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

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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-␬B activation nor granulocyte/macrophage-CSF secretion. Possibly, ad- http://www.jimmunol.org/ ditional factors different from TRAF2 are involved in TNF-mediated NF-␬B 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-␤ 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 ␬ 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-␬B activation (1, 11, 12). 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-␬B 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 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 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-␬B activation by their ability to recruit restriction site, removal of the protruding 3Ј 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 (⌬304–345) 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 (⌬288–340) was constructed by deleting an XmaI fragment in the intracellular receptor part. M5 (⌬403- 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. The medium (RPMI 1640) supplemented with FCS. Wild-type and mutant caspase-8 molecule binds with its N-terminal part, by means of a TNF-R expression plasmids (20 ␮g), together with 1 ␮g of a selection homologous death effector domain, to the N-terminally located plasmid pSV2Neo (G418 selection), or a murine TRAF2-DN expression plasmid pRK5TRAF2-DN (20 ␮g), a generous gift of Dr. D. V. Goeddel death effector domain of FADD (41, 42). Expression of caspase-8 Downloaded from mutants lacking enzymatic activity block both TNF-R55- and Fas/ (25), together with the selection plasmid pUTSV1 (Cayla, Toulouse, France; phleomycin selection) were added to the cells, and the mixture was Apo-1-mediated cytotoxicity (41). The mechanism by which pro- exposed to a single electropulse (1500 ␮F, 300 V). Two days later, G418 caspase-8 becomes activated after recruitment in the death-induc- (1500 ␮g; Life Technologies, Paisley, U.K.) or phleomycin (200 ␮g; ing signaling complex is still unclear (43); but once activated, Cayla) was added, and cells were cloned by limiting dilution. Cell lines caspase-8 is able to activate downstream caspases (44). derived from transfected PC60 R55/8 or PC60 R75/24 were named PC60 R55NR75 and PC60 R75NR55, respectively.

These findings indicate that members of the TNF-R family share http://www.jimmunol.org/ various intracellular signaling proteins to activate similar trans- Immunofluorescence and propidium iodide (PI) exclusion assays duction pathways. This sheds new on the possible mech- TNF-R75 expression on transfected cells was analyzed by staining for 45 anism of TNF-R55 and TNF-R75 cooperation. Several recent min with 1 ␮g utr-1/ml, a mAb against TNF-R75 (50), and subsequent reports have strongly suggested cooperation between TNF-R55 incubation with a fluorescein-conjugated goat anti-mouse Ab, followed by fluorocytometric detection with a Coulter Epics 753 (Coulter, Hialeah, FL). and TNF-R75, especially in the case of induction of cytotoxic- The number of apoptotic cells in a given culture was measured by a PI ity (11, 12, 21, 45, 46). exclusion assay as described (11). PI was added at a final concentration of We developed a thymoma system, based on the introduction of 30 ␮M, and cells were analyzed on an Epics 753 fluorometer. human TNF-R in the rat/mouse T cell hybridoma PC60, to inves- Immunoprecipitation and immunoblotting tigate the role of TNF-R55 and TNF-R75 in TNF responses. Pre- by guest on September 25, 2021 7 viously, we have shown that both receptors are signal-transducing PC60 cells (10 ) expressing different TNF-R combinations were washed twice with ice-cold PBS A and lysed (15 min) in 1 ml lysis buffer (20 mM in these cells (11, 12, 47). Remarkably, only PC60 cells expressing Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 100 ␮g/ml both of the receptor types were sensitive to TNF-induced apopto- PMSF, 0.27 trypsin-inhibitory U/ml aprotinin, 10 ␮g/ml leupeptin, and 2 sis. Furthermore, we reported that the intracellular domain of TNF- mM pefabloc) (27). After centrifugation at 14,000 ϫ g (10 min), 20 ␮gof R75 is required to mediate this receptor cooperation, thereby ex- the TNF-R75-specific Ab utr-4 (50) was added to the supernatant and left for1hat4°C, then mixed with 40 ␮l of a 1:1 slurry of protein A-Sepharose cluding a role for ligand passing (48). In the present report, we for another 1.5 h. Beads were washed twice with lysis buffer, twice with show the existence of a bidirectional TNF-R55 and TNF-R75 co- high salt (1 M) lysis buffer, and again twice with low salt lysis buffer. operation at the level of apoptosis induction and a unidirectional Proteins were eluted with Laemmli gel-loading buffer containing 4.2% ligand-independent cooperation of TNF-R55 in TNF-R75-induced 2-ME, loaded on a 10% SDS-polyacrylamide gel, and after electrophoresis electroblotted on a nitrocellulose membrane. TNF-R75 proteins were re- granulocyte/macrophage-CSF (GM-CSF) secretion. Our studies vealed using a rabbit polyclonal anti-TNF-R75 antiserum (a gift of Dr. with TNF-R75 mutants suggest a role for the TNF-R75-associated W. A. Buurman, University of Maortricht) and enhanced chemilumines- TRAF1/TRAF2 complex in the observed TNF-R55 cooperation in cence (ECL; Amersham Life Science, Amersham, U.K.). For coimmuno- apoptosis. precipitations, the parental PC60 R55R75/5 and PC60 R55R75T2DN cells (108) were washed with warm growth medium (37°C) and incubated for different time intervals in the presence or absence of TNF (1 ␮g/ml) in a 10-ml volume on a rotating wheel, then lysed with 4 ml lysis buffer (150 Materials and Methods mM NaCl) further supplemented with 30 mM NaF and 2 mM Na-pyro- Cytokines and cytokine assays phosphate. The lysate was further incubated with a combination of utr-4 Ab (50) and protein A-Trisacryl beads or with protein A-Trisacryl beads alone Purified E. coli-derived human TNF, the human TNF-R55-specific mutein and further treated as above (27). Immunoblots were developed with anti- R32WS86T, and the human TNF-R75-specific mutein D143F were pre- murine TRAF2 (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-TNF- pared in our laboratory as described (11, 48). GM-CSF was quantified, R75 antisera using ECL. using the murine GM-CSF preparation of the National Institute for Bio- logical Standards (Potters Bar, U.K.) as a reference, in an FDCp1 prolif- Results eration assay (49). TNF-R55 and TNF-R75 cooperation in PC60 cells Previously, we have shown that PC60 cells transfected with both Cells human TNF-R were sensitive to TNF-mediated apoptosis, in con- trast to cells expressing only one TNF-R type. Both TNF-R55 and The hybridoma PC60.21.14.4, provided by Dr. M. Nabholz (Swiss Institute TNF-R75 were able to elicit intermediate apoptosis levels in the of Experimental Cancer Research, Epalinges, Switzerland), was transfected with cDNAs coding for TNF-R55, TNF-R75, or both, as previously re- double transfectants when separately triggered by agonistic Abs or ported (11). The representative cell clones used for each type of transfec- receptor-specific TNF muteins (11). The percentage of PI-positive tant were PC60 R55/8, PC60 R75/24, and PC60 R55R75/5, respectively. cells was taken as a measure for apoptotic death in these cultures. 392 TNF-R75 TRAF-BINDING DOMAIN AND TNF-R COOPERATION Downloaded from

FIGURE 2. The presence of unliganded TNF-R55 is sufficient to coop- http://www.jimmunol.org/ erate with TNF-R75-induced GM-CSF secretion. PC60 R75/24 cells were transfected with a selection plasmid (PC60 R75/24 Neo) or with a selection plasmid plus a TNF-R55 expression plasmid (PC60 R75NR55). These cell lines (5 ϫ 104 cells/well) were stimulated with medium (control) or with the TNF-R75-specific TNF mutein D143F. 48 h later, supernatant was collected and the GM-CSF content was measured in an FDCp1 assay. by guest on September 25, 2021 of TNF-R55 and TNF-R75 was as cytotoxic as stimulation with wt-TNF. Specific stimulation of TNF-R75 in PC60 R75 cells in- duced consistently low, but reproducible GM-CSF levels (Ref. 47; FIGURE 1. The presence of both TNF-R types is required for TNF- Fig. 2). After introduction of TNF-R55 in these cells, treatment mediated induction of apoptosis in PC60 cells, but separate triggering of TNF-R55 and TNF-R75 is sufficient to mediate apoptosis. A and B, PC60 with the TNF-R75-specific agent D143F resulted in considerably cells expressing only one TNF-R type, i.e., PC60 R55/8 and PC60 R75/24, enhanced GM-CSF production (Fig. 2). Since single-transfected ϩ were transfected with expression plasmids containing TNF-R75 and TNF- TNF-R55 cells already produced elevated levels of GM-CSF af- R55 cDNAs, resulting in PC60 R55NR75 and PC60 R75NR55 cell lines, ter specific receptor stimulation and since the levels of induced respectively (representative clones are used). Cell lines transfected with cytokine varied between different clones, we were not able to con- only the selection plasmid were named PC60 R55/8 Neo and PC60 R75/24 clude unambiguously whether, reciprocally, the presence of TNF- 4 Neo. Cells (5 ϫ 10 /well) were cultured in the absence (control) or pres- R75 facilitated the R55 GM-CSF-inducing capacity (see below ence of saturating concentrations of TNF (100 ng/ml) or TNF-R55 and data not shown). These results confirm and extend our previ- (R32WS86T)- or TNF-R75-specific (D143F) TNF muteins (500 ng/ml). ous observations indicating cooperation by even an unliganded After 20 h, the percentage of PI-positive cells was determined by cytoflu- second receptor in PC60 cells (11). Our data show a bidirectional orometric analysis. cooperation between both TNF-R in inducing apoptosis and a clear help of the TNF-R55 presence in TNF-R75-signaling to GM-CSF To further confirm these results, we transfected TNF-R55ϩ and secretion. ϩ TNF-R75 single transfectant PC60 cells with TNF-R75 or TNF- ϩ R55 expression plasmids, respectively. Cell lines expressing both Expression of mutant TNF-R75 in PC60 R55 cells TNF-R were selected by immunostaining, and subsequent cytoflu- To further dissect the mechanism of TNF-R cooperation in PC60 orometric analysis in the case of TNF-R75, or by treating cells cells, we characterized the intracellular part of TNF-R75 respon- with the TNF-R55-specific TNF mutein R32WS86T and scoring sible for cooperation with TNF-R55. Therefore, several TNF-R75 the GM-CSF levels induced in the case of TNF-R55 (data not deletion mutants were constructed and expressed in PC60 R55ϩ shown). PC60 R55NR75 and PC60 R75NR55 cells were the new cells. Comparison of the human and mouse TNF-R75 intracellular double transfectants so obtained. When treated with TNF, both cell sequences revealed two major boxes of homology: a conserved clones responded in an apoptotic way (Fig. 1A). This response was domain containing a sequence of six consecutive serine residues in contrast to cells transfected only with the selection plasmid and the TRAF-binding region (Fig. 3A). We decided to make de- (PC60 R55/8 Neo and PC60 R75/24 Neo, respectively). Separate letion mutants in which the serine-rich region (mutants M3 clustering of receptors by the TNF muteins R32WS86T (TNF- (⌬304–345) and M4 (⌬288–340)) or the TRAF-binding domain R55) or D143F (TNF-R75) resulted in intermediate levels of ap- (M1, ⌬313-end) were removed (Fig. 3B). Rothe et al. previously optosis in R55R75ϩ cells (Fig. 1B), while simultaneous activation reported that deletion of the 37 C-terminal amino acids resulted in The Journal of Immunology 393

FIGURE 3. Expression of mutant Downloaded from TNF-R75 in PC60 R55ϩ cells. A, Com- parison of the intracellular sequences of human (h) and murine (m) TNF-R75. The TRAF-binding domain is shown in italics. Additionally, the positions of the Ser box, a putative TRAF-binding site, and the positions of the TNF-R75 mu- http://www.jimmunol.org/ tant constructs described here are indi- cated. B, Schematic representation of the TNF-R75 mutants constructed. TM, transmembrane region; EC, extracellu- lar receptor part; IC, intracellular re- ceptor part. The Ser box contains six consecutive Ser residues. C, PC60 R55/ 8-derived cell lines expressing different

TNF-R75 constructs (see B) were incu- by guest on September 25, 2021 bated with control (thin line) or TNF- R75-specific utr-1 (thick line) Abs. The second Ab used was FITC-labeled goat anti-mouse Ab. TNF-R75 expression levels were analyzed on a Coulter Epics 753 cytofluorometer. D, PC60 R55/8- derived cell lines expressing different TNF-R75 constructs (see B), were lysed and the cleared lysates were treated with TNF-R75-specific utr-4 Ab. The resulting immunoprecipitates were sub- jected to PAGE and electroblotted. The blots were developed using a polyclonal rabbit antiserum raised against TNF- R75 and ECL. 394 TNF-R75 TRAF-BINDING DOMAIN AND TNF-R COOPERATION

Deletion of the serine box-containing domain has no influence on the functionality of TNF-R75 PC60 R55NR75 M3 (⌬304–345) and PC60 R55NR75 M4 (⌬288– 340) cells expressing TNF-R75 lacking the Ser box were stimu- lated with TNF-R-specific muteins or TNF, and the levels of GM- CSF induced were quantified. From Figure 4A it was clear that all cell lines responded to TNF-R55-specific triggering (R32WS86T), thereby showing the presence of TNF-R55 on the transfected PC60 cells. D143F stimulation of the mutant TNF-R75, M3 and M4, stimulated the secretion of similar amounts of GM-CSF as activa- tion of wt-TNF-R75 in PC60 R55R75/5 cells. Simultaneous acti- vation of both TNF-R55 and TNF-R75 resulted in a synergistic production of GM-CSF in wt as well as in R75 M3- and R75 M4-coexpressing cells. This indicated that the gene-inducing ca- pacity of TNF-R75 was not affected by removal of the conserved Ser box. When these cultures were analyzed for the percentage of apoptotic cells induced by TNF-R triggering, it was clear that R75

M3 and R75 M4 were still able to cooperate with R55 to render Downloaded from PC60 cells susceptible to TNF-R-mediated cell death, as did wt- R75 (Fig. 4B). Additionally, M3 and M4 R75 mutants induced apoptosis after specific stimulation with D143F. Parental PC60 R55/8 cells and control PC60 R55/8 Neo transfectants showed no apoptotic cells after mutant TNF treatment. Hence, we concluded

that the TNF-R75 Ser box region was not involved in the process http://www.jimmunol.org/ of receptor cooperation, nor did deletion of this R75 region influ- ence its apoptosis- or gene-inducing capability.

Disruption of the TRAF-binding domain in TNF-R75 abrogates its functionality and cooperation in TNF-R55-mediated apoptosis Complete deletion of the TNF-R75 TRAF-binding domain (M1, ⌬313-end) resulted in loss of receptor functionality. TNF-R75- specific triggering of PC60 R55NR75 M1 cells was not able to by guest on September 25, 2021 induce any GM-CSF secretion neither on its own, nor in synergy with TNF-R55 stimulation (Fig. 5A). Moreover, this mutated re- FIGURE 4. Deletion of the Ser box-containing domain has no influence ceptor was no longer capable of transducing a signal required for on the functionality of TNF-R75. PC60 cells expressing TNF-R55 (PC60 receptor cooperation in inducing apoptosis by TNF-R55 (Fig. 5B). R55/8) were transfected with TNF-R75 M3 (⌬304–345), or TNF-R75 M4 Removal of the 37 C-terminal amino acids of the intracellular (⌬288–340) mutants, lacking the domain containing a conserved Ser box, TNF-R75 domain (⌬403-end), which disturbs the TRAF complex just upstream of the TRAF-binding region. Cell lines (5 ϫ 104 cells/well) binding to this receptor (25), had identical effects. PC60 R55NR75 were cultured in the absence (control) or presence of saturating concen- M5 cells stimulated with D143F did not produce GM-CSF and did trations of TNF (100 ng/ml) or TNF-R55 (R32WS86T)- or TNF-R75-spe- not show an apoptotic response even when stimulated with TNF cific (D143F) TNF muteins (500 ng/ml). A, After 48 h of incubation, su- (Fig. 5). These results led us to postulate an involvement of the pernatants of these cultures were collected and the amount of GM-CSF secreted determined in an FDCp1 assay. B, After 20 h, the percentage of TRAF-binding domain, and hence the TRAF1/TRAF2 complex, PI-positive cells was determined by cytofluorometric analysis. not only in R75-mediated GM-CSF induction but also in the co- operation that allows TNF-R55 to induce apoptosis.

Overexpression of a TRAF2-DN molecule does not inhibit ␬ an inactive receptor due to the inability to bind the TRAF1/TRAF2 receptor cooperation nor NF- B activation in PC60 R55R75 complex (25). This deletion mutant was also included in our struc- cells ture-function studies (M5, ⌬403-end). Since our results with the TNF-R75 M5 mutant pointed to a role After selection and isolation of transfected cell lines by limiting for the TRAF1/TRAF2 complex in receptor cooperation in PC60 dilution, the appropriate PC60 clones were selected on the basis of R55R75 cells, we transfected these cells with a TRAF2-DN mol- similar levels of TNF-R75 expression as the original PC60 ecule lacking its RING-finger domain. This mutant TRAF2 was R55R75/5 cells, as determined by flow fluorocytometry (Fig. 3C). previously shown to be able to inhibit TNF-R55-, TNF-R75-, and Since the levels of transfected TNF-R55 in PC60 cells are ex- CD40-mediated NF-␬B activation after transient overexpression tremely low (11), TNF-R55 presence could be evaluated only on (26, 27). The function of TRAF1 is still unresolved. PC60 the basis of functionality, viz the induction of GM-CSF by R55- R55R75T2DN clones expressing high levels of the TRAF2-DN specific stimulation (data not shown, see further). To demonstrate protein were selected (Fig. 6A). Functional analysis of TRAF2- that the transfected cell lines expressed the mutant TNF-R75 gene DN-expressing PC60 R55R75 cells, however, revealed that there products, the receptors were immunoprecipitated from the respec- was no influence on the induction of apoptosis by TNF-R55 and/or tive cell lines using the mAb utr-4 (50), then further analyzed by TNF-R75 (Fig. 6B). PC60 R55R75/5 and PC60 R55R75T2DN immunoblotting (Fig. 3D). cells were both equally sensitive to different concentrations of TNF The Journal of Immunology 395

cells. In PC60 R55R75/5 cells, we could show a TNF-dependent recruitment of endogenous wt TRAF2 (Fig. 6D). This recruitment seemed to be an early event in signal transduction, since TRAF2 could already be detected in the TNF-R75 immunoprecipitates af- ter 1 min of TNF treatment. A maximum TRAF2 recruitment was observed after 15 min of TNF treatment. In contrast, PC60 R55R75T2DN cells showed no enodogenous wt TRAF2 recruit- ment to TNF-R75 upon TNF triggering. Instead, the overexpressed TRAF2-DN molecule was recruited to TNF-R75 in a TNF-depen- dent way. We could even detect a small amount of TRAF-2DN in untreated cells, probably due to an excess of overexpressed TRAF2-DN molecules, which are recruited with low efficiency to TNF-R75 dimerized by means of the immunoprecipitating utr-4 mAb. Immunoprecipitations with beads alone did not reveal TRAF2 or TRAF2-DN molecules. Taken together, these observa- tions show that the level of TRAF2-DN expression in PC60 R55R75T2DN cells was sufficient to completely inhibit the recruit- ment of functional endogenous TRAF2 to the TNF-R75 signaling Downloaded from complex, in this way blocking TRAF2-dependent signaling path- ways. Therefore, it is conceivable that PC60 R55R75 cells might use other receptor-associated molecules leading to NF-␬B activa- tion. Considering the clear association of TRAF2-DN with TNF- R75 in PC60 R55R75T2DN cells, we conclude that in PC60 cells no known TRAF2-mediated activities are implicated in receptor cooperation leading to apoptosis and activation of NF-␬B. http://www.jimmunol.org/

Discussion We have previously shown a role for unliganded TNF-R cooper- ation in PC60 cells transfected with TNF-R55 and/or TNF-R75 (11). In these cells, the presence of both receptor types was an absolute requirement for sensitivity to specific TNF-R55- or TNF-

R75-mediated apoptosis. Further studies indicated that introduc- by guest on September 25, 2021 tion of only the intracellular domain of TNF-R75 in TNF-R55- expressing cells was sufficient to render these cells susceptible to FIGURE 5. Disruption of the TRAF-binding domain in TNF-R75 ab- TNF-R55-induced cell death (48). To further dissect the mecha- rogates its functionality and cooperation in TNF-R55-mediated apoptosis. nism of receptor cooperation in apoptosis and to identify the in- PC60 cells expressing TNF-R55 (PC60 R55/8) were transfected with TNF- tracellular domains involved, we constructed several TNF-R75 de- R75 mutants: PC60 R55NR75 M1 cells expressed a TNF-R75, which letion mutants and introduced them into PC60 R55/8 cells, already lacked the complete TRAF1/TRAF2-binding domain (⌬313-end), while expressing TNF-R55. A similar approach for TNF-R55 was ham- PC60 R55NR75 M5 cells expressed a TNF-R75 mutant of which the 37 pered by the very low expression levels of TNF-R55 in PC60 cells ϫ 4 C-terminal amino acids were deleted. Cell lines (5 10 cells/well) were (Ͻ50 receptors/cell (11)). This is probably due to regulatory mech- cultured in the absence (control) or presence of saturating concentrations of anisms that keep the number of TNF-R55 molecules per cell very TNF (100 ng/ml) or TNF-R55 (R32WS86T)- or TNF-R75-specific (D143F) TNF muteins (500 ng/ml). A, After 48 h of incubation, superna- low in PC60 cells, since elevated TNF-R55 expression levels may tants of these cultures were collected, and the amount of secreted GM-CSF cause self-association through their DDs and subsequent activation was determined in an FDCp1 assay. B, After 20 h, the percentage of PI- of the death pathway (52). By transfecting the missing wt-TNF-R positive cells was determined by cytofluorometric analysis. in PC60 R55 and PC60 R75 cells and thus making the cells sen- sitive to TNF killing, we could prove that PC60 R55/8 and PC60 R75/24 cells were not just cell lines coincidentally blocked in their (data not shown). Surprisingly, we were also unable to show any apoptotic capacities. The many different PC60 R55/8 Neo and inhibitory effect on the stimulation of GM-CSF secretion or NF-␬B PC60 R75/24 Neo clones that were tested never showed apoptotic activation (Fig. 6C and data not shown), although a strong involve- behavior after TNF treatment. Unliganded TNF-R55 not only co- ment of NF-␬B in GM-CSF promoter activity has been reported operated with TNF-R75 in the process of apoptosis, but the levels (51). This lack of effect was not because we had for some reason of TNF-R75-induced GM-CSF secretion in PC60 R75NR55 cells been working with an inappropriate TRAF2-DN molecule, since were also strongly enhanced as compared with the consistently DNA sequence analysis revealed the correct sequence (data not low, induced GM-CSF levels in PC60 R75 cell lines expressing shown) and transient overexpression in HEK293 cells could block only human TNF-R75. Nevertheless, TNF-R75-induced NF-␬B TNF-R55-mediated activation of NF-␬B determined by a lucif- activation, as measured by gel shift assay, was similar in PC60 erase reporter gene construct (data not shown), as reported cells carrying one or both TNF-R types (G.D., unpublished obser- previously (27). vation). The molecular basis for this cooperation is still unknown. To further investigate the reason for the lack of inhibition of Most cases of receptor cooperation are a result of simultaneous NF-␬B activation by TRAF2-DN overexpression, immunopre- receptor triggering, although the observation of unliganded recep- cipitations of TNF-R75 were conducted to examine whether tor cooperation is not restricted to PC60 R55R75 cells. Indeed, it TRAF2-DN indeed interacted with this receptor in PC60 R55R75 was reported that selective loss of TNF-R75 expression can result 396 TNF-R75 TRAF-BINDING DOMAIN AND TNF-R COOPERATION

FIGURE 6. Overexpression of a TRAF2-DN Downloaded from molecule inhibits neither receptor cooperation in ap- optosis nor TNF-R55- or TNF-R75-induced GM- CSF secretion in PC60 R55R75 cells. A, PC60 R55R75/5 cells were transfected with the pRK- TRAF2-DN plasmid (24) together with a selection plasmid pUTSV-1 coding for the phleomycin (Phl) http://www.jimmunol.org/ resistance gene. Cells were lysed, and 200 ␮g of pro- tein was subjected to SDS-PAGE and electroblotted. Immunoblots were revealed with a rabbit polyclonal anti-mouse TRAF2 serum and ECL. B, Control cell lines (PC60 R55R75/5 and PC60 R55R75 Phl) and cells expressing the TRAF2-DN molecule (PC60 R55R75T2DN) were cultured in the presence of me- dium (control), of saturating concentrations of TNF (100 ng/ml), or of TNF-R55 (R32WS86T)- or TNF-

R75-specific (D143F) TNF muteins (500 ng/ml). Af- by guest on September 25, 2021 ter 20 h of incubation, the percentage of PI-positive cells was determined by cytofluorometric analysis. C, Same as in B, but after 48 h of incubation, super- natants of these cultures were collected and the amount of secreted GM-CSF was determined in an FDCp1 assay. D, Parental PC60 R55R75/5 and PC60 R55R75T2DN cells (108) were washed with growth medium and incubated for different time intervals at 37°C in the presence or absence of TNF (1 ␮g/ml), then subsequently were lysed. The soluble fraction of the extracts was immunoprecipitated with 20 ␮gofa nonagonistic utr-4 Ab specific for the TNF-R75 ex- tracellular domain. Immunoblots were developed with anti-mouse TRAF2 using ECL. The Journal of Immunology 397 in resistance to TNF-mediated cytotoxicity in KYM-1-derived cell rapid ligand-dependent association of TRAF2 with TNF-R75 lines (45). In this study, a role for ligand passing was excluded by within 1 min following TNF stimulation is also seen in the case of making use of TNF-R-specific Abs. FADD or TRAF3 targeting to the trimerized Fas receptor or Our structure/function studies with TNF-R75 deletion mutants LT␤-R, respectively (36, 57). The low level of TNF-independent focused on two conserved regions in the intracellular part of the TRAF2-DN recruitment to TNF-R75 in PC60 R55R75T2DN cells receptor, namely a conserved region upstream of the TRAF com- is probably caused by the bivalent immunoprecipitating Ab and plex-binding domain containing a Ser box of six consecutive indicates that TRAF2 also can bind to dimerized TNF-R75. This serine residues, and the TRAF1/TRAF2-binding domain itself. No phenomenon is also observed in experiments showing ligand-de- role for the Ser box region in receptor cooperation was found. pendent TRAF3 association with the LT␤-R (57). However, en- These data are consistent with the earlier observation that this re- dogenous TRAF2 recruitment in PC60 R55R75/5 control immu- gion is not involved in TNF-R75-mediated NF-␬B activation (25). noprecipitations could not be observed, even after overexposition Disruption of the TNF-R75 TRAF complex-binding domain (ami- of the ECL Western blot. Previously, TRAF2 was postulated as an no acids 346–423) resulted in loss of receptor functionality. Com- essential molecule in TNF-R-mediated NF-␬B activation by means plete deletion of this region (PC60 R55NR75 M1 cells, ⌬313-end) of transient overexpression experiments (26, 27). Recently, reports or removal of the TNF-R75 37 C-terminal amino acids (PC60 on the basis of TRAF2-DN transgene and TRAF2Ϫ/Ϫ mice indi- R55NR75 M5) abrogated TNF-R75-mediated induction of GM- cated the existence of TRAF2-independent pathways leading to the CSF and apoptosis, in addition to TNF-R55-induced apoptosis. activation of NF-␬B (59, 60). Our results confirm these observa- Rothe et al. (25) showed that this TNF-R75 mutant could no longer tions. Hence, TNF-R complex-associated proteins that overrule the Downloaded from signal for proliferation and NF-␬B activation in CT6 T cells, pre- need for TRAF2 in NF-␬B activation remain to be identified. RIP, sumably due to the absence of TRAF1/TRAF2 binding. This TNF- a signaling molecule that interacts with TRADD and TRAF2 (35), R75 M5 mutation deletes part of a putative TRAF interaction site seems to be a good candidate to fulfill this function, since a RIP- (53). Hence, our results with PC60 R55NR75 M1 and R55NR75 deficient Jurkat cell line is defective in TNF-induced NF-␬B acti- M5 cells strongly point to a role for the TRAF1/TRAF2 complex vation (38). This model can be true for TNF-R55-induced NF-␬B in apoptosis, both by induction of apoptosis by TNF-R75 itself and activation, since TRADD can recruit RIP to this signaling com- by receptor cooperation in TNF-R55. The function of TRAF1 is plex. On the other hand, we were unable to show RIP association http://www.jimmunol.org/ still unclear, while TRAF2, a molecule involved in both TNF-R55 to the TNF-R75 by its affinity for TRAF1 and/or TRAF2 in over- and TNF-R75 signal transduction (26, 27), might be a likely can- expression experiments (W.D., unpublished observation). Alterna- didate for the molecule involved in receptor cooperation in PC60 tively, TNF may activate sphingomyelinases, leading to ceramide R55R75 cells. Furthermore, recent data suggest a link between generation and subsequent NF-␬B activation (61), although acidic TRAF molecules and death signaling. First, CD40, which binds sphingomyelinases do not seem to be involved in TNF-induced directly to TRAF2, TRAF3, and TRAF5 (26, 28, 29, 54), but pre- NF-␬B activation (62). In addition, stable overexpression of mu- sumably not to the DD-containing proteins TRADD, FADD, or tant TRAF family members TRAF3 and TRAF5 block CD40-me-

RIP (29), is also capable of inducing apoptosis in transformed cells diated induction of CD23 expression (28, 29), although experi- by guest on September 25, 2021 (26, 28, 29, 54, 55). Second, signals mediated by CD30 in com- ments with TRAF3Ϫ/Ϫcells could not confirm a role for TRAF3 in bination with signals transduced by the TCR induce Fas-indepen- CD40 signaling (63). dent cell death in T cell hybridomas (56). Deletion analysis shows The observation that both TNF-R55 and TNF-R75 share the that the 66 C-terminal amino acids of the CD30 cytoplasmic do- same intracellular signaling molecules brings us back to the ques- main, necessary for binding TRAF1 and TRAF2, are required to tion of why the presence of both TNF-R types is required for induce cell death. Third, overexpression of an N-terminal trun- induction of apoptosis in PC60 cells. A possible explanation could cated TRAF3 molecule abrogated LT␤-R-induced cell death, but be the formation of heteromeric receptor complexes, which need to not activation of NF-␬B, in HT29 cells (57). Fourth, transient contain both TNF-R55 and TNF-R75 to be fully active. Since we transfection of a TRAF2-DN mutant in HeLa cells renders these were able to induce apoptosis in PC60 R55R75/5 cells making use cells more susceptible to TNF-mediated apoptosis, pointing to a of TNF-R-specific muteins or TNF-R-specific Abs (11), such a protective role of TRAF2 in apoptosis (58). Fifth, the TNF-R75 receptor complex could not be formed by simultaneous interaction TRAF2 association site seems to be required for TNF-R75-depen- of both TNF-R with the same ligand molecule. Possibly, mixed dent enhancement of TNF-R55-mediated cytotoxicity in HeLa TNF-R complex formation might be due to bridging between as- cells (46). sociated molecules. However, ligand-induced conformational To test the possible involvement of TRAF2 in receptor cooper- changes, enabling TNF-Rs to interact with each other, cannot be ation in PC60 R55R75 cells, we transfected this cell line stably excluded. Alternatively, a low level of spontaneous homomeric with an expression vector coding for a murine TRAF2(87–501) TNF-R aggregation resulting in low level signal transduction suf- mutant, which having lost its biologic functions acts as a DN mol- ficient for receptor cooperation in PC60 cells is another possible ecule. This mutant is still capable of binding the TRAF domain explanation. This synergizing process would be abrogated if one of of R75, while it blocks NF-␬B, JNK, and p38 MAPK activation the receptors was nonfunctional. We should stress that the neces- (25, 26, 58). Although a clear inhibition of the TNF-dependent sity for both TNF-R types to allow TNF-R75-induced apoptosis recruitment of functional, endogenous wt TRAF2 to the TNF-R75 seems not to be a general mechanism, since TNF-R75 has been signaling complex by overexpression of a TRAF2-DN mutant shown able to induce apoptosis in a subset of T cells in TNF- could be shown, PC60 R55R75T2DN cells were neither impaired R55Ϫ/Ϫ mice (22). However, a similar mechanism of receptor co- in TNF-R75-mediated apoptosis nor in cooperation involved in operation probably exists in HeLa cells (46). TNF-R55-dependent apoptosis. The observation that PC60 In summary, our data indicate the existence of bidirectional R55R75T2DN cells were as sensitive to TNF as PC60 R55R75/5 TNF-R55 and TNF-R75 cooperation in PC60 cells in the case of cells indicated that TRAF2 was not involved in providing anti- apoptosis. We were also able to demonstrate ligand-independent apoptotic signals in these cells. The kinetics of TRAF2 recruitment TNF-R55-mediated cooperation in TNF-R75-induced GM-CSF to the TNF-R75 were comparable with these of TRADD recruit- secretion, but not the reverse. Mutational analysis of the TNF-R75 ment to the TNF-R55 signaling complex (27). Additionally, the intracellular domain revealed the involvement of the TRAF1/ 398 TNF-R75 TRAF-BINDING DOMAIN AND TNF-R COOPERATION

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