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Cathepsin B Is Involved in the Trafficking of TNF--Containing Vesicles to the Plasma Membrane in Macrophages¹

Soon-Duck Ha^*,, Andrew Martins^*,, Khashayarsha Khazaie, Jiahuai Han, Bosco M. C. Chan^* and Sung Ouk Kim^2,*,

^* Department of Microbiology and Immunology, and Infectious Diseases Research Group, Siebens-Drake Research Institute, University of Western Ontario, London, Ontario, Canada; Department of Microbiology-Immunology, Northwestern University, Chicago, IL 60611-3015; and The Scripps Research Institute, La Jolla, CA 92037

TNF- 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-, we used a functional gene identification method using retroviral integration-mediated mutagenesis, followed by LPS-stimulated TNF- production analysis in macrophages. We found that cathepsin B, a lysosomal cysteine proteinase, was required for optimal posttranslational processing of TNF- 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- 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--containing vesicle trafficking to the plasma membrane.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Research Grant MOP68841 from the Canadian Institute of Health (to S.O.K.) and by National Institutes of Health Research Grant R01-CA104547-01 (to K.K.).

² Address correspondence and reprint requests to Dr. Sung Ouk Kim, Infectious Diseases Research Group, Siebens-Drake Research Institute, Room 119, University of Western Ontario, 1400 Western Road, London, Ontario N6G 2V4, Canada. E-mail address: sung.kim{at}schulich.uwo.ca

³ Abbreviations used in this paper: SNAP, soluble-N-ethylmaleimide-sensitive factor-attachment protein; SNARE, SNAP receptor; siRNA, small interfering RNA; BMDIM, bone marrow-derived immortalized macrophage.