TWEAK Inhibits TRAF2-Mediated CD40 Signaling by Destabilization of CD40 Signaling Complexes This information is current as Steffen Salzmann, Isabell Lang, Alevtina Rosenthal, of October 1, 2021. Viktoria Schäfer, Daniela Weisenberger, José Antonio Carmona Arana, Johannes Trebing, Daniela Siegmund, Manfred Neumann and Harald Wajant J Immunol 2013; 191:2308-2318; Prepublished online 5 August 2013; doi: 10.4049/jimmunol.1202899 Downloaded from http://www.jimmunol.org/content/191/5/2308 Supplementary http://www.jimmunol.org/content/suppl/2013/08/06/jimmunol.120289 http://www.jimmunol.org/ Material 9.DC1 References This article cites 56 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/191/5/2308.full#ref-list-1 Why The JI? Submit online. by guest on October 1, 2021 • 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 *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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology TWEAK Inhibits TRAF2-Mediated CD40 Signaling by Destabilization of CD40 Signaling Complexes Steffen Salzmann, Isabell Lang, Alevtina Rosenthal, Viktoria Scha¨fer, Daniela Weisenberger, Jose´ Antonio Carmona Arana, Johannes Trebing, Daniela Siegmund, Manfred Neumann,1 and Harald Wajant1 We found recently that TNF-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor–inducible-14 (Fn14) by virtue of their strong capability to reduce the freely available cytoplasmic pool of TNFR-associated factor (TRAF)2 and cellular inhibitors of apoptosis (cIAPs) antagonize the functions of these molecules in TNFR1 signaling, resulting in sensitization for apoptosis and inhibition of classical NF-kB signaling. In this study, we demonstrate that priming of cells with TWEAK also interferes with activation of the classical NF-kB pathway by CD40. Likewise, there was strong inhibition of CD40 ligand (CD40L)–induced activation of MAPKs in TWEAK-primed cells. FACS analysis and CD40L binding studies revealed unchanged Downloaded from CD40 expression and normal CD40L–CD40 interaction in TWEAK-primed cells. CD40L immunoprecipitates, however, showed severely reduced amounts of CD40 and CD40-associated proteins, indicating impaired formation or reduced stability of CD40L– CD40 signaling complexes. The previously described inhibitory effect of TWEAK on TNFR1 signaling has been traced back to reduced activity of the TNFR1-associated TRAF2–cIAP1/2 ubiquitinase complex and did not affect the stability of the immuno- precipitable TNFR1 receptor complex. Thus, the inhibitory effect of TWEAK on CD40 signaling must be based at least partly on other mechanisms. In line with this, signaling by the CD40-related TRAF2-interacting receptor TNFR2 was also attenuated but http://www.jimmunol.org/ still immunoprecipitable in TWEAK-primed cells. Collectively, we show that Fn14 activation by soluble TWEAK impairs CD40L–CD40 signaling complex formation and inhibits CD40 signaling and thus identify the Fn14-TWEAK system as a potential novel regulator of CD40-related cellular functions. The Journal of Immunology, 2013, 191: 2308–2318. umor necrosis factor–like weak inducer of apoptosis intervention, or tumor development (1, 2). Indeed, Fn14 has origi- (TWEAK) is a typical representative of the TNF ligand nally been identified as a growth factor–inducible protein (3). T family and as such it is initially expressed as a type 2 Although expression of TWEAK has been demonstrated at the mRNA level in a variety of tissue and cell lines, relatively little transmembrane protein (1, 2). Membrane-bound TWEAK (mem- by guest on October 1, 2021 TWEAK) can be cleaved by furin proteases in the stalk region is known about TWEAK expression at the protein level. The full- separating the transmembrane domain from the conserved extra- length membrane-bound form of TWEAK has been detected only cellular C-terminal TNF homology domain, resulting in the re- in IFN-g–treated monocytes, macrophages, dendritic cells, and a lease of soluble TWEAK. The only known unquestionable verified few breast and hepatocellular cancer cell lines (1, 2, 4, 5). It has signaling-competent receptor of TWEAK is fibroblast growth also been recognized that membrane-bound TWEAK is efficiently factor–inducible-14 (Fn14), an unusual small member of the TNF processed by furin proteases, but production of soluble TWEAK receptor family (1, 2). Fn14 is highly expressed on most tumor cell has been barely addressed in vitro in quantitative and cell type– lines but rarely on leukemia or lymphoma cell lines. Immuno- related terms. Thus, it is currently unclear whether the lack of histological studies further suggest that in vivo Fn14 expression is detectable expression of membrane TWEAK in cells positive low in healthy homeostatic tissue but induced in scenarios asso- for TWEAK mRNA is related to highly efficient furin-mediated ciated with tissue damage such as ischemia, cachexia, surgical processing of memTWEAK to soluble TWEAK or to inadequate translation of TWEAK mRNA. Similar to Fn14, immunohistology data indicate that TWEAK protein expression is upregulated in Division of Molecular Internal Medicine, Department of Internal Medicine II, Uni- versity Hospital Wu¨rzburg, 97070 Wu¨rzburg, Germany damaged tissue and development, but the corresponding studies 1M.N. and H.W. are cosenior authors. give typically no information about the cellular source of TWEAK Received for publication October 17, 2012. Accepted for publication June 5, 2013. and its degree of processing. Nevertheless, it appears that the TWEAK-Fn14 system is active in processes related to growth and This work was supported by Deutsche Forschungsgemeinschaft Grants Wa 1025/19-2 and Wa 1025/21-1 and by Deutsche Krebshilfe Grant 109922. remodeling of tissue and organs during development and tissue Address correspondence and reprint requests to Prof. Harald Wajant, Division of repair. In accordance with this, animal studies implicated the Molecular Internal Medicine, Department of Internal Medicine II, University Hospi- TWEAK-Fn14 system in liver progenitor cell proliferation (6, 7), tal Wu¨rzburg, Ro¨ntgenring 11, 97070 Wu¨rzburg, Germany. E-mail address: harald. regulation of muscle development and muscle regeneration (8–11), [email protected] tumor-associated angiogenesis (12), and in various inflammation- The online version of this article contains supplemental material. related pathologies including graft-versus-host disease, systemic Abbreviations used in this article: CD40L, CD40 ligand; cIAP, cellular inhibitor of apoptosis; Fn14, fibroblast growth factor–inducible-14; GpL, Gaussia princeps lu- lupus erythematosus–related nephritis (13), 2,4,6-trinitrobenzene ciferase; IKK, IkB kinase; memTWEAK, membrane-bound TNF-like weak inducer sulfonic acid-induced colitis (14), renal and cerebral ischemia (15– of apoptosis; NIK, NF-kB–inducing kinase; TRAF, TNFR-associated factor; 18), and collagen-induced arthritis (19, 20). TWEAK, TNF-like weak inducer of apoptosis. Similar to most other TNF receptors, activation of Fn14 typically Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 results in stimulation of the classical and/or alternative NF-kB www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202899 The Journal of Immunology 2309 pathway. Activation of various MAPKs, the PI3K/Akt pathway, site preceding the Flag epitope in pCR3-GpL-Flag-CD40L. Fc-Flag-scTNF and of apoptotic and necrotic cell death programs has also been (143N/145R) was generated by fusing the recently described single chain– reported (1, 2). In several examples of Fn14-mediated cell death, encoded TNFR2-specific TNF variant Flag-scTNF(143/145R) (35) C- terminally to an expression cassette for the Fc region of human IgG. the cytotoxic effect has been traced back to the induction of TNF The NF-kB GpL reporter plasmid pNF-kB-GpL-reporter was obtained and subsequent activation of TNFR1, which in turn is able to by exchange of the Metridia longa luciferase gene in the commercially trigger Fas-associated death domain protein– and caspase-8– available NF-kB reporter plasmid pNF-kB-MetLuc2-reporter (Clontech) mediated apoptosis or receptor interacting protein 1–mediated nec- by a GpL containing amplicon generated with GpL-specific primers having an Age1 and Not1 site in their overhang and using a synthetic gene encoding roptosis (21–23). However, in some studies there is also evidence GpL as a template. for a TNF-independent mode of TWEAK/Fn14-mediated apo- ptosis, although no data are yet available concerning the under- Western blotting lying molecular mechanisms (21). Indeed, in general only little is Cells were directly lysed in Laemmli sample buffer and incubated for 5– known with respect to the role that the various Fn14-asscociated 10 min at 96˚C. SDS-PAGE and Western blot analyses were performed by signaling pathways play in the