Tumor Necrosis Factor-α-Mediated Lysosomal Permeabilization Is FAN and Caspase 8/Bid-Dependent

TNFα cytotoxic signaling involves lysosomal permeabilization with release of the lysosomal protease cathepsin B (ctsb) into the cytosol. However, the mechanisms mediating lysosomal breakdown remain unclear. As caspase-8 and factor associated with neutral sphingomyelinase activation (FAN) have been implicated as proximal mediators of TNFαassociated apoptosis, their role in lysosomal permeabilization was examined. The cellular distribution of cathepsin B-green fluorescent protein (ctsb-GFP) in a rat hepatoma cell line was imaged by confocal microscopy. Ctsb-GFP fluorescence was punctate under basal conditions but became diffuse following treatment with TNFα/actinomycin D. This cellular redistribution of ctsb-GFP was blocked by transfection with a vector expressing a dominant negative Fasassociated protein with death domain (∆FADD), CrmA or a pharmacologic caspase-8 inhibitor, IETD-fmk. Consistent with the concept that caspase 8-mediated apoptosis is also Bid-dependent in hepatocytes, ctsb-GFP release from lysosomes was reduced in hepatocytes from Bid -/mice. Interestingly, transfection with a vector expressing a dominant negative FAN (∆FAN) also blocked ctsb-GFP release and caspase-8 activation. Paradigms that inhibited ctsb-GFP release from lysosomes also reduced apoptosis as assessed by morphology and biochemical criteria. In conclusion, these studies suggest FAN is upstream of caspase-8/Bid in a signaling cascade culminating in lysosomal permeabilization.