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

99 Citations (Scopus)

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)
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume287
Issue number2 50-2
DOIs
StatePublished - Aug 2004
Externally publishedYes

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Cathepsin B
Caspase 8
Apoptosis
Lysosomes
Transfection
Hepatocytes
Fas-Associated Death Domain Protein
Sphingomyelin Phosphodiesterase
Caspase Inhibitors
Dactinomycin
Green Fluorescent Proteins
Confocal Microscopy
Cytosol
Hepatocellular Carcinoma
Peptide Hydrolases
Fluorescence
Pharmacology
Cytokines
Cell Line

Keywords

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

ASJC Scopus subject areas

  • Gastroenterology
  • Physiology

Cite this

TNF-α-mediated lysosomal permeabilization is FAN and caspase 8/Bid dependent. / Werneburg, Nate; Guicciardi, M. Eugenia; Yin, Xiao-Ming; Gores, Gregory J.

In: American Journal of Physiology - Gastrointestinal and Liver Physiology, Vol. 287, No. 2 50-2, 08.2004.

Research output: Contribution to journalArticle

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