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Fas and tumor necrosis factor receptor-mediated cell death: similarities and distinctions
1Department of Pathology, Massachusetts General Hospital, Charlestown.
Abstract:
Fas antigen and two tumor necrosis factor receptors (TNFR), p55 and p75, are implicated in the triggering of cell death upon stimulation by natural ligands and specific monoclonal antibodies. However, the relative efficiency of each receptor, the mechanisms that regulate their function and the signaling pathways they employ, remain to be elucidated. In this study, fusion proteins, composed of the extracellular domain of CD40 and the intracellular and transmembrane domains of Fas, TNFRp55 and TNFRp75, were stably expressed in a human melanoma cell line that is deficient in Fas and TNFR expression. Transfectants were stimulated by a soluble recombinant form of the CD40 ligand gp39, and the effect on cell viability determined. Engagement of all three fusion proteins by the gp39 ligand induced lethal signals, but the rate at which cell death occurred was distinct. Fas-derived signals were observed to have the most rapid effect, killing most cells within hours of stimulation, whereas TNFRp55- and TNFRp75-associated signals resulted in cell death within 2-3 d after engagement by ligand. It is interesting to note that optimal cell killing by all three fusion proteins was dependent on a critical, low to intermediate, cell surface expression level. High levels of fusion protein expression, on the other hand, were associated with inhibition of cell death. Our results provide a model to study Fas and TNFR-mediated cell death and suggest a novel mechanism for the regulation of death signals triggered by members of the TNFR family.
Insights
Tumor necrosis factor receptors (TNFR) and Fas antigen trigger cell death. This study used fusion proteins to show Fas signals were fastest, while TNFR signals were slower, with optimal cell killing dependent on expression levels.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Fas antigen and tumor necrosis factor receptors (TNFR) mediate cell death.
- Mechanisms and signaling pathways of TNFR family-mediated apoptosis require further elucidation.
Purpose of the Study:
- To investigate the relative efficiency and regulation of Fas and TNFR signaling pathways.
- To establish a model system for studying TNFR family-mediated cell death.
Main Methods:
- Engineered fusion proteins combining CD40 extracellular domains with intracellular/transmembrane domains of Fas, TNFRp55, and TNFRp75.
- Stable expression in a human melanoma cell line lacking Fas and TNFR.
- Stimulation with soluble CD40 ligand (gp39) to assess cell viability.
Main Results:
- Engagement of all three fusion proteins induced lethal signals.
- Fas-derived signals resulted in rapid cell death (hours), while TNFRp55 and TNFRp75 signals led to death in 2-3 days.
- Optimal cell killing required low to intermediate fusion protein expression; high expression inhibited cell death.
Conclusions:
- Fas and TNFR signaling pathways exhibit distinct kinetics in inducing cell death.
- Cell surface expression levels critically regulate TNFR family-mediated apoptosis.
- A novel regulatory mechanism for death signals in the TNFR family was identified.