Introduction
Rheumatoid arthritis (RA) is the most common inflammatory disorder of the joints. It is characterized by chronic inflammation, autoimmune phenomena and synovial hyperplasia, which lead to the progressive destruction of articular structures [
1]. Alterations in synovial cell apoptosis, which regulate tissue composition and homeostasis, affect the pathogenesis of rheumatoid arthritis [
2,
3]. These changes lead to synovial cell activation and contribute to both chronic inflammation and hyperplasia. The resistance of rheumatoid arthritis synovial fibroblasts to apoptosis is closely linked to the progressive destruction of articular cartilage. However, the detailed mechanisms that prevent rheumatoid arthritis-associated cells from undergoing programmed cell death are unclear.
The endoplasmic reticulum (ER) plays an important role in secretory cells, including synovial fibroblasts. Adaptive responses to the accumulation of misfolded proteins in the ER (namely ER stress) provide protection from cell death, as gene transfer-mediated overexpression of GRP78 reduces cell death induced by oxidative stress and Ca
2+ disturbances [
4]. Persistent, excessive ER stress triggers cell death [
5,
6] via the initiation of apoptosis and the induction of CHOP or by activation of caspase-12-dependent pathways [
7,
8]. CHOP mRNA is transcribed mainly during ER stress [
8,
9] and leads to apoptosis [
10]. The ER stress also can contribute to autoimmune disease [
11]. ER stress is studied in collagen-induced rheumatoid arthritis joints [
12]. To study the role of ER stress in rheumatoid arthritis, we used synovial fibroblasts from rheumatoid arthritis patients, categorized according to ACR (American College of Rheumatology) classification criteria [
13], to study apoptosis. In this study, ER stress response was examined in relation to the resistance characteristics in rheumatoid arthritis synovial fibroblasts (RASF).
Autophagy is implicated in various diseases, including cancer and neurodegenerative diseases [
14‐
16]. During autophagy, a single-membrane structure (isolation membrane) surrounds a portion of the cytoplasm and organelles [
14]. Autophagy can protect cells from ER stress-induced cell death [
17]. Another explanation for apoptosis resistance in RASF could be that the unique cellular phenotype induced by autophagy protects against apoptotic stress. Here, we compare the response to ER stress and autophagy induction between synovial fibroblasts from rheumatoid arthritis and those from osteoarthritis.
Materials and methods
Cell cultures
Synovial fibroblasts were isolated from surgical samples from 13 rheumatoid arthritis and 8 osteoarthritis patients. Informed patient consents were obtained for isolation of fibroblasts. Cells were obtained by enzymatic digestion as described before [
18]. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal calf serum (Gibco-BRL, Grand Island, NY, USA). The fibroblasts were cultured for six to eight passages. All studies were approved by the Chonbuk National Hospital ethics committee.
Cell viability
Fibroblasts were assessed microscopically for dead cells by trypan blue exclusion. Cell viability was calculated by dividing the non-stained (viable) cell count by the total cell count. The number of cells was determined by averaging the number of cells in four squares and multiplying this average by a dilution factor.
Measurement of autophagy
Autophagy was analyzed as described before [
19]. Synovial fibroblasts from osteoarthritis and rheumatoid arthritis patients were plated at 2 × 10
5 on glass coverslips in six-well plates and cultured to 70% confluence. Cells were transfected with GFP-LC3 plasmid DNA (kindly provided by Dr. T. Yoshimori, Osaka University, Japan) for 16 h and then treated with thapsigargin or tunicamycin for various times. Transfection was performed using an Amaxa Nucleofector apparatus (Amaxa, Cologne, Germany). Five μg of plasmid DNA were mixed with 0.1 ml of cell suspension, transferred to a 2.0-mm electroporation cuvette, and transfected using an Amaxa Nucleofector apparatus (Amaxa, Cologne, Germany) according to the manufacturer's protocol. The DNA quantity, cell concentration and buffer volume were kept constant throughout the experiments. After electroporation, the cells were transferred immediately to 2.0 ml of complete medium and cultured in six-well plates at 37°C until needed. Microphotographs of GFP-LC3 fluorescence were obtained with a fluorescence microscope. The detection of punctuated staining of GFP-LC3 from diffuse staining indicated the formation of autophagosomes. The punctuated stained cells were compared to the total number of GFP-transfected cells to calculate percents.
Determination of caspase-3 activity
Fibroblasts (3 × 106) were washed with phosphate buffered saline (PBS) and incubated for 30 minutes on ice with 100 ml of lysis buffer (10 mM Tris-HCl, 10 mM NaH2PO4/NaHPO4, pH 7.5, 130 mM NaCl, 1% Triton1 X-100, and 10 mM sodium pyrophosphate). Cell lysates were spun down, supernatants were collected, and protein concentrations were determined using the BCA method. For each reaction, 30 μg of protein was added to 1 ml of freshly prepared protease assay buffer (20 mM HEPES pH 7.5, 10% glycerol, 2 mM dithiothreitol) containing 20 mM of AC-DEVD-AMC (Sigma-Aldrich). Reaction mixtures without cellular extracts were used as negative controls. Reaction mixtures were incubated for 1 h at 37°C and the aminomethyl-coumarin liberated from AC-DEVD-AMC was determined by spectrofluorometry (Hitachi F-2500, Hitachi, Tokyo, Japan) at 380 nmexcitation and 400 to 550 nmemission. Readings were corrected for background fluorescence.
Western blotting
Western blotting was performed using the protocol described previously [
19]. The total protein was resolved in pre-casted 4 to 12% SDS-PAGE gradient gels. Immunoblotting was performed using the indicated antibodies. ECL reagents (Amersham Biosciences, Piscataway, NJ, USA) were used to visualize signals.
siRNA transfection
siRNAs were synthesized in duplex and purified using Bioneer technology (Daejon, South Korea). Double-stranded small interfering RNA (siRNA) targeting CHOP (SC-35437) was obtained from Santacruz company (Santa Cruz, California, USA) with control siRNA (SC-37007). For Beclin siRNA, 5'-CAGUUACAGAUGGAGCUAAtt-3' and for non-specific siRNA, 5'-CUUACGCUGAGUACUUCGAtt-3' were transfected into OASF and RASF using Amaxa Nucleofector (Amaxa, Gaithersburg, MD, USA). Briefly, confluent cells were trypsinized and resuspended in Amaxa Nucleofector solution at a density of 2 × 105 cells per 100 μl of solution, and each siRNA was added. Cells were transfected by electroporation using the A24 pulsing program.
Statistical analysis
The data were analyzed by analysis of variance (ANOVA) in dose-response experiments, or by two-tailed Student's t-tests. A P value < 0.05 was considered significant. In each case, the statistical test used is indicated, and the number of experiments is stated in figure legends.
Discussion
The present study investigated the effects of ER stress on cell death in rheumatoid arthritis synovial fibroblasts (RASF). When exposed to ER stress, cell death and expression of the pro-apoptotic ER stress protein, CHOP, were lower in RASF than in OASF (Figure
1a and
1b). Furthermore, autophagy was significantly higher in RASF (Figure
3a, 3b, and
3c) than in OASF. Beclin siRNA transfection also showed that the formation of autophagosomes is related to the protective effect against ER stress in RASF (Figure
4b and
4c). CHOP siRNA protected cells from ER stress, showing that induction of CHOP explains cell death in RASF as well as in OASF (Figure
5d). In RASF, the knock-down of CHOP increased autophagy induction, which was related to cell protection (Figure
5c and
5d). ER stress in RASF showed autophagy and lower CHOP expression, increasing resistance to death.
Autophagy is a protective mechanism against apoptotic stimuli [
26,
29,
30]. ER stress, which induces autophagy and apoptosis, is a pathological mechanism for disease [
31‐
33]. GRP78, an ER stress protein, is associated with collagen-induced rheumatoid arthritis [
34]. Normally, CHOP is ubiquitously expressed at very low levels [
35], but is robustly expressed when perturbations induce stress [
35], and CHOP
-/- cells are resistant to ER-stress-mediated apoptosis [
36,
37]. In OASF, CHOP expression was significantly increased at 2 h after treatment with thapsigargin, and reached a maximum after 8 h. In OASF, its expression was significantly increased at both 12 h and 36 h (Figure
2b), causing failure of the defense mechanisms and subsequent cell death.
To show direct evidence for the role of CHOP in ER stress-induced cell death, CHOP siRNA transfection was compared between OASF and RASF. As expected, the knock-down of CHOP increased cell viability, especially in OASF (Figure
4d). Because ER stress rarely affects autophagy in OASF, CHOP inhibition did not affect autophagy formation in OASF (Figure.
4c). These results are consistent with other studies that show CHOP as a pro-apoptotic protein [
38,
39]. In addition, CHOP expression after treatment with thapsigargin is lower in RASF than OASF, suggesting resistance against ER stress-induced cell death.
To explain the mechanism of the findings (that is, the increased autophagy in RASF), we compared the characteristics of OASF and RASF when exposed to ER stress. First, pro-inflammatory cytokines, including IL-6, were significantly higher in RASF. However, neutralizing antibodies for the cytokines did not affect autophagosome formation (data not shown). Second, there was no difference in intra-ER calcium between OASF and RASF when exposed to thapsigargin [
40]. Therefore, the role of CHOP as a pro-apoptotic protein is more convincing than other possibilities. This is the first study on the induction of autophagy and ER stress that compares OASF and RASF. Increased autophagy induction and CHOP underexpression could explain the anti-apoptotic characteristics of RASF, at least when exposed to ER stress. An in depth study of CHOP will clarify the resistance to apoptosis in rheumatoid arthritis.
Conclusions
RASF resists apoptosis following ER stress, such as Ca2+ disturbances, by autophagy formation, which may contribute to resistance against rheumatoid arthritis treatments. A better understanding of the mechanisms contributing to apoptosis resistance through autophagy will provide better insight into the mechanisms of rheumatoid arthritis and help to identify targets for the development of novel, more effective and long-lasting therapies for the treatment of rheumatoid arthritis.
Acknowledgements
This work was partly supported by grants from the Korea Research Foundation (2007-531-E00015, 2007-314-E00111, 2008-E00540) and supported by a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare and Family Affairs (A084144).
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YS participated in the design of the study and the experiments. SH performed autophagy experiments and autophagy mechanism studies. DK and GL carried out cell viability experiments. WY participated in the design of the study and provided fibroblasts. YK, JC, YL, SP, SJ, SC and HRK contributed to the experimental designs and the interpretation of the data. HTK performed electron microscopy experiments. HJ performed Western blotting experiments. HC supervised all of the experiments. All experiments were performed and supervised in HC's laboratory. All authors read and approved the final manuscript.