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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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Thapsigargin increases autophagy in RASF. OASF and RASF were incubated with thapsigargin (0, 0.1, 0.5, 1 or 5 μM) for 60 h. Autophagic cell number was determined by autophagic vesicles. Data represent means ± S.E. (n = 4) (a). Thapsigargin (5 μM) was added for 0, 12, 24, 36 or 48 h. After incubation, total protein was extracted. SDS-PAGE and immunoblotting were performed with anti-beclin, LC3 or actin antibody (b: upper). The expression of beclin and LC3-II was quantified, compared with the expression of actin (b: lower). OASF and RASF were treated with thapsigargin (5 μM) for 60 h. Crystal violet-stained cells are shown by light microscopy (upper panel) and GFP-LC3-transfected cells are shown by fluorescent microscopy (lower panel) (c). (a) to (b): *P < 0.05, versus non-treated OASF. #P < 0.05, versus OASF with same treatments.
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Figure 3: Thapsigargin increases autophagy in RASF. OASF and RASF were incubated with thapsigargin (0, 0.1, 0.5, 1 or 5 μM) for 60 h. Autophagic cell number was determined by autophagic vesicles. Data represent means ± S.E. (n = 4) (a). Thapsigargin (5 μM) was added for 0, 12, 24, 36 or 48 h. After incubation, total protein was extracted. SDS-PAGE and immunoblotting were performed with anti-beclin, LC3 or actin antibody (b: upper). The expression of beclin and LC3-II was quantified, compared with the expression of actin (b: lower). OASF and RASF were treated with thapsigargin (5 μM) for 60 h. Crystal violet-stained cells are shown by light microscopy (upper panel) and GFP-LC3-transfected cells are shown by fluorescent microscopy (lower panel) (c). (a) to (b): *P < 0.05, versus non-treated OASF. #P < 0.05, versus OASF with same treatments.

Mentions: When misfolded proteins accumulate in the ER, this stress activates the unfolded protein response (UPR) to induce expression of chaperones and proteins involved in the recovery process [23]. Under conditions of ER stress, pre-autophagosomal structures are assembled, and autophagosomal transport to vacuoles is stimulated [24]. In this study, we examined whether ER stress induces autophagy in either OASF or RASF. RASF showed high levels of autophagy at relatively low doses of thapsigargin (1 μM) (Figure 3a) and forms autophagosomes (Supplementary Figure 1). ER stress also increased the expression of beclin, an autophagy marker protein, and LC3-II more in RASF than in OASF (Figure 3b). When autophagy is induced, intra-lumenal LC3 is degraded by lysosomal proteases, forming an 18 kDa form (LC3-I) and subsequently being processed to a membrane-bound form (LC3-II, 16 kDa) [25]. To verify autophagy, we measured crystal violet-stained vacuoles under a light microscope (Figure 3c). A GFP-LC3 (LC3, mammalian homolog of yeast Atg8) fusion gene was transfected into OASF and RASF to measure changes in autophagosome numbers after treatment. The classical expression pattern of processed LC3-II was more evident in RASF, indicating autophagy vesicle formation. The expression was quantified in the lower panel of Figure 3c.


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)

Thapsigargin increases autophagy in RASF. OASF and RASF were incubated with thapsigargin (0, 0.1, 0.5, 1 or 5 μM) for 60 h. Autophagic cell number was determined by autophagic vesicles. Data represent means ± S.E. (n = 4) (a). Thapsigargin (5 μM) was added for 0, 12, 24, 36 or 48 h. After incubation, total protein was extracted. SDS-PAGE and immunoblotting were performed with anti-beclin, LC3 or actin antibody (b: upper). The expression of beclin and LC3-II was quantified, compared with the expression of actin (b: lower). OASF and RASF were treated with thapsigargin (5 μM) for 60 h. Crystal violet-stained cells are shown by light microscopy (upper panel) and GFP-LC3-transfected cells are shown by fluorescent microscopy (lower panel) (c). (a) to (b): *P < 0.05, versus non-treated OASF. #P < 0.05, versus OASF with same treatments.
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Figure 3: Thapsigargin increases autophagy in RASF. OASF and RASF were incubated with thapsigargin (0, 0.1, 0.5, 1 or 5 μM) for 60 h. Autophagic cell number was determined by autophagic vesicles. Data represent means ± S.E. (n = 4) (a). Thapsigargin (5 μM) was added for 0, 12, 24, 36 or 48 h. After incubation, total protein was extracted. SDS-PAGE and immunoblotting were performed with anti-beclin, LC3 or actin antibody (b: upper). The expression of beclin and LC3-II was quantified, compared with the expression of actin (b: lower). OASF and RASF were treated with thapsigargin (5 μM) for 60 h. Crystal violet-stained cells are shown by light microscopy (upper panel) and GFP-LC3-transfected cells are shown by fluorescent microscopy (lower panel) (c). (a) to (b): *P < 0.05, versus non-treated OASF. #P < 0.05, versus OASF with same treatments.
Mentions: When misfolded proteins accumulate in the ER, this stress activates the unfolded protein response (UPR) to induce expression of chaperones and proteins involved in the recovery process [23]. Under conditions of ER stress, pre-autophagosomal structures are assembled, and autophagosomal transport to vacuoles is stimulated [24]. In this study, we examined whether ER stress induces autophagy in either OASF or RASF. RASF showed high levels of autophagy at relatively low doses of thapsigargin (1 μM) (Figure 3a) and forms autophagosomes (Supplementary Figure 1). ER stress also increased the expression of beclin, an autophagy marker protein, and LC3-II more in RASF than in OASF (Figure 3b). When autophagy is induced, intra-lumenal LC3 is degraded by lysosomal proteases, forming an 18 kDa form (LC3-I) and subsequently being processed to a membrane-bound form (LC3-II, 16 kDa) [25]. To verify autophagy, we measured crystal violet-stained vacuoles under a light microscope (Figure 3c). A GFP-LC3 (LC3, mammalian homolog of yeast Atg8) fusion gene was transfected into OASF and RASF to measure changes in autophagosome numbers after treatment. The classical expression pattern of processed LC3-II was more evident in RASF, indicating autophagy vesicle formation. The expression was quantified in the lower panel of Figure 3c.

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