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Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase
M G Cifone1, R De Maria, P Roncaioli
1Department of Experimental Medicine and Biochemical Sciences, University of Rome, Tor Vergata, Italy.
Abstract:
Intracellular pathways leading from membrane receptor engagement to apoptotic cell death are still poorly characterized. We investigated the intracellular signaling generated after cross-linking of CD95 (Fas/Apo-1 antigen), a broadly expressed cell surface receptor whose engagement results in triggering of cellular apoptotic programs. DX2, a new functional anti-CD95 monoclonal antibody was produced by immunizing mice with human CD95-transfected L cells. Crosslinking of CD95 with DX2 resulted in the activation of a sphingomyelinase (SMase) in promyelocytic U937 cells, as well as in other human tumor cell lines and in CD95-transfected murine cells, as demonstrated by induction of in vivo sphingomyelin (SM) hydrolysis and generation of ceramide. Direct in vitro measurement of enzymatic activity within CD95-stimulated U937 cell extracts, using labeled SM vesicles as substrates, showed strong SMase activity, which required pH 5.0 for optimal substrate hydrolysis. Finally, all CD95-sensitive cell lines tested could be induced to undergo apoptosis after exposure to cell-permeant C2-ceramide. These data indicate that CD95 cross-linking induces SM breakdown and ceramide production through an acidic SMase, thus providing the first information regarding early signal generation from CD95, and may be relevant in defining the biochemical nature of intracellular messengers leading to apoptotic cell death.
Insights
Engagement of the CD95 receptor triggers apoptosis by activating sphingomyelinase (SMase). This enzyme breaks down sphingomyelin (SM), producing ceramide, a key messenger in programmed cell death.
Area of Science:
- Cell Biology
- Biochemistry
- Immunology
Background:
- Intracellular signaling pathways initiating apoptosis after membrane receptor activation are not fully understood.
- CD95 (Fas/Apo-1 antigen) is a cell surface receptor crucial for initiating apoptotic cell death programs.
Purpose of the Study:
- To investigate the intracellular signaling cascade triggered by CD95 receptor cross-linking.
- To identify early molecular events and signaling molecules involved in CD95-mediated apoptosis.
Main Methods:
- Production of a functional anti-CD95 monoclonal antibody (DX2).
- Assessment of sphingomyelinase (SMase) activity and ceramide generation in CD95-stimulated cells.
- In vitro enzymatic assays using labeled sphingomyelin (SM) vesicles.
- Induction of apoptosis using cell-permeant C2-ceramide.
Main Results:
- CD95 cross-linking with DX2 activated SMase in various human and murine cell lines.
- Demonstrated in vivo SM hydrolysis and subsequent ceramide production.
- Identified optimal acidic pH (5.0) for SMase activity in vitro.
- Exogenous C2-ceramide induced apoptosis in CD95-sensitive cell lines.
Conclusions:
- CD95 engagement initiates apoptosis through the activation of an acidic SMase, leading to ceramide production.
- This study provides novel insights into the early signaling events downstream of CD95.
- Ceramide generation is a critical intracellular messenger in CD95-induced apoptotic pathways.