Bid is upstream of lysosome-mediated caspase 2 activation in tumor necrosis factor alpha-induced hepatocyte apoptosis.
OBJECTIVE
During tumor necrosis factor alpha-mediated hepatocyte cytotoxicity, cathepsin B is released from lysosomes and contributes to apoptosis by indirectly promoting mitochondrial dysfunction. How this lysosomal pathway mediates mitochondrial dysfunction is unclear. Because Bcl-2 family proteins and caspase 2 have been implicated in proximal apoptosis-signaling pathways, we examined the role of these proteins in tumor necrosis factor alpha-induced lysosomal permeabilization and cathepsin B-mediated mitochondrial dysfunction.


METHODS
Studies were performed in primary hepatocytes from wild-type cathepsin B knockout, Bid knockout, and caspase 2 knockout mice and in the rat hepatoma cell line McArdle7777 by using tumor necrosis factor alpha/actinomycin D.


RESULTS
Studies in wild-type and Bid knockout hepatocytes showed that tumor necrosis factor alpha-mediated lysosomal permeabilization is Bid dependent. After tumor necrosis factor alpha/actinomycin D treatment, caspase 2 activity increased severalfold in wild-type hepatocytes, whereas minimal activity was observed in hepatocytes from cathepsin B knockout mice or in hepatoma cells treated with a cathepsin B inhibitor. In contrast, Bax was activated independently of cathepsin B. Pharmacological, genetic, or small interfering RNA-mediated inhibition of caspase 2 attenuated tumor necrosis factor alpha-mediated mitochondrial dysfunction, downstream caspase activation, and hepatocyte apoptosis.


CONCLUSIONS
These data suggest that tumor necrosis factor alpha triggers Bid-dependent lysosomal permeabilization, followed by release of cathepsin B into the cytosol and activation of caspase 2. Caspase 2 then facilitates efficient mitochondrial cytochrome c release and apoptosis.
Guicciardi
M Eugenia
ME
Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
Bronk
Steven F
SF
Werneburg
Nathan W
NW
Yin
Xiao-Ming
XM
Gores
Gregory J
GJ
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000378:metabolism*; D053143:Caspase 2; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002401:Cathepsin B / Q000378:metabolism; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D051023:BH3 Interacting Domain Death Agonist Protein; C494815:Bid protein, mouse; D002352:Carrier Proteins; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D045304:Cytochromes c; D053143:Caspase 2; D020169:Caspases; D002401:Cathepsin B