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Necrotic death pathway in Fas receptor signaling
H Matsumura1, Y Shimizu, Y Ohsawa
1Department of Genetics, Osaka University Medical School, Osaka 565-0871, Japan.
Abstract:
A caspase 8-deficient subline (JB6) of human Jurkat cells can be killed by the oligomerization of Fas-associated protein with death domain (FADD). This cell death process is not accompanied by caspase activation, but by necrotic morphological changes. Here, we show that the death effector domain of FADD is responsible for the FADD-mediated necrotic pathway. This process was accompanied by a loss of mitochondrial transmembrane potential (DeltaPsim), but not by the release of cytochrome c from mitochondria. Pyrrolidine dithiocarbamate, a metal chelator and antioxidant, efficiently inhibited the FADD-induced reduction of DeltaPsim and necrotic cell death. When human Jurkat, or its transformants, expressing mouse Fas were treated with Fas ligand or anti-mouse Fas antibodies, the cells died, showing characteristics of apoptosis. A broad caspase inhibitor (z-VAD-fmk) blocked the apoptotic morphological changes and the release of cytochrome c. However, the cells still died, and this cell death process was accompanied by a strong reduction in DeltaPsim, as well as necrotic morphological changes. The presence of z-VAD-fmk and pyrrolidine dithiocarbamate together blocked cell death, suggesting that both apoptotic and necrotic pathways can be activated through the Fas death receptor.
Insights
Fas-associated protein with death domain (FADD) can trigger necrotic cell death independently of caspases. Pyrrolidine dithiocarbamate inhibits this FADD-mediated necrosis, suggesting dual apoptotic and necrotic pathways via the Fas receptor.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Human Jurkat cells, a T-cell line, are susceptible to apoptosis via the Fas receptor.
- Caspase 8-deficient Jurkat cells (JB6) exhibit a necrotic pathway upon Fas-associated protein with death domain (FADD) oligomerization.
- FADD-mediated cell death in caspase 8-deficient cells bypasses caspase activation and displays necrotic morphology.
Purpose of the Study:
- To investigate the role of the FADD death effector domain in mediating necrotic cell death.
- To elucidate the mechanisms underlying FADD-induced necrosis, including mitochondrial involvement.
- To determine if the Fas death receptor can activate both apoptotic and necrotic pathways.
Main Methods:
- Utilizing caspase 8-deficient human Jurkat cells (JB6) to study FADD-mediated cell death.
- Assessing mitochondrial transmembrane potential (DeltaPsim) and cytochrome c release.
- Employing pyrrolidine dithiocarbamate (an antioxidant and metal chelator) and a broad caspase inhibitor (z-VAD-fmk) to modulate cell death pathways.
- Treating Fas-expressing Jurkat cells with Fas ligand or anti-Fas antibodies.
Main Results:
- The death effector domain of FADD is crucial for the necrotic pathway, characterized by loss of DeltaPsim but not cytochrome c release.
- Pyrrolidine dithiocarbamate effectively inhibited FADD-induced DeltaPsim reduction and necrosis.
- In Fas-expressing cells, z-VAD-fmk blocked apoptosis but did not prevent cell death, which proceeded with reduced DeltaPsim and necrotic morphology.
- Combined treatment with z-VAD-fmk and pyrrolidine dithiocarbamate abolished Fas-induced cell death.
Conclusions:
- FADD can directly induce necrotic cell death through its death effector domain, involving mitochondrial dysfunction.
- Pyrrolidine dithiocarbamate acts as an inhibitor of this FADD-mediated necrotic pathway.
- The Fas death receptor is capable of activating both caspase-dependent apoptosis and caspase-independent necrosis, which can be modulated by inhibitors.