OBJECTIVE: To investigate the role of autophagy in the regulation of cell death in rheumatoid arthritis synovial fibroblasts (RASF).
METHODS: RASF and osteoarthritis synovial fibroblasts (OASF) were treated with thapsigargin (TG), an inducer of ER stress, and MG132, a proteasome inhibitor. 3-methyladenine was used as an autophagy inhibitor and bafilomycin A1 as a lysosomal inhibitor. Poly-ubiquitinated proteins, p62 and autophagy induction were evaluated by immunoblotting, immunofluorescence microscopy and immunohistochemistry. OASF were transfected with siRNA targeting autophagy-linked FYVE protein (ALFY). Cell death was evaluated by flow cytometry and a caspase-3 activity assay.
RESULTS: In RASF, the induction of autophagy by TG and MG132 was increased compared to OASF. Whereas autophagy promoted a caspase-3 independent induction of cell death under ER stress, autophagy had a protective role in apoptosis induced by proteasome inhibition. Treatment of RASF with 3-methyladenine blocked cell death induced by TG. ER stress induced a strong accumulation of p62-positive poly-ubiquitinated protein aggregates accompanied by the formation of large vacuoles in RASF but not in OASF. Furthermore, TG-induced p62 protein expression was increased, whereas TG-induced ALFY expression was reduced in RASF compared to OASF. ALFY knockdown promoted the accumulation of p62, the formation of poly-ubiquitinated protein aggregates and cell death.
CONCLUSION: Our data provides the first evidence of a dual role of autophagy in the regulation of death pathways in RASF. A reduced expression of ALFY and the formation of p62-positive poly-ubiquitinated protein aggregates promote cell death in RASF under severe ER stress. © 2013 American College of Rheumatology.