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

Nate Werneburg, M. Eugenia Guicciardi, Xiao Ming Yin, Gregory J. Gores

Research output: Contribution to journalArticle

100 Scopus citations

Abstract

TNF-α 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-α-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-α/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 (ΔFADD), 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 (ΔFAN) 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.

Original languageEnglish (US)
Pages (from-to)G436-G443
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume287
Issue number2 50-2
DOIs
StatePublished - Aug 1 2004

Keywords

  • Cathepsin B
  • Dominant-negative Fas-associated protein with death domain
  • Dominant-negative factor associated with neutral sphingomyelinase activation
  • Green fluorescent protein

ASJC Scopus subject areas

  • Physiology
  • Hepatology
  • Gastroenterology
  • Physiology (medical)

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