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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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CHOP plays an important role in autophagy in RASF and in cell death in OASF. OASF and RASF were treated with thapsigargin (5 μM) or tunicamycin (5 μg/ml) for 60 h. Autophagy formation was measured as described in methods *P < 0.05, significantly different from thapsigargin-treated OASF, #P < 0.05, significantly different from tunicamycin-treated OASF (a). Non-specific or CHOP siRNA was transfected into OASF and RASF. After 16 h, cells were treated with thapsigargin (5 μM) or tunicamycin (5 μg/ml) for 4 h. Immunoblotting was performed with anti-CHOP or actin antibody (b). Autophagy formation (c) and cell death (d) were measured. *P < 0.05, significantly different from non-specific siRNA-transfected OASF in the presence of thapsigargin. #P < 0.05, significantly different from non-specific siRNA-transfected OASF in the presence of tunicamycin, $P < 0.05, significantly different from non-specific siRNA-transfected RASF in the presence of thapsigargin. &P < 0.05, significantly different from non-specific siRNA-transfected RASF in the presence of tunicamycin.
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Figure 5: CHOP plays an important role in autophagy in RASF and in cell death in OASF. OASF and RASF were treated with thapsigargin (5 μM) or tunicamycin (5 μg/ml) for 60 h. Autophagy formation was measured as described in methods *P < 0.05, significantly different from thapsigargin-treated OASF, #P < 0.05, significantly different from tunicamycin-treated OASF (a). Non-specific or CHOP siRNA was transfected into OASF and RASF. After 16 h, cells were treated with thapsigargin (5 μM) or tunicamycin (5 μg/ml) for 4 h. Immunoblotting was performed with anti-CHOP or actin antibody (b). Autophagy formation (c) and cell death (d) were measured. *P < 0.05, significantly different from non-specific siRNA-transfected OASF in the presence of thapsigargin. #P < 0.05, significantly different from non-specific siRNA-transfected OASF in the presence of tunicamycin, $P < 0.05, significantly different from non-specific siRNA-transfected RASF in the presence of thapsigargin. &P < 0.05, significantly different from non-specific siRNA-transfected RASF in the presence of tunicamycin.

Mentions: Autophagy plays an important role in the characteristics of RASF. In light of this, we examined other ER stress agents in OASF and RASF. First, we treated cells with tunicamycin, an N-acetyl glycosylation inhibitor. In RASF, tunicamycin increased autophagy in a similar manner as thapsigargin (Figure 5a). Using this model, we studied the effect of CHOP. First, CHOP siRNA was transfected into OASF and RASF. CHOP expression was barely detected in either OASF or RASF (Figure 2a). To show transfection efficiency, immunoblots were performed under ER stress-treated conditions. The siRNA knock-down approach successfully inhibited CHOP expression in both thapsigargin and tunicamycin-treated OASF and RASF (Figure 5b). CHOP inhibition significantly increased autophagy in RASF (Figure 5c) and increased cell viability (Figure 5d). Inhibition of CHOP increases cell viability in OASF, clearly showing the pro-apoptotic role of CHOP in OASF as well as in RASF (Figure 5d). These data suggest an inverse relation between CHOP expression and autophagy induction to increase cell resistance against ER stress in RASF.


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)

CHOP plays an important role in autophagy in RASF and in cell death in OASF. OASF and RASF were treated with thapsigargin (5 μM) or tunicamycin (5 μg/ml) for 60 h. Autophagy formation was measured as described in methods *P < 0.05, significantly different from thapsigargin-treated OASF, #P < 0.05, significantly different from tunicamycin-treated OASF (a). Non-specific or CHOP siRNA was transfected into OASF and RASF. After 16 h, cells were treated with thapsigargin (5 μM) or tunicamycin (5 μg/ml) for 4 h. Immunoblotting was performed with anti-CHOP or actin antibody (b). Autophagy formation (c) and cell death (d) were measured. *P < 0.05, significantly different from non-specific siRNA-transfected OASF in the presence of thapsigargin. #P < 0.05, significantly different from non-specific siRNA-transfected OASF in the presence of tunicamycin, $P < 0.05, significantly different from non-specific siRNA-transfected RASF in the presence of thapsigargin. &P < 0.05, significantly different from non-specific siRNA-transfected RASF in the presence of tunicamycin.
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Figure 5: CHOP plays an important role in autophagy in RASF and in cell death in OASF. OASF and RASF were treated with thapsigargin (5 μM) or tunicamycin (5 μg/ml) for 60 h. Autophagy formation was measured as described in methods *P < 0.05, significantly different from thapsigargin-treated OASF, #P < 0.05, significantly different from tunicamycin-treated OASF (a). Non-specific or CHOP siRNA was transfected into OASF and RASF. After 16 h, cells were treated with thapsigargin (5 μM) or tunicamycin (5 μg/ml) for 4 h. Immunoblotting was performed with anti-CHOP or actin antibody (b). Autophagy formation (c) and cell death (d) were measured. *P < 0.05, significantly different from non-specific siRNA-transfected OASF in the presence of thapsigargin. #P < 0.05, significantly different from non-specific siRNA-transfected OASF in the presence of tunicamycin, $P < 0.05, significantly different from non-specific siRNA-transfected RASF in the presence of thapsigargin. &P < 0.05, significantly different from non-specific siRNA-transfected RASF in the presence of tunicamycin.
Mentions: Autophagy plays an important role in the characteristics of RASF. In light of this, we examined other ER stress agents in OASF and RASF. First, we treated cells with tunicamycin, an N-acetyl glycosylation inhibitor. In RASF, tunicamycin increased autophagy in a similar manner as thapsigargin (Figure 5a). Using this model, we studied the effect of CHOP. First, CHOP siRNA was transfected into OASF and RASF. CHOP expression was barely detected in either OASF or RASF (Figure 2a). To show transfection efficiency, immunoblots were performed under ER stress-treated conditions. The siRNA knock-down approach successfully inhibited CHOP expression in both thapsigargin and tunicamycin-treated OASF and RASF (Figure 5b). CHOP inhibition significantly increased autophagy in RASF (Figure 5c) and increased cell viability (Figure 5d). Inhibition of CHOP increases cell viability in OASF, clearly showing the pro-apoptotic role of CHOP in OASF as well as in RASF (Figure 5d). These data suggest an inverse relation between CHOP expression and autophagy induction to increase cell resistance against ER stress in RASF.

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