TNFα modulates protein degradation pathways in rheumatoid arthritis synovial fibroblasts

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic inflammatory and destructive disease of the joint. The synovial lining consists of two main types of cells: synovial fibroblasts and macrophages. The macrophage-derived cytokine TNFα stimulates RA synovial fibroblasts to proliferate and produce growth factors, chemokines, proteinases and adhesion molecules, making them key players in the RA disease process. If proteins are not correctly folded, cellular stress occurs that can be relieved in part by increased degradation of the aberrant proteins by the proteasome or autophagy. We hypothesized that the activity of the protein degradation pathways would be increased in response to TNFα stimulation in RA synovial fibroblasts compared with control fibroblasts. METHODS: Endoplasmic reticulum (ER) stress markers were examined in synovial fibroblasts by immunoblotting and PCR. Use of the autophagy and proteasome protein degradation pathways in response to TNFα stimulation was determined using a combination of experiments involving chemical inhibition of the autophagy or proteasome pathways followed by immunoblotting for the autophagy marker LC3, measurement of proteasome activity and long-lived protein degradation, and determination of cellular viability. RESULTS: RA synovial fibroblasts are under acute ER stress, and the stress is increased in the presence of TNFα. Autophagy is the main pathway used to relieve the ER stress in unstimulated fibroblasts, and both autophagy and the proteasome are more active in RA synovial fibroblasts compared with control fibroblasts. In response to TNFα, the autophagy pathway but not the proteasome is consistently stimulated, yet there is an increased dependence on the proteasome for cell viability. If autophagy is blocked in the presence of TNFα, an increase in proteasome activity occurs in RA synovial fibroblasts but not in control cells. CONCLUSIONS: TNFα stimulation of synovial fibroblasts results in increased expression of ER stress markers. Survival of synovial fibroblasts is dependent on continuous removal of proteins by both the lysosome/autophagy and ubiquitin/proteasome protein degradation pathways. Both pathways are more active in RA synovial fibroblasts compared with control fibroblasts. These results may provide a better understanding of the mechanism of TNFα on prolonging the survival of synovial fibroblasts in RA tissue.