Abstract
IntroductionRheumatoid 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.MethodsEndoplasmic 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.ResultsRA 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.ConclusionsTNFα 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.
Highlights
Rheumatoid arthritis (RA) is a chronic inflammatory and destructive disease of the joint
RA synovial fibroblasts are under acute endoplasmic reticulum (ER) stress, and the stress is increased in the presence of TNFa
These included eukaryotic initiation factor 2a (eIF2a) that is phosphorylated by active protein kinase-like endoplasmic reticulum kinase (PERK), Xbp1 mRNA that has an intron removed by active inositol-requiring transmembrane kinase and endonuclease 1a (IRE1a), and activation of transcription factor 6 (ATF6) protein that is proteolytically processed to its active form in the golgi in response to ER stress
Summary
Rheumatoid arthritis (RA) is a chronic inflammatory and destructive disease of the joint. ER stress occurs when levels of misfolded proteins exceed the capacity of the protein folding and endoplasmic reticulum-associated degradation systems, or when there is a change in the calcium regulation or oxidative stress in the ER In this case, the unfolded protein response (UPR) is triggered. The UPR involves phosphorylation of the translation initiation factor eukaryotic initiation factor 2a (eIF2a), resulting in inhibition of most new protein synthesis, activation of the transcription factor XBP-1 and increased expression of ER chaperone proteins such as Bip/GRP78. These changes enable the cell to repair misfolded proteins and upregulate the proteasomal degradation system to eliminate aberrant proteins [4]
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