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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 expression is decreased in thapsigargin-treated RASF. Thapsigargin (5 μM) was added for 0, 0.5, 1, 2, 4 and 8 h and SDS-PAGE and immunoblotting was performed with anti-GRP78, GRP94, CHOP, p21, p53 or Bax antibody (a: upper). The expression of GRP78 and CHOP was quantified (a: lower). OASF and RASF were treated with thapsigargin (5 μM) for 0, 12, 36, 48 and 60 h. After incubation, total protein was extracted. SDS-PAGE was performed and GRP78 and CHOP expression was analyzed (b: upper). The expression of CHOP was quantified (b: lower) *P < 0.05, versus non-treated OASF. #P < 0.05, versus OASF with same treatments.
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Figure 2: CHOP expression is decreased in thapsigargin-treated RASF. Thapsigargin (5 μM) was added for 0, 0.5, 1, 2, 4 and 8 h and SDS-PAGE and immunoblotting was performed with anti-GRP78, GRP94, CHOP, p21, p53 or Bax antibody (a: upper). The expression of GRP78 and CHOP was quantified (a: lower). OASF and RASF were treated with thapsigargin (5 μM) for 0, 12, 36, 48 and 60 h. After incubation, total protein was extracted. SDS-PAGE was performed and GRP78 and CHOP expression was analyzed (b: upper). The expression of CHOP was quantified (b: lower) *P < 0.05, versus non-treated OASF. #P < 0.05, versus OASF with same treatments.

Mentions: ER stress increases levels of stress proteins such as GRP78 or CHOP, as well as adaptation or cell death pathways [22]. In this study, we determined the expression levels of proteins involved in ER-stress-induced cell death in OASF and RASF. Intriguingly, the expression level of the pro-apoptotic protein, CHOP, was significantly decreased in RASF (Figure 2a). However, the expression of glucose response protein 78 (GRP78), also involved in ER stress responses, was similar in OASF and RASF. In addition, elongation initiation factor-1α (eIF-1α), the downstream protein of PERK, a PKR (RNA-dependent protein kinase)-like ER kinase that attenuates protein translation in response to ER stress, was similar in OASF and RASF (data not shown). When stress was prolonged more than 24 h, CHOP expression remained lower in RASF than OASF (Figure 2b). These results indicate that CHOP expression is regulated in RASF, which could explain resistance to ER stress-induced cell death.


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 expression is decreased in thapsigargin-treated RASF. Thapsigargin (5 μM) was added for 0, 0.5, 1, 2, 4 and 8 h and SDS-PAGE and immunoblotting was performed with anti-GRP78, GRP94, CHOP, p21, p53 or Bax antibody (a: upper). The expression of GRP78 and CHOP was quantified (a: lower). OASF and RASF were treated with thapsigargin (5 μM) for 0, 12, 36, 48 and 60 h. After incubation, total protein was extracted. SDS-PAGE was performed and GRP78 and CHOP expression was analyzed (b: upper). The expression of CHOP was quantified (b: lower) *P < 0.05, versus non-treated OASF. #P < 0.05, versus OASF with same treatments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2875648&req=5

Figure 2: CHOP expression is decreased in thapsigargin-treated RASF. Thapsigargin (5 μM) was added for 0, 0.5, 1, 2, 4 and 8 h and SDS-PAGE and immunoblotting was performed with anti-GRP78, GRP94, CHOP, p21, p53 or Bax antibody (a: upper). The expression of GRP78 and CHOP was quantified (a: lower). OASF and RASF were treated with thapsigargin (5 μM) for 0, 12, 36, 48 and 60 h. After incubation, total protein was extracted. SDS-PAGE was performed and GRP78 and CHOP expression was analyzed (b: upper). The expression of CHOP was quantified (b: lower) *P < 0.05, versus non-treated OASF. #P < 0.05, versus OASF with same treatments.
Mentions: ER stress increases levels of stress proteins such as GRP78 or CHOP, as well as adaptation or cell death pathways [22]. In this study, we determined the expression levels of proteins involved in ER-stress-induced cell death in OASF and RASF. Intriguingly, the expression level of the pro-apoptotic protein, CHOP, was significantly decreased in RASF (Figure 2a). However, the expression of glucose response protein 78 (GRP78), also involved in ER stress responses, was similar in OASF and RASF. In addition, elongation initiation factor-1α (eIF-1α), the downstream protein of PERK, a PKR (RNA-dependent protein kinase)-like ER kinase that attenuates protein translation in response to ER stress, was similar in OASF and RASF (data not shown). When stress was prolonged more than 24 h, CHOP expression remained lower in RASF than OASF (Figure 2b). These results indicate that CHOP expression is regulated in RASF, which could explain resistance to ER stress-induced cell death.

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