TNF- -mediated lysosomal permeabilization is FAN and caspase 8/Bid dependent

Werneburg, Nate, M. Eugenia Guicciardi, Xiao-Ming Yin, and Gregory J. Gores. TNF-mediated lysosomal permeabilization is FAN and caspase 8/Bid dependent. Am J Physiol Gastrointest Liver Physiol 287: G436–G443, 2004. First published April 8, 2004; 10.1152/ajpgi.00019.2004.—TNFcytotoxic signaling involves lysosomal permeabilization with release of the lysosomal protease cathepsin B (ctsb) into the cytosol. However, the mechanisms mediating lysosomal breakdown remain unclear. Because caspase-8 and factor associated with neutral sphingomyelinase activation (FAN) have been implicated as proximal mediators of TNF-associated apoptosis, their role in lysosomal permeabilization was examined. Cellular distribution of ctsb-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 after treatment with TNF/actinomycin D. This cellular redistribution of ctsb-GFP was blocked by transfection with a vector expressing a dominant-negative Fas-associated protein with death domain ( FADD), cytokine response modifier A, or a pharmacological 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.