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


Kato, Masaru; Ospelt, Caroline; Gay, Renate E; Gay, Steffen; Klein, Kerstin (2014). Dual role of autophagy in stress-induced cell death in rheumatoid arthritis synovial fibroblasts. Arthritis & Rheumatism, 66(1):40-48.

Abstract

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.

Abstract

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Rheumatology Clinic and Institute of Physical Medicine
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:2014
Deposited On:06 Jan 2014 12:04
Last Modified:05 Apr 2016 17:19
Publisher:Wiley-Blackwell
ISSN:0004-3591
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1002/art.38190
PubMed ID:24022587

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