytokines responsible for the increased levels of tumor necrosis factor receptor (TNFR) in the joints of patients with rheumatoid arthritis. Methods. Antibodies to TNFR types were used both to inhibit ligand cell binding and to quantify released receptors in rheumatoid synovial fibroblasts. Results. Binding by and shedding of the p75 TNFR was affected by interleukin-1 (IL-1), IL-4, and interferon-γ. Conclusion. IL-1 could cause increased TNFα binding and TNFR shedding in inflamed joints.     

Suppression of Hyaluronan Synthesis Alleviates Inflammatory Responses in Murine Arthritis and in Human Rheumatoid Synovial Fibroblasts

Objective

To clarify the roles of hyaluronan (HA) in joint inflammation and the process of joint destruction, using 4‐methylumbelliferone (4‐MU), an inhibitor of HA synthesis, in a mouse model of collagen‐induced arthritis (CIA) and in a monolayer culture of fibroblast‐like synoviocytes (FLS) derived from patients with rheumatoid arthritis.

Methods

DAB/1J mice were immunized with type II collagen. The effects of 4‐MU were evaluated by the physiologic arthritis score, paw swelling, the histologic arthritis score, and expression of matrix metalloproteinase 3 (MMP‐3) and MMP‐13 in chondrocytes and synovial tissue. In vitro, the effect of 4‐MU on messenger RNA and protein expression of MMP‐1 and MMP‐3 was determined. The effects of 4‐MU on HA deposition and on serum/medium concentrations of HA were analyzed using biotinylated HA binding protein staining and an HA binding assay, respectively.

Results

Treatment with 4‐MU in mice with CIA dramatically decreased the severity of arthritis (based on the arthritis score), paw thickness, and histopathologic changes. MMP‐3 and MMP‐13 expression in chondrocytes and synovial cells was significantly inhibited by 4‐MU in vivo. Treatment with 4‐MU also inhibited MMP‐1 and MMP‐3 expression in tumor necrosis factor α–stimulated FLS, in a dose‐dependent manner. The 4‐MU–induced decreases in the serum HA concentration in mice with CIA and in “medium” and “pericellular” HA concentrations in cultured FLS support the contention that the inhibitory mechanism of 4‐MU is mediated by HA suppression.

Conclusion

Reduced disease activity induced by 4‐MU in mice with CIA revealed HA to be a crucial regulator in the course of arthritis. Therefore, 4‐MU is a potential therapeutic agent in arthritis, and its inhibitory mechanism is possibly mediated by suppression of HA synthesis.