Dual role of autophagy in stress-induced cell death in rheumatoid arthritis synovial fibroblasts.
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.


CONCLUSIONS
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.
Center of Experimental Rheumatology, University Hospital Zurich, Zurich, Switzerland.
Masaru
M
Kato
Caroline
C
Ospelt
Renate E
RE
Gay
Steffen
S
Gay
Kerstin
K
Klein
D048868:Adaptor Proteins, Signal Transducing / Q000187:drug effects / Q000378:metabolism; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D001172:Arthritis, Rheumatoid / Q000503:physiopathology*; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D000071183:Autophagy-Related Proteins; D053148:Caspase 3 / Q000187:drug effects / Q000378:metabolism; D016923:Cell Death / Q000502:physiology; D002478:Cells, Cultured; D059865:Endoplasmic Reticulum Stress / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000502:physiology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D008247:Lysosomes / Q000187:drug effects / Q000502:physiology; D018942:Macrolides / Q000494:pharmacology; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010003:Osteoarthritis / Q000503:physiopathology; D025821:Polyubiquitin / Q000187:drug effects / Q000378:metabolism; D000071456:Sequestosome-1 Protein; D013583:Synovial Membrane / Q000166:cytology*; D014157:Transcription Factors / Q000187:drug effects / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D000071183:Autophagy-Related Proteins; D004791:Enzyme Inhibitors; D018942:Macrolides; D008565:Membrane Proteins; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D014157:Transcription Factors; C499825:WDFY3 protein, human; D025821:Polyubiquitin; C025946:3-methyladenine; C040929:bafilomycin A1; C505407:CASP3 protein, human; D053148:Caspase 3; D000225:Adenine