Sphingosine-induced apoptosis is dependent on lysosomal proteases

We propose a new mechanism for sphingosine-induced apoptosis, involving rel ocation of lysosomal hydrolases to the cytosol. Owing to its lysosomotropic properties, sphingosine, which is also a detergent, especially when proton ated, accumulates by proton trapping within the acidic vacuolar apparatus, where most of its action as a deter-ent would be exerted. When sphingosine was added in low-to-moderate concentrations to Jurkat and J774 cells, parti al lysosomal rupture occurred dose-dependently, starting within a few minut es, This phenomenon preceded caspase activation, as well as changes of mito chondrial membrane potential. High sphingosine doses rapidly caused extensi ve lysosomal rupture and ensuing necrosis, without antecedent apoptosis or caspase activation. The sphingosine effect was prevented by pre-treatment w ith another, non-toxic, lysosomotropic base, ammonium chloride, at 10 mM. T he lysosomal protease inhibitors, pepstatin A and epoxysuccinyl-L-leucyl-am ido-3-methyl-butane ethyl ester ('E-64d'), inhibited markedly sphingosine-i nduced caspase activity to almost the same degree as the general caspase in hibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone ('Z-VAD-FMK'), although they did not by themselves inhibit caspases. We conclude that cath epsin D and one or more cysteine proteases, such as cathepsins B or L, are important mediators of sphingosine-induced apoptosis, working upstream of t he caspase cascade and mitochondrial membrane-potential changes.