Granulysin-Induced Apoptosis. I. Involvement of at Least Two Distinct Pathways

Granulysin is a newly described cytolytic molecule released by CTL and NK cells via granule-mediated exocytosis. It shares homology with saposin-like proteins, including NK-lysin and amoebapores, and has been implicated in the lysis of tumor cells and microbes. In the present study we show that recombinant granulysin alone induces apoptosis of Jurkat cells. This apoptosis is associated with a sixfold increase in the ceramide/sphingomyelin ratio, implicating the activation of sphingomyelinases. Granulysin- and ceramide-induced apoptosis are similar in that they both are only minimally inhibited by the more selective cysteine protease p32 (caspase 3)-like caspase inhibitor N-acetyl-Asp-Glu-Val-Asp aldehyde, while they are significantly inhibited by the more general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk). Nevertheless, while Z-VAD-fmk almost completely inhibits ceramide-induced apoptosis, a Z-VAD-fmk-resistant component was observed using granulysin. Granulysin also causes apoptosis in cells depleted of sphingomyelin by prolonged treatment with the ceramide synthase inhibitor fumonisin B1. These data indicate that granulysin induces target cell death by both ceramide- and caspase-dependent and -independent pathways.