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  • Open Access

Long-term exposure of rheumatoid arthritis synovial fibroblasts to tumor necrosis factor alpha inhibits FasL-mediated apoptosis through activation of NF-κB and upregulation of the small ubiquitin-like modifier SUMO-1

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Arthritis Res Ther20046 (Suppl 3) :98

  • Published:


  • Tumor Necrosis Factor Alpha
  • Synovial Tissue
  • Electrophoretic Mobility Shift Assay
  • TUNEL Staining
  • Sustained Activation


There is accumulating evidence that rheumatoid arthritis synovial fibroblasts (RA-SF) are resistant to FasL-induced apoptosis despite the abundant expression of Fas. Tumor necrosis factor alpha (TNF-α) has been suggested to contribute to this process mainly through the transient activation of transcription factors such as NF-κB. However, in addition to short-term induction of transcriptional regulators, long-term activation of RA-SF has gained increasing interest. In this context the small ubiquitin-like modifier SUMO-1 appears to be of importance, and some data indicate that increased levels of SUMO-1 are linked to the resistance of RA-SF against programmed cell death. However, little is known about the regulation of SUMO-1 in fibroblast-like cells. Here, we investigated the effects of long-term stimulation of RA-SF with TNF-α on the activation of NF-κB, the expression of SUMO-1, and on spontaneous as well as FasL-induced cell death.


Synovial tissues were obtained from patients with rheumatoid arthritis at joint replacement surgery, and synovial fibroblasts were isolated by enzymatic digestion. Long-term effects of TNF-α were analyzed by stimulation of RA-SF with 10 or 100 ng/ml TNF-α for 24 hours. Nuclear binding of NF-κB was assessed by electrophoretic mobility shift assay. The expression of SUMO-1 in TNF-α-stimulated and unstimulated RA-SF was determined by quantitative real-time PCR and western blot. To induce apoptosis, TNF-α pretreated and untreated RA-SF were stimulated with recombinant human FasL (100 ng/ml) for 16 hours. Apoptosis was measured by a histone fragmentation assay (Cell Death ELISA; Roche Diagnostics, Mannheim, Germany) and confirmed by FACS analysis with intercellular TUNEL staining (Apo-BRDU™ Kit; BD Biosciences, Heidelberg, Germany).


Treatment of RA-SF with TNF-α over 24 hours did not induce cell death but slightly reduced the rate of spontaneous apoptosis. More significantly, long-term exposure of RA-SF to TNF-α clearly prevented the induction of apoptosis by recombinant human FasL in a dose-dependent manner. This was accompanied not only by a sustained activation of NF-κB, but also by a significant increase in the expression of SUMO-1. The induction of SUMO-1 by TNF-α was dose dependent and seen both at the mRNA and the protein level.


The data suggest that long-term exposure of RA-SF to TNF-α inhibits FasL-induced apoptosis not only through sustained activation of NF-κB, but also through upregulation of the small ubiquitin-like modifier SUMO-1. Although SUMO-1 has been demonstrated to be elevated in RA-SF in the absence of continuous stimulation with inflammatory cytokines and to be part of the stable activation of RA-SF, TNF-α in the inflamed synovium may enhance further the expression of SUMO-1 and, thus, contribute to the resistance of RA-SF against apoptosis.

Authors’ Affiliations

Division of Experimental Rheumatology and Orthopedics, University Hospital Magdeburg, Magdeburg, Germany
Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, Switzerland
Institute of Experimental Internal Medicine, University Hospital Magdeburg, Magdeburg, Germany


© The Author(s) 2004