Cooperation of Both TNF Receptors in Inducing Apoptosis: Involvement of the TNF Receptor-Associated Factor Binding Domain of the TNF Receptor 75 This information is current as of September 25, 2021. Wim Declercq, Geertrui Denecker, Walter Fiers and Peter Vandenabeele J Immunol 1998; 161:390-399; ; http://www.jimmunol.org/content/161/1/390 Downloaded from References This article cites 63 articles, 30 of which you can access for free at: http://www.jimmunol.org/content/161/1/390.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 25, 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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cooperation of Both TNF Receptors in Inducing Apoptosis: Involvement of the TNF Receptor-Associated Factor Binding Domain of the TNF Receptor 751 Wim Declercq, Geertrui Denecker, Walter Fiers, and Peter Vandenabeele2 TNF-R55 is the main receptor mediating TNF-induced cytotoxicity. However, in some cells TNF-R75 also signals cell death. In PC60 cells, the presence of both receptor types is required to induce apoptosis following either specific TNF-R55 or TNF-R75 triggering, pointing to a mechanism of receptor cooperation. In this study, we extend previous observations and show that TNF-R55 and TNF-R75 cooperation in the case of apoptosis in PC60 cells is bidirectional. We also demonstrate ligand-indepen- dent TNF-R55-mediated cooperation in TNF-R75-induced granulocyte/macrophage-CSF secretion, but not vice versa. To deter- mine which part of the intracellular TNF-R75 sequence was responsible for the observed receptor cooperation in apoptosis, we Downloaded from introduced different TNF-R75 mutant constructs in PC60 cells already expressing TNF-R55. Our data indicate that an intact TNF-R-associated factors 1 and 2 (TRAF1/TRAF2)-binding domain is required for receptor cooperation. These findings suggest a role for the TRAF complex in TNF-R cooperation in the induction of cell death in PC60 cells. Nevertheless, introduction of a dominant negative (DN) TRAF2 molecule was not able to affect receptor cooperation. Remarkably, TRAF2-DN overexpression, which was found to inhibit the TNF-dependent recruitment of endogenous wild-type TRAF2 to the TNF-R75 signaling complex, could neither block TNF-R55- or TNF-R75-induced NF-kB activation nor granulocyte/macrophage-CSF secretion. Possibly, ad- http://www.jimmunol.org/ ditional factors different from TRAF2 are involved in TNF-mediated NF-kB activation. The Journal of Immunology, 1998, 161: 390–399. umor necrosis factor is produced mainly by activated The role of TNF-R75 in cell death has long been controversial, macrophages and exerts its function through binding to and its activity was proposed to be independent of signal trans- T two different receptors, TNF-R55 and TNF-R75 (1). The duction (13–18). The ligand-passing model reconciled many seem- TNF-R superfamily, related by the presence of a conserved cys- ingly conflicting data and showed that, due to its higher affinity and teine-rich repeat in its extracellular domains, harbors several rapid dissociation kinetics, TNF-R75 could regulate the rate of by guest on September 25, 2021 death-inducing members such as TNF-R55, TNF-R75, Fas/Apo-1, TNF association with TNF-R55 (17). However, in the past few lymphotoxin-b receptor, and the recently described DR3, DR4, years it has become clear that TNF-R75-specific triggering has a and DR5 receptors (2–9). TNF-R55, Fas/Apo-1, and DR3–5 con- signaling role for apoptosis in PC60, KYM-1, HeLa, Colo205, and 3 tain a so-called death domain (DD) in their intracellular regions; mature T cells (11, 15, 19–22). this DD signals to the pathways leading to apoptosis and, in the In recent years, the signaling events leading to TNF-R-mediated k case of TNF-R55 and DR3–5, also to NF- B activation (4–6, cell death have been partially elucidated (1, 23, 24). Mainly by the 10). Although the intracellular domains of TNF-R55 and TNF- use of two-hybrid-based strategies, several intracellular TNF- R75 are completely unrelated, they share certain activities, such R55-, TNF-R75-, and Fas/Apo-1-signaling complex-constituting as induction of apoptosis and NF-kB activation (1, 11, 12). proteins were identified. These molecules belong to four novel families of signal-transducing effectors. First, the TNF-R-associ- ated factors 1 and 2 (TRAF1 and TRAF2) were cloned on the basis Laboratory of Molecular Biology, Flanders Interuniversity Institute for Biotechnology of their associating capacity with TNF-R75 (25). TRAFs associate and University of Ghent, Ghent, Belgium by means of a conserved C-terminal TRAF domain with members Received for publication August 22, 1997. Accepted for publication February of the TNF-R superfamily. A 78-residue C-terminal region of 20, 1998. TNF-R75, the TRAF-binding region, interacts with these signal The costs of publication of this article were defrayed in part by the payment of page transducers. TRAF2 mediates NF-kB activation by TNF-R55, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. TNF-R75, and CD40 (26, 27). In general, TRAF family members 1 This work was supported by the Fonds voor Geneeskundig Wetenschappelijk seem to be involved in cytokine-induced gene activation (26–30). Onderzoek, the Interuniversitaire Attractiepolen, and the Vlaams Interuniversitair In- Second, the TNF-R55- and Fas-associated DD proteins TRADD, stituut voor Biotechnologie, as well as by a European Community Biomed-2 Program Grant (No. BMH4-CT96-0300). G.D. is a research assistant and P.V. a postdoctoral FADD/MORT1, and RIP, together with the TNF-R family mem- research assistant with the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. bers TNF-R55, Fas, and the DR3–5, constitute a family of mole- 2 Address correspondence and reprint requests to Dr. P. Vandenabeele, Laboratory of cules containing a DD motif, which is involved in homotypic and Molecular Biology, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium. E-mail ad- heterotypic protein/protein interactions (7–10, 31–34). Ligand-in- dress: [email protected] duced receptor aggregation targets these DD proteins to their re- 3 Abbreviations used in this paper: DD, death domain; DN, dominant negative; PI, propidium iodide; TRAF, TNF-R-associated factor; wt, wild-type; GM-CSF, granu- spective receptors, i.e., TRADD/FADD/RIP to TNF-R55 (al- locyte/macrophage-CSF; ECL, enhanced chemiluminescence; TRADD, TNF-R1-as- though FADD recruitment to the endogenous TNF-R55 has not sociated DD protein; FADD, Fas-associating protein with DD; ICE/CED, IL-1D- converting enzyme/Caenorhabditis elegans death gene; C-IAP, cellular inhibitor of been shown yet) and FADD to Fas/Apo-1 (27, 35–37). Overex- apoptosis; RIP, receptor-interacting protein. pression of these DD molecules leads to cell death, probably by Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 391 mimicking the formation of an intracellular ligand-induced signal- Construction of mutant TNF-R75 and electroporation of PC60 ing complex. A FADD molecule lacking its N-terminal region cells functions as a dominant negative (DN) mutant able to inhibit TNF- The starting plasmid for TNF-R75 mutagenesis, containing wild-type (wt)- R55-, Fas/Apo-1-, and DR3-induced apoptosis (5, 27, 37). TNF-R75 under control of the SV40 early promoter, was previously de- TRADD and RIP, besides their presumed role in cell death, are scribed (11). TNF-R75 M1 was generated by digestion of the unique BstXI also involved in NF-kB activation by their ability to recruit restriction site, removal of the protruding 39 sticky end with T4 polymer- ase, and ligation to the XbaI linker CCTCTAGAGG, generating an in- TRAF2 (27, 35, 38). A third family of TNF-R complex-constitut- frame stop codon. Due to the cloning procedure, an additional stretch of ing proteins involves the baculoviral IAP-related inhibitors of ap- five irrelevant amino acids was added. The M3 mutant (D304–345) re- optosis c-IAP1 and c-IAP2 (39, 40). Although it is known that sulted from the removal of the sequence between two SacI restriction sites c-IAP1 and -2 participate in the TNF-R75 complex and that c- present in the cytoplasmic domain. R75 M4 (D288–340) was constructed by deleting an XmaI fragment in the intracellular receptor part. M5 (D403- IAP1 can be recruited to TNF-R55, in both cases via their affinity end) was generated by introducing an in-frame stop codon at the indicated for TRAF1 and/or TRAF2, their function in signal transduction is position by PCR, thereby deleting the sequence coding for the 37 C-ter- still unclear. A fourth class of proteins links the receptor-associ- minal amino acids. Mutant receptors were verified using dsDNA sequenc- ating death-inducing FADD molecule to ICE/CED-3-like cysteine ing. PC60 R55/8, PC60 R75/24, or PC60 R55R75/5 cells were transfected by electroporation as reported (48). Briefly, cells were washed with growth proteases (caspases) involved in the process of apoptosis.