Cathepsin B is involved in the trafficking of TNF-alpha-containing vesicles to the plasma membrane in macrophages.
TNF-alpha is a potent proinflammatory cytokine, essential for initiating innate immune responses against invading microbes and a key mediator involved in the pathogenesis of acute and chronic inflammatory diseases. To identify molecules involved in the production of TNF-alpha, we used a functional gene identification method using retroviral integration-mediated mutagenesis, followed by LPS-stimulated TNF-alpha production analysis in macrophages. We found that cathepsin B, a lysosomal cysteine proteinase, was required for optimal posttranslational processing of TNF-alpha in response to the bacterial cell wall component LPS. Mouse bone marrow-derived macrophages from cathepsin B-deficient mice and macrophages treated with the cathepsin B-specific chemical inhibitor CA074 methyl ester or small interfering RNA against cathepsin B secreted significantly less TNF-alpha than wild-type or nontreated macrophages. We further showed that the inhibition of cathepsin B caused accumulation of 26-kDa pro-TNF-containing vesicles. Ectopic expression of GFP-conjugated pro-TNF further suggests that pro-TNF failed to reach the plasma membrane without intracellular cathepsin B activity. Altogether, these data suggest that intracellular cathepsin B activity is involved in the TNF-alpha-containing vesicle trafficking to the plasma membrane.
Ha
Soon-Duck
SD
Department of Microbiology and Immunology, Siebens-Drake Research Institute, University of Western Ontario, London, Ontario, Canada.
Martins
Andrew
A
Khazaie
Khashayarsha
K
Han
Jiahuai
J
Chan
Bosco M C
BM
Kim
Sung Ouk
SO
D000818:Animals; D001483:Base Sequence; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D004151:Dipeptides / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
C400541:CA 074 methyl ester; D004151:Dipeptides; D004791:Enzyme Inhibitors; D014409:Tumor Necrosis Factor-alpha; D002401:Cathepsin B