ag-490 and Arthritis--Rheumatoid

Although conventional combination therapy is effective for most patients with rheumatoid arthritis (RA), many still do not respond to current therapies. Therefore, novel combination regimens that better target cellular processes involved in RA pathogenesis are required. Preliminary studies have demonstrated the beneficial effects of a combination of cyclophosphamide (CTX) and methotrexate (MTX) in models of RA. Using western blotting, real-time polymerase chain reaction, enzyme-linked immunosorbent assays, and immunofluorescent staining, we demonstrated that the combination of 4-hydroperoxy CTX (4-H-CTX) and MTX inhibited the expression of receptor activator of nuclear factor-κB ligand (RANKL) in fibroblast-like synoviocytes (FLS) treated with the interleukin (IL)-6/soluble IL-6 receptor (sIL-6R) complex. To elucidate the mechanisms underlying this effect, we treated RA-FLS with the JAK2/STAT3 inhibitor AG490 or p38MAPK inhibitor SB203580. The results showed that IL-6/sIL-6R-induced RANKL upregulation required phosphorylation-mediated activation of STAT3 and p38 signaling, and that 4-H-CTX and/or MTX inhibited RANKL expression in IL-6/sIL-6R-stimulated FLS by suppressing JAK2/STAT3 and p38MAPK signaling. This study demonstrated for the first time the inhibitory effects of 4-H-CTX and MTX on RANKL expression in IL-6/sIL-6R-stimulated FLS via suppression of STAT3 and p38MAPK phosphorylation. These results identify promising therapeutic agents that might have clinical applications in patients with RA who are at high risk of bone erosion or do not respond well to conventional therapy.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Cyclophosphamide; Drug Therapy, Combination; Fibroblasts; Gene Expression Regulation; Humans; Imidazoles; Interleukin-6; Janus Kinase 2; Methotrexate; p38 Mitogen-Activated Protein Kinases; Pyridines; RANK Ligand; Receptors, Interleukin-6; Signal Transduction; STAT3 Transcription Factor; Synoviocytes; Tyrphostins

To clarify the mechanism by which interleukin?22 (IL?22) promotes the proliferation of fibroblast?like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).. FLS were isolated from the synovial tissues of patients with RA and identified by immunohistochemistry for vimentin/CD68. The cells were subcultured and incubated with different concentrations of IL?22 for 24, 48, or 72 h, and their proliferation was examined using MTT assay. After treatment of the cells with IL?22 and AG490, alone or in combination, the expressions of the total and phosphorylated proteins of STAT3, ERK1/2 and P38 were detected with Western blotting.. IL?22 significantly increased the proliferation of FLS in a dose?dependent manner (P<0.05). The total protein of STAT3 in the cells showed no significant changes with extended time of IL?22 treatment (P=0.68), but the expression of phosphorylated STAT3 protein increased significantly (P<0.001). The total and phosphorylated proteins of ERK1/2 and P38 underwent no significant changes after IL?22 treatment (P>0.05). A combined treatment with 50 ng/mL IL?22 and 100 µmol/L AG490 resulted in a significant decrease in the proliferation of FLS as compared with IL?22 treatment alone (P<0.01).. IL?22 can dose?dependently promote the proliferation of FLS from patients with RA by inducing phosphorylation of STAT3 protein but not through ERK1/2 or P38 signal pathway.

Topics: Arthritis, Rheumatoid; Cell Proliferation; Cells, Cultured; Fibroblasts; Humans; Interleukin-22; Interleukins; MAP Kinase Signaling System; Phosphorylation; STAT3 Transcription Factor; Synovial Membrane; Synoviocytes; Tyrphostins

IL-6-mediated STAT3 signaling is essential for Th17 differentiation and plays a central role in the pathogenesis of rheumatoid arthritis. To investigate the molecular mechanism underlying the antirheumatic effects and T cell regulatory effects of STAT3 inhibition, we studied the effects of the JAK 2 inhibitor AG490 on Th17 cell/regulatory T cell (Treg) balance and osteoclastogenesis. AG490 was administered to mice with collagen-induced arthritis (CIA) via i.p. injection, and its in vivo effects were determined. Differential expression of proinflammatory cytokines, including IL-17A, IL-1β, and IL-6, was analyzed by immunohistochemistry. Levels of phosphorylated STAT3 and STAT5 and differentiation of Th17 cells and Tregs after AG490 treatment in our CIA model were analyzed by immunostaining. In vitro development of Th17 cells and Tregs was analyzed by flow cytometry and real-time PCR. AG490 ameliorated the arthritic phenotype in CIA and increased the proportion of Foxp3(+) Tregs. In contrast, the proportion of IL-17A-producing T cells and levels of inflammatory markers were reduced in AG490-treated mice. Numbers of p-STAT3(+) CD4(+) T cells and p-STAT5(+) CD4(+) T cells were reduced and elevated, respectively, after treatment with AG490. Furthermore, AG490 markedly increased the expression of molecules associated with Treg development (ICOS, programmed cell death protein 1, ICAM-1, and CD103). The development and function of osteoclasts were suppressed by AG490 treatment. Our results suggest that AG490, specifically regulating the JAK2/STAT3 pathway, may be a promising treatment for rheumatoid arthritis.

Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Cell Differentiation; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Immunoblotting; Immunohistochemistry; Janus Kinase 2; Mice; Microscopy, Confocal; Osteoclasts; Real-Time Polymerase Chain Reaction; Signal Transduction; STAT3 Transcription Factor; T-Lymphocytes, Regulatory; Th17 Cells; Tyrphostins

Interleukin (IL)-23 stimulates T lymphocytes to produce inflammatory molecules, which can cause inflammatory arthritis. This study was undertaken to explore the role of IL-23 in stimulating the expression of the receptor activator of the nuclear factor kappa B (NF-kappaB) ligand (RANKL) and osteoclastogenic activity in human fibroblast-like synoviocytes (FLS). These cells were separated from the synovium of patients with rheumatoid arthritis (RA-FLS) and osteoarthritis (OA-FLS) and stimulated with IL-23. RANKL expression was measured by real-time polymerase chain reaction (PCR) amplification and immunostaining. Osteoclast precursor cells were cocultured with IL-23-stimulated RA-FLS and OA-FLS and subsequently stained for tartrate-resistant acid phosphatase (TRAP) activity. IL-23 upregulated RANKL expression in RA-FLS. The expression of RANKL mRNA and protein was blocked completely by inhibitors of NF-kappaB (parthenolide) or of the JAK II-STAT3 pathway (AG490), showing that the RANKL expression pathway is mediated by NF-kappaB and STAT3. TRAP-positive osteoclastogenesis was enhanced in IL-23-stimulated FLS. RA-FLS were more responsive to IL-23 in terms of their RANKL expression than OA-FLS or normal FLS. Thus, IL-23 appears to induce joint inflammation and bone destruction by stimulating RANKL expression in RA-FLS. These interactions between IL-23 and FLS indicate possible new therapeutic approaches for treating bone destruction in patients with inflammatory diseases.

Topics: Adult; Aged; Arthritis, Rheumatoid; Cell Differentiation; Female; Fibroblasts; Gene Expression Regulation; Humans; Interleukin-23; Male; Middle Aged; NF-kappa B; Osteoarthritis; Osteoclasts; RANK Ligand; Sesquiterpenes; Signal Transduction; STAT3 Transcription Factor; Synovial Membrane; Tyrphostins