TY - JOUR
T1 - Dual role of autophagy in stress-induced cell death in rheumatoid arthritis synovial fibroblasts
AU - Kato, Masaru
AU - Ospelt, Caroline
AU - Gay, Renate E.
AU - Gay, Steffen
AU - Klein, Kerstin
PY - 2014/1
Y1 - 2014/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84897018493&partnerID=8YFLogxK
U2 - 10.1002/art.38190
DO - 10.1002/art.38190
M3 - 学術論文
C2 - 24449574
AN - SCOPUS:84897018493
SN - 2326-5191
VL - 66
SP - 40
EP - 48
JO - Arthritis and Rheumatology
JF - Arthritis and Rheumatology
IS - 1
ER -