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

Objective

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.

Methods

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).

Results

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.

Conclusions

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.