Abstract
Tumor necrosis factor (TNF) induces a typical apoptotic cell death program in various cell lines by interacting with the p55 tumor necrosis factor receptor (TNF-R55). In contrast, triggering of the fibrosarcoma cell line L929sA gives rise to characteristic cellular changes resulting in necrosis. The intracellular domain of TNF-R55 can be subdivided into two parts: a membrane-proximal domain (amino acids 202-325) and a C-terminal death domain (DD) (amino acids 326-413), which has been shown to be necessary and sufficient for apoptosis. Structure/function analysis of TNF-R55-mediated necrosis in L929sA cells demonstrated that initiation of necrotic cell death, as defined by swelling of the cells, rapid membrane permeabilization, absence of nuclear condensation, absence of DNA hypoploidy, and generation of mitochondrial reactive oxygen intermediates, is also confined to the DD. The striking synergistic effect of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone on TNF-induced necrosis was also observed with receptors solely containing the DD. TNF-R55-mediated necrosis is not affected by the dominant negative deletion mutant of the Fas-associated death domain (FADD-(80-205)) that lacks the N-terminal death effector domain. Moreover, overexpression of FADD-(80-205) in L929sA is cytotoxic and insensitive to CrmA, while the cytotoxicity due to overexpression of the deletion mutant FADD-(1-111) lacking the DD is prevented by CrmA. These results demonstrate that the death domain of FADD can elicit an active necrotic cell death pathway.
Highlights
Tumor necrosis factor (TNF) induces a typical apoptotic cell death program in various cell lines by interacting with the p55 tumor necrosis factor receptor (TNF-R55)
The specific activity amounted to 2.2 ϫ 108 IU/mg, as determined in a standardized cytotoxicity assay on L929sA cells. htr1 and htr9 are agonistic mouse monoclonal antibodies directed against the extracellular domain of the p55 human tumor necrosis factor receptor and was generously provided by Dr M
The death domain (DD) of hTNF-R55 Is Required for Induction of Necrosis— L929sA cells were stably transfected with cDNAs encoding different hTNF-R55 variants (Fig. 1A) and a pSV2neo selection plasmid
Summary
Tumor necrosis factor (TNF) induces a typical apoptotic cell death program in various cell lines by interacting with the p55 tumor necrosis factor receptor (TNF-R55). Overexpression of FADD-(80 –205) in L929sA is cytotoxic and insensitive to CrmA, while the cytotoxicity due to overexpression of the deletion mutant FADD-(1–111) lacking the DD is prevented by CrmA These results demonstrate that the death domain of FADD can elicit an active necrotic cell death pathway. A key step in the pathway to apoptosis is activation of procaspases Activation of these cysteinyl aspartate-specific proteases is initiated by formation of a death-inducing signaling complex (DISC) after oligomerization of the p55 TNF receptor (TNF-R55) or the Fas receptor by the respective ligands [9, 10]. In contrast to apoptotic systems, overexpression of FADD-(80 –205) lacking the DED is cytotoxic for L929sA cells in a CrmA-insensitive way, while overexpression of a FADD-(1–111) mutant containing the DED is cytotoxic in a CrmA-inhibitory way This indicates that the death domain of FADD might be implicated in TNF-R55-mediated necrosis
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