title: Tumor necrosis factor-alpha-associated lysosomal permeabilization is cathepsin B dependent.
abstract: Cathepsin B (Cat B) is released from lysososomes during tumor necrosis factor-alpha (TNF-alpha) cytotoxic signaling in hepatocytes and contributes to cell death. Sphingosine has recently been implicated in lysosomal permeabilization and is increased in the liver by TNF-alpha. Thus the aims of this study were to examine the mechanisms involved in TNF-alpha-associated lysosomal permeabilization, especially the role of sphingosine. Confocal microscopy demonstrated Cat B-green fluorescent protein and LysoTracker Red were both released from lysosomes after treatment of McNtcp.24 cells with TNF-alpha/actinomycin D, a finding compatible with lysosomal destabilization. In contrast, endosomes labeled with Texas Red dextran remained intact, suggesting lysosomes were specifically targeted for permeabilization. LysoTracker Red was released from lysosomes in hepatocytes treated with TNF-alpha or sphingosine in Cat B(+/+) but not Cat B(-/-) hepatocytes, as assessed by a fluorescence-based assay. With the use of a calcein release assay in isolated lysosomes, sphingosine permeabilized liver lysosomes isolated from Cat B(+/+) but not Cat B(-/-) liver. C(6) ceramide did not permeabilize lysosomes. In conclusion, these data implicate a sphingosine-Cat B interaction inducing lysosomal destabilization during TNF-alpha cytotoxic signaling.
authors.affiliation: Division of Gastroenterology and Hepatology, Mayo Medical School, Clinic, and Foundation, Rochester, Minnesota 55905, USA.
authors.forename: Nathan W
authors.initials: NW
authors.lastname: Werneburg
authors.forename: M Eugenia
authors.initials: ME
authors.lastname: Guicciardi
authors.forename: Steven F
authors.initials: SF
authors.lastname: Bronk
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Gores
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D017209:Apoptosis; D002401:Cathepsin B / Q000235:genetics / Q000502:physiology*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003609:Dactinomycin / Q000494:pharmacology; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D022781:Hepatocytes / Q000378:metabolism / Q000648:ultrastructure; D008114:Liver Neoplasms, Experimental; D008164:Luminescent Proteins / Q000235:genetics; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013110:Sphingosine / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D015703:Antigens, CD; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D003609:Dactinomycin; D002401:Cathepsin B; D013110:Sphingosine