Cathepsin B Is Involved in the Trafficking of TNF- α-Containing Vesicles to the Plasma Membrane in Macrophages This information is current as Soon-Duck Ha, Andrew Martins, Khashayarsha Khazaie, of September 27, 2021. Jiahuai Han, Bosco M. C. Chan and Sung Ouk Kim J Immunol 2008; 181:690-697; ; doi: 10.4049/jimmunol.181.1.690 http://www.jimmunol.org/content/181/1/690 Downloaded from References This article cites 47 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/181/1/690.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology 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 Kim2*† 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 produc- tion 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 Downloaded from 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-con- taining vesicles. Ectopic expression of GFP-conjugated pro-TNF further suggests that pro-TNF failed to reach the plasma mem- brane 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 Journal of Immunology, 2008, 181: 690–697. http://www.jimmunol.org/ roduction of the potent inflammatory cytokine TNF-␣ in cellular Ag-1 that promotes mRNA degradation or inhibits protein response to invading microbes is a crucial step for mount- translation, respectively (6). P ing initial innate immune responses. However, uncon- TNF-␣ is encoded as a 26-kDa type II transmembrane precursor trolled production of TNF-␣ is linked to the pathogenesis of severe (pro-TNF), which is transported from the trans-Golgi network to acute and chronic inflammatory diseases (1). Thus, the biosynthe- the recycling endosome (7), then delivered to the cell surface sis and release of TNF-␣ should be tightly regulated at different where pro-TNF undergoes proteolytic cleavage by the TNF-␣-con- levels to prevent inadvertent production under normal condition verting enzyme (8–11). Membrane fusion between the TNF-␣- (1–4). Activation of macrophages by the bacterial cell wall com- containing vesicles from trans-Golgi network and the recycling by guest on September 27, 2021 ponent LPS is mediated through TLR4, resulting in the recruitment endosomes, and between the recycling endosomes and the cell of signaling adaptors such as MyD88 and TRIF (5). This recruit- surface membrane was shown to be mediated through interactions ment of signaling adaptors to TLR4 induces activation of signaling between various trans-SNARE (soluble-N-ethylmaleimide-sensi- cascades involving multiple signaling molecules, including the tive factor-attachment protein (SNAP)3 receptor) family members family of protein serine kinases IL-1R-associated kinase 1 and (4). Recent studies have shown that TNF-␣ surface delivery and kinase 4 and the adaptor molecule TNFR-associated factor 6. Sub- secretion is regulated through a process involving cholesterol-de- sequent activation of MAPKs and transcription factor NF-␬B oc- pendent lipid raft formation at the phagocytic cup of activated curs, which controls transcription of cytokines including TNF-␣ macrophages (7, 12); however, it is unknown how TNF-␣ surface (5). Additional layers of TNF-␣ production control occur at the delivery is regulated and whether it can be facilitated upon TLR level of translation. TNF-␣ mRNA contains an AU-rich element in activation. We used a functional gene identification procedure us- its 3Ј untranslated region, which determines its half-life and trans- ing retrovirus integration-generated mutagenesis (13–15) and identi- lational efficiency through binding tristetraprolin or T cell intra- fied that cathepsin B activity was required for optimal TNF-␣ trans- portation to the plasma membrane. Cathepsin B is a lysosomal cysteine protease involved in the degradation of cellular proteins in lysosomes. Unlike other cathepsins, cathepsin B functions as an en- *Department of Microbiology and Immunology, and †Infectious Diseases Research Group, Siebens-Drake Research Institute, University of Western Ontario, London, dopeptidase at neutral pH and is also found in extralysosomal sites, Ontario, Canada; ‡Department of Microbiology-Immunology, Northwestern Univer- including the cytosol, the plasma membrane, and pericellular spaces, sity, Chicago, IL 60611-3015; and §The Scripps Research Institute, La Jolla, CA where it participates in various cellular processes including cancer 92037 metastasis, inflammation, myoblast differentiation, IL-1␤ production, Received for publication February 27, 2008. Accepted for publication April 27, 2008. and cell death (16–21). In this study, we demonstrate that cathepsin B The costs of publication of this article were defrayed in part by the payment of page is involved in the posttranslational process of TNF-␣, likely in the charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. trafficking of TNF-␣-containing vesicles to the plasma membrane. 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.). 3 Abbreviations used in this paper: SNAP, soluble-N-ethylmaleimide-sensitive factor- 2 Address correspondence and reprint requests to Dr. Sung Ouk Kim, Infectious Dis- attachment protein; SNARE, SNAP receptor; siRNA, small interfering RNA; eases Research Group, Siebens-Drake Research Institute, Room 119, University of BMDIM, bone marrow-derived immortalized macrophage. Western Ontario, 1400 Western Road, London, Ontario N6G 2V4, Canada. E-mail address: [email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 691 Materials and Methods room temperature Nucleofector solution V to a final concentration of 2.0 ϫ 6 ␮ Materials and reagents 10 cells/100 l, and mixed with siRNA (Dharmacon), for human cathep- sin B (NM_001908, ON-TARGET Plus Duplex J-004266-13-0005). Cathepsin B inhibitor III, cathepsin B inhibitor IV, cathepsin K inhibitor Transfection was performed using the Nucleofector I device (Amaxa Bio- III, cathepsin G inhibitor I, and calpain inhibitor III (zVF-Cho) were pur- systems). At 48 h after nucleofection, cells were plated and stimulated with chased from Calbiochem (EMD Bioscience). Cathepsin B and cathepsin L LPS for TNF-␣ assay. Other cells were harvested for cathepsin B protein inhibitor (zFF-fmk) was purchased from Sigma-Aldrich. Abs for p38 knockdown analysis. The verification of cathepsin B knockdown was per- MAPK and cathepsin B were obtained from Cell Signaling Technology formed by Western blot analysis using anti-human cathepsin B Ab and Calbiochem, respectively. Mouse and human TNF-␣ Abs were pur- (eBioscience). chased from eBioscience. Total cell lysate preparation and immunoblotting Cell culture Total cell lysate preparation and immunoblotting procedures were per- Mouse bone marrow-derived immortalized macrophages (BMDIM) were formed as previously described (25). Briefly, cells were lysed in ice-cold prepared as previously described (22–24) from C57BL/6 mice. Bone mar- lysis buffer (20 mM MOPS, 2 mM EGTA, 5 mM EDTA, 1 mM Na3VO4, row-derived macrophages from cathepsin B gene (Ctsb)-deficient 40 mM ␤-glycerophosphate, 30 mM NaF, and 20 mM sodium pyrophos- (CtsbϪ/Ϫ) or control strain C57BL/6 (Ctsbϩ/ϩ) mice or from the human phate (pH 7.2)) containing a protease inhibitor cocktail (Roche). The cell monocytic cell line THP-1 were cultured in RPMI 1640 medium contain- lysates were incubated on ice for 10 min and centrifuged