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Dual Role of Autophagy in Stress‐Induced Cell Death in Rheumatoid Arthritis Synovial Fibroblasts
Author(s) -
Kato Masaru,
Ospelt Caroline,
Gay Renate E.,
Gay Steffen,
Klein Kerstin
Publication year - 2014
Publication title -
arthritis and rheumatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 4.106
H-Index - 314
eISSN - 2326-5205
pISSN - 2326-5191
DOI - 10.1002/art.38190
Subject(s) - autophagy , unfolded protein response , programmed cell death , microbiology and biotechnology , mg132 , endoplasmic reticulum , vacuole , apoptosis , thapsigargin , biology , proteasome , chemistry , proteasome inhibitor , biochemistry , cytoplasm
Objective To investigate the role of autophagy in the regulation of cell death in rheumatoid arthritis synovial fibroblasts (RASFs). Methods RASFs and osteoarthritis synovial fibroblasts (OASFs) were treated with thapsigargin (TG), an inducer of endoplasmic reticulum (ER) stress, and MG132, a proteasome inhibitor. Then, 3‐methyladenine was used as an autophagy inhibitor and bafilomycin A1 as a lysosome inhibitor. Polyubiquitinated proteins, p62, and autophagy induction were evaluated by immunoblotting, immunofluorescence microscopy, and immunohistochemistry, respectively. OASFs were transfected with small interfering RNA targeting autophagy‐linked FYVE protein (ALFY). Cell death was evaluated by flow cytometry and a caspase 3 activity assay. Results In RASFs, the induction of autophagy by TG and MG132 was increased compared to that in OASFs. 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 RASFs with 3‐methyladenine blocked TG‐induced cell death. ER stress induced a strong accumulation of p62‐positive polyubiquitinated protein aggregates, accompanied by the formation of large vacuoles in RASFs but not OASFs. Furthermore, TG‐induced p62 protein expression was increased, whereas TG‐induced ALFY expression was reduced, in RASFs compared to OASFs. ALFY knockdown promoted the accumulation of p62, the formation of polyubiquitinated protein aggregates, and cell death. Conclusion Our data provide the first evidence of a dual role of autophagy in the regulation of death pathways in RASFs. A reduced expression of ALFY and the formation of p62‐positive polyubiquitinated protein aggregates promote cell death in RASFs under severe ER stress.