Autophagy induction and CHOP under-expression promotes survival of fibroblasts from rheumatoid arthritis patients under endoplasmic reticulum stress.
BACKGROUND
Synovial fibroblasts from rheumatoid arthritis show resistance to apoptotic stimuli, indicating they may be difficult to treat. To clearly understand these mechanisms of resistance, rheumatoid and osteoarthritis synovial fibroblasts (RASF and OASF) were exposed to endoplasmic reticulum (ER) stress such as thapsigargin, Ca2+-ATPase inhibitor.


METHODS
Fibroblasts were assessed microscopically for cell viability by trypan blue exclusion and for autophagic cells by LC-3II formation. Caspase-3 activity was measured as aminomethyl-coumarin (AMC) liberated from AC-DEVD-AMC. Immunoblotting was performed to compare protein expression in OASF and RASF.


RESULTS
ER stress caused cell death in OASF but not in RASF. Thapsigargin, a Ca2+-ATPase inhibitor, did not change the expression of GRP78, an ER chaperone in OASF and RASF, but induced another ER stress protein, CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) differently, showing high levels in OASF and low levels in RASF. Thapsigargin increased the autophagy response in RASF, with autophagosome formation, beclin expression, and LC3-II conversion. Transfection with beclin siRNA inhibited autophagy and increased the susceptibility to ER stress-induced cell death. On the other hand, CHOP siRNA increased autophagy and improved cell survival, especially in RASF, indicating that CHOP is involved in regulation of autophagy and cell death, but that low expression of CHOP protects RASF from apoptosis.


CONCLUSIONS
Autophagy induction and CHOP under-expression increases cell resistance against ER stress-induced cell death in fibroblasts from rheumatoid arthritis patients.
Shin
Yong-Joo
YJ
Department of Rheumatology, Medical School, the Catholic University of Korea, Seoul, Republic of Korea, 150-713. yjs@catholic.ac.kr
Han
Song-Hee
SH
Kim
Do-Sung
DS
Lee
Geum-Hwa
GH
Yoo
Wan-Hee
WH
Kang
Yong-Mo
YM
Choi
Je-Yong
JY
Lee
Yong Chul
YC
Park
Seoung Ju
SJ
Jeong
Seul-Ki
SK
Kim
Hyung-Tae
HT
Chae
Soo-Wan
SW
Jeong
Hyun-Ja
HJ
Kim
Hyung-Ryong
HR
Chae
Han-Jung
HJ
D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D015153:Blotting, Western; D053148:Caspase 3 / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D015536:Down-Regulation; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors / Q000633:toxicity; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D010003:Osteoarthritis / Q000378:metabolism; D034741:RNA, Small Interfering; D013583:Synovial Membrane / Q000187:drug effects / Q000378:metabolism; D019284:Thapsigargin / Q000633:toxicity; D051742:Transcription Factor CHOP / Q000096:biosynthesis* / Q000187:drug effects; D014162:Transfection
C497249:DDIT3 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors; C000717247:HSPA5 protein, human; D034741:RNA, Small Interfering; D051742:Transcription Factor CHOP; D019284:Thapsigargin; D053148:Caspase 3