Susceptibility of rheumatoid arthritis synovial fibroblasts to FasL- and TRAIL-induced apoptosis is cell cycle-dependent

被引:35
|
作者
Pundt, Noreen [1 ]
Peters, Marvin A. [1 ]
Wunrau, Christina [1 ]
Strietholt, Simon [1 ]
Fehrmann, Carsten [2 ]
Neugebauer, Katja [1 ]
Seyfert, Christine [3 ]
van Valen, Frans [1 ]
Pap, Thomas [1 ]
Meinecke, Ingmar [1 ,4 ]
机构
[1] Univ Hosp Muenster, Inst Expt Musculoskeletal Med, D-48149 Munster, Germany
[2] Univ Hosp Muenster, Inst Med Microbiol, D-48149 Munster, Germany
[3] Zeisigwaldkliniken Bethanien Chemnitz, Dept Orthopaed Surg, D-09130 Chemnitz, Germany
[4] Pk Krankenhaus Leipzig Suedost GmBH, Dept Orthopaed Surg, D-04289 Leipzig, Germany
关键词
NECROSIS-FACTOR-ALPHA; MEDIATED APOPTOSIS; LIGAND TRAIL; JOINT DESTRUCTION; GROWTH-FACTOR; IN-VITRO; EXPRESSION; RECEPTOR; FAMILY; SYNOVIOCYTES;
D O I
10.1186/ar2607
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
Introduction The rheumatoid arthritis (RA) synovium is characterised by the presence of an aggressive population of activated synovial fibroblasts (RASFs) that are prominently involved in the destruction of articular cartilage and bone. Accumulating evidence suggests that RASFs are relatively resistant to Fas-ligand (FasL)-induced apoptosis, but the data concerning tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) have been conflicting. Here, we hypothesise that the susceptibility of RASFs to receptor-mediated apoptosis depends on the proliferation status of these cells and therefore analysed the cell cycle dependency of FasL- and TRAIL-induced programmed cell death of RASFs in vitro. Methods Synovial fibroblasts were isolated from patients with RA by enzymatic digestion and cultured under standard conditions. Cell cycle analysis was performed using flow cytometry and staining with propidium iodide. RASFs were synchronised or arrested in various phases of the cell cycle with 0.5 mM hydroxyurea or 2.5 mu g/ml nocodazol and with foetal calf serum-free insulin-transferrin-sodium selenite supplemented medium. Apoptosis was induced by stimulation with 100 ng/ml FasL or 100 ng/ ml TRAIL over 18 hours. The apoptotic response was measured using the Apo-ONE (R) Homogenous Caspase-3/7 Assay (Promega GmbH, Mannheim, Germany) and the Cell Death Detection (ELISA(Plus)) (enzyme-linked immunosorbent assay) (Roche Diagnostics GmbH, Mannheim, Germany). Staurosporin-treated cells (1 mu g/ml) served as a positive control. Expression of Fas and TRAIL receptors (TRAILR1-4) was determined by fluorescence-activated cell sorting analysis. Results Freshly isolated RASFs showed only low proliferation in vitro, and the rate decreased further over time, particularly when RASFs became confluent. RASFs expressed Fas, TRAIL receptor-1, and TRAIL receptor-2, and the expression levels were independent of the cell cycle. However, the proliferation rate significantly influenced the susceptibility to FasL- and TRAIL-induced apoptosis. Specifically, proliferating RASFs were less sensitive to FasL- and TRAIL-induced apoptosis than RASFs with a decreased proliferation rate. Furthermore, RASFs that were synchronised in S phase or G(2)/M phase were less sensitive to TRAIL-induced apoptosis than synchronised RASFs in G(0)/G(1) phase. Conclusions Our data indicate that the susceptibility of RASFs to FasL- and TRAIL-induced apoptosis depends on the cell cycle. These results may explain some conflicting data on the ability of RASFs to undergo FasL- and TRAIL-mediated cell death and suggest that strategies to sensitise RASFs to apoptosis may include the targeting of cell cycle-regulating genes.
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页数:10
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