Involvement of FAN in TNF-induced apoptosis
Bruno Ségui, … , Martin Krönke, Thierry Levade
Bruno Ségui, … , Martin Krönke, Thierry Levade
Published July 1, 2001
Citation Information: J Clin Invest. 2001;108(1):143-151. https://doi.org/10.1172/JCI11498.
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Article

Involvement of FAN in TNF-induced apoptosis

Abstract

TNF-α is a pleiotropic cytokine activating several signaling pathways initiated at distinct intracellular domains of the TNF receptors. Although the C-terminal region is believed to be responsible for apoptosis induction, the functions of more membrane-proximal domains, including the domain that couples to neutral sphingomyelinase activation, are not yet fully elucidated. The roles of this region and of the associated adapter protein FAN (factor associated with neutral SMase activation) in the cytotoxic response to TNF have been investigated. We have now shown that stable expression in human fibroblasts of a dominant negative form of FAN abrogates TNF-induced ceramide generation from sphingomyelin hydrolysis and reduces caspase processing, thus markedly inhibiting TNF-triggered apoptosis. However, the cytotoxic responses to daunorubicin and exogenous ceramide remain unaltered, as do the TNF-induced p42/p44 MAPK activation and CD54 expression. Fibroblasts from FAN-knockout mice also proved to be resistant to TNF toxicity. These findings highlight the previously unrecognized role of the adapter protein FAN in signaling cell death induction by TNF.

Authors

Bruno Ségui, Olivier Cuvillier, Sabine Adam-Klages, Virginie Garcia, Sophie Malagarie-Cazenave, Sophie Lévêque, Sylvie Caspar-Bauguil, Jérôme Coudert, Robert Salvayre, Martin Krönke, Thierry Levade

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TNF-induced cell death, but not NF-κB activation or CD54 expression, is ...
TNF-induced cell death, but not NF-κB activation or CD54 expression, is impaired in human fibroblasts expressing a truncated FAN. (a and b) Toxicity of TNF, daunorubicin, and Cer on human fibroblasts. Fibroblasts transfected with empty vector (pcDNA3) or a vector encoding the truncated FAN (ΔFAN) were treated for 24 hours with the indicated concentration of TNF (a) or 1 μM daunorubicin (DNR) or 25 μM C2-Cer (Cer) (b). For the experiments with TNF and Cer, 50 μg/ml of cycloheximide was added to the medium. Cell viability was estimated by the MTT test (mean ± SE of three independent experiments performed in duplicate). (c) TNF-induced nuclear translocation of NF-κB in human transformed fibroblasts. pcDNA3 and ΔFAN-transfected cells were treated for the indicated times with TNF (50 ng/ml). Nuclear extracts were incubated with a NF-κB oligoprobe and analyzed by a gel shift assay. The arrow indicates the retarded NF-κB-probe complex. (d) TNF-induced CD54 expression by human fibroblasts. Cells were incubated for 24 hours in the presence (filled histograms) or absence (open histograms) of TNF (100 ng/ml). CD54 expression was assessed by flow cytometry. Similar data were obtained in two additional independent experiments and using 50 ng/ml TNF.