title: TNF-alpha-mediated lysosomal permeabilization is FAN and caspase 8/Bid dependent.
abstract: TNF-alpha 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. Because caspase-8 and factor associated with neutral sphingomyelinase activation (FAN) have been implicated as proximal mediators of TNF-alpha-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-alpha/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 (DeltaFADD), 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 (DeltaFAN) 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.
authors.affiliation: Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905, USA.
authors.forename: Nate
authors.initials: N
authors.lastname: Werneburg
authors.forename: M Eugenia
authors.initials: ME
authors.lastname: Guicciardi
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Yin
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Gores
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism / Q000502:physiology*; D002401:Cathepsin B / Q000378:metabolism; D002478:Cells, Cultured; D003609:Dactinomycin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D053200:Fas-Associated Death Domain Protein; D049452:Green Fluorescent Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D010539:Permeability / Q000187:drug effects; D011506:Proteins / Q000502:physiology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051023:BH3 Interacting Domain Death Agonist Protein; C494815:Bid protein, mouse; C494816:Bid protein, rat; D002352:Carrier Proteins; C505534:Fadd protein, mouse; C505535:Fadd protein, rat; D053200:Fas-Associated Death Domain Protein; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; C491112:Nsmaf protein, mouse; C491113:Nsmaf protein, rat; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D003609:Dactinomycin; C505526:Casp8 protein, mouse; C505528:Casp8 protein, rat; D053181:Caspase 8; D020169:Caspases; D002401:Cathepsin B