Background and Objectives Aberrant regulation of proliferation and survival of immune and stromal cells contributes to the pathogenesis of rheumatoid arthritis (RA). Forkhead box O (FoxO) transcription factors integrate extracellular signals to modulate expression of genes regulating cell cycle and apoptosis, and alterations in activity and expression of FoxOs have been reported in several inflammatory diseases, including RA. In this study, we examined the relationships between inflammation and FoxO expression in RA, and analysed the mechanisms and biological consequences of cytokine-mediated regulation of FoxO1 expression in RA fibroblast-like synoviocytes (FLS).
Materials and Methods RNA was isolated from synovial biopsies obtained by arthroscopy from 20 RA patients and expression of FoxO1, FoxO3a, FoxO4 and IL-6 was measured by quantitative PCR (qPCR). FoxO1 DNA binding, FoxO1 expression and mRNA stability were measured by ELISA-based assays and qPCR in RA FLS stimulated with IL-1β, TNFα, or LPS in the absence or presence of mitogen-activated protein kinase (MAPK) or protein kinase B (PKB) inhibitors. RA FLS were transduced with adenovirus encoding control GFP or constitutively active FoxO1ADA to examine the effects on cell viability and gene expression.
Results In RA synovial tissue expression of FoxO1 negatively correlated with clinical parameters of disease activity: serum C-reactive protein (R = –0.771, P = 0.0008), erythrocyte sedimentation rate (R = –0.739, P = 0.0003), and DAS28 (R = –0.575, P = 0.01), as well as synovial IL-6 mRNA levels (R = –0.628, P = 0.004). In vitro, RA FLS stimulation with IL-1β or TNFα caused rapid downregulation of FoxO1 mRNA levels, followed by reduction of FoxO1 protein expression and DNA binding. This effect was independent of PKB signalling, and was associated with acceleration of FoxO1 mRNA degradation in the presence of IL-1β. Inhibition of c-Jun N-terminal kinase (JNK), but not other MAPKs, prevented downregulation of FoxO1 expression and binding by IL-1β, and blocked IL-1β-induced reduction of FoxO1 mRNA stability. Overexpression of constitutively active FoxO1 in RA FLS induced apoptosis associated with altered expression of genes regulating cell cycle and apoptosis: BIM and p27Kip1 were induced while expression of Bcl-XL was suppressed in cells expressing active FoxO1.
Conclusions Collectively, our findings suggest that suppressed synovial FoxO1 expression is strongly associated with RA pathology and demonstrate that reduction of FoxO1 expression might contribute to perpetuation of inflammation in RA by promoting FLS survival and proliferation. Our data also identify JNK-mediated modulation of FoxO1 mRNA stability as an important mechanism underlying regulation of FoxO1 by inflammatory cytokines.