Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase

Maria Grazia Cifone, Ruggero De Maria, Paola Roncaioli, Maria Rita Rippo, Miyuki Azuma, Lewis L. Lanier, Angela Santoni, Roberto Testi

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Pages (from-to)1547-1552
Number of pages6
JournalJournal of Experimental Medicine
Issue number4
Publication statusPublished - Oct 1 1994

ASJC Scopus subject areas

  • Immunology


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