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Autophagy induction and CHOP under-expression promotes survival of fibroblasts from rheumatoid arthritis patients under endoplasmic reticulum stress.

Shin YJ, Han SH, Kim DS, Lee GH, Yoo WH, Kang YM, Choi JY, Lee YC, Park SJ, Jeong SK, Kim HT, Chae SW, Jeong HJ, Kim HR, Chae HJ - Arthritis Res. Ther. (2010)

Bottom Line: Caspase-3 activity was measured as aminomethyl-coumarin (AMC) liberated from AC-DEVD-AMC.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.Autophagy induction and CHOP under-expression increases cell resistance against ER stress-induced cell death in fibroblasts from rheumatoid arthritis patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Rheumatology, Medical School, the Catholic University of Korea, Seoul, Republic of Korea, 150-713. yjs@catholic.ac.kr

ABSTRACT

Introduction: 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.

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Autophagy protects against ER stress in RASF. OASF and RASF were transfected with non-specific or beclin siRNA, followed by thapsigargin treatment for 60 h. Non-specific and beclin siRNA were transfected into OASF and RASF. Sixteen hours later, total protein was extracted. SDS-PAGE and immunoblotting were performed with anti-beclin or actin antibody (a). Autophagosomes (b) and dead cells (c) were counted as previously described. Data represent means ± S.E. (n = 6). *P < 0.05, versus non-specific siRNA-transfected OASF with thapsigargin. #P < 0.05, versus beclin siRNA-transfected OASF with thapsigargin.
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Figure 4: Autophagy protects against ER stress in RASF. OASF and RASF were transfected with non-specific or beclin siRNA, followed by thapsigargin treatment for 60 h. Non-specific and beclin siRNA were transfected into OASF and RASF. Sixteen hours later, total protein was extracted. SDS-PAGE and immunoblotting were performed with anti-beclin or actin antibody (a). Autophagosomes (b) and dead cells (c) were counted as previously described. Data represent means ± S.E. (n = 6). *P < 0.05, versus non-specific siRNA-transfected OASF with thapsigargin. #P < 0.05, versus beclin siRNA-transfected OASF with thapsigargin.

Mentions: Autophagy is induced under ER stress conditions to protect against cell death [26,27]. In this study, we examined the role of ER stress-induced autophagy via knock-down of the autophagy marker, beclin. In OASF and RASF, transfection of beclin siRNA inhibited the expression of beclin, showing efficient transfection (Figure 4a). In RASF, the beclin siRNA decreased autophagy (Figure 4b) and increased cell death (Figure 4c). Beclin siRNA transfection did not affect cell death in OASF (Figure 4c). To understand the pathological meaning of autophagy, we tested its regulatory effect in RASF. In RASF, Ca2+-induced autophagy is regulated by hydroxychloroquine, a routinely used Disease-Modifying Anti-Rheumatic Drug (DMARD) that inhibits autophagy [28]. Hydroxychloroquine also increased susceptibility to cell death in thapsigargin-treated RASF (data not shown).


Autophagy induction and CHOP under-expression promotes survival of fibroblasts from rheumatoid arthritis patients under endoplasmic reticulum stress.

Shin YJ, Han SH, Kim DS, Lee GH, Yoo WH, Kang YM, Choi JY, Lee YC, Park SJ, Jeong SK, Kim HT, Chae SW, Jeong HJ, Kim HR, Chae HJ - Arthritis Res. Ther. (2010)

Autophagy protects against ER stress in RASF. OASF and RASF were transfected with non-specific or beclin siRNA, followed by thapsigargin treatment for 60 h. Non-specific and beclin siRNA were transfected into OASF and RASF. Sixteen hours later, total protein was extracted. SDS-PAGE and immunoblotting were performed with anti-beclin or actin antibody (a). Autophagosomes (b) and dead cells (c) were counted as previously described. Data represent means ± S.E. (n = 6). *P < 0.05, versus non-specific siRNA-transfected OASF with thapsigargin. #P < 0.05, versus beclin siRNA-transfected OASF with thapsigargin.
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Figure 4: Autophagy protects against ER stress in RASF. OASF and RASF were transfected with non-specific or beclin siRNA, followed by thapsigargin treatment for 60 h. Non-specific and beclin siRNA were transfected into OASF and RASF. Sixteen hours later, total protein was extracted. SDS-PAGE and immunoblotting were performed with anti-beclin or actin antibody (a). Autophagosomes (b) and dead cells (c) were counted as previously described. Data represent means ± S.E. (n = 6). *P < 0.05, versus non-specific siRNA-transfected OASF with thapsigargin. #P < 0.05, versus beclin siRNA-transfected OASF with thapsigargin.
Mentions: Autophagy is induced under ER stress conditions to protect against cell death [26,27]. In this study, we examined the role of ER stress-induced autophagy via knock-down of the autophagy marker, beclin. In OASF and RASF, transfection of beclin siRNA inhibited the expression of beclin, showing efficient transfection (Figure 4a). In RASF, the beclin siRNA decreased autophagy (Figure 4b) and increased cell death (Figure 4c). Beclin siRNA transfection did not affect cell death in OASF (Figure 4c). To understand the pathological meaning of autophagy, we tested its regulatory effect in RASF. In RASF, Ca2+-induced autophagy is regulated by hydroxychloroquine, a routinely used Disease-Modifying Anti-Rheumatic Drug (DMARD) that inhibits autophagy [28]. Hydroxychloroquine also increased susceptibility to cell death in thapsigargin-treated RASF (data not shown).

Bottom Line: Caspase-3 activity was measured as aminomethyl-coumarin (AMC) liberated from AC-DEVD-AMC.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.Autophagy induction and CHOP under-expression increases cell resistance against ER stress-induced cell death in fibroblasts from rheumatoid arthritis patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Rheumatology, Medical School, the Catholic University of Korea, Seoul, Republic of Korea, 150-713. yjs@catholic.ac.kr

ABSTRACT

Introduction: 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.

Show MeSH
Related in: MedlinePlus