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Apoptotic crosstalk of TNF receptors: TNF-R2-induces depletion of TRAF2 and IAP proteins and accelerates TNF-R1-dependent activation of caspase-8

Mariola Fotin-Mleczek^1,*, Frank Henkler^1,*, Dierk Samel¹, Monica Reichwein¹, Angelika Hausser¹, Ingela Parmryd², Peter Scheurich¹, Johannes A. Schmid³ and Harald Wajant^1,

¹ Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
² National Institute for Medical Research, Division of Membrane Biology, The Ridgeway, Mill Hill, London NW7 1AA, UK
³ Institut für Gefäßbiologie und Thromboseforschung, University of Vienna, 1235 Vienna, Austria
^* These authors contributed equally to this work

View larger version (41K): [in a new window]   Fig. 1. Enhancement of TNF-R1-induced cell death by costimulation of TNF-R2, CD40 or CD30. (A) HeLa transfectants stably expressing TNF-R2, CD40 or CD30 were analyzed for expression of these receptors by FACS analysis with receptor-specific monoclonal antibodies. (B,C) HeLa-TNF-R2, -CD40 and -CD30 cells were grown in 96-well microtiter plates (15x103 cells/well) and cultured overnight at 37°C. The next day the cells were treated overnight in triplicates with TNF (B), crosslinked FasL (C) or crosslinked TRAIL (D) in the presence of CHX (2.5 µg/ml) with ([UNK]) or without () costimulation of the respective TRAF2-interacting receptor. TNF-R2 was triggered with a polyclonal TNF-R2-specific IgG preparation (2 µg/ml), CD30 was stimulated with the agonistic mAb Ki-1 (3 µg/ml) and CD40 was activated with soluble Flag-tagged CD40L (100 ng/ml) crosslinked with the anti-Flag mAb M2 (1 µg/ml). Finally, viable cells were quantified by crystal violet staining.

View larger version (31K): [in a new window]   Fig. 2. Enhancement of TNF-R1-induced cell death by costimulation of TNF-R2 occurs in various cell lines. (A) Jurkat cells stably transfected with TNF-R2 were seeded in 96-well microtiter plates at a density of 50x103 cells/well and were treated overnight in triplicates with TNF, crosslinked FasL or crosslinked TRAIL with ([UNK]) or without () costimulation of TNF-R2. (B) Colo205 cells were seeded in 96-well microtiter plates at a density of 10x103 cells/well and cultured overnight at 37°C. The next day the cells were treated for 48 hours in triplicates with TNF or crosslinked TRAIL with ([UNK]) or without () costimulation of TNF-R2. (C) Kym1 cells were seeded in 96-well microtiter plates at a density of 15x103 cells/well and cultured overnight at 37°C. The next day the cells were treated overnight in triplicates with the TNF-R1-specific agonistic mAb Htr1 with () or without () costimulation of TNF-R2. As stimulation of TNF-R2 results in Kym1 cells in significant induction of endogenous TNF (Grell et al., 1999), the action of endogenous TNF was suppressed by addition of TNF-R1-Fc (10 µg/ml) and TNF-R1 triggering was aquired by the agonistic mAb Htr1c. Viable cells were quantified in all experiments by the MTT method.

View larger version (33K): [in a new window]   Fig. 3. Receptor-induced depletion of cytoplasmic TRAF2. (A) Cell lysate (200 µl) of untreated HeLa-TNF-R2 cells were separated by size exclusion chromatography on a HR10/30 Superdex 200 column and the fractions (0.5 ml) were analyzed by immunoblotting with a polyclonal TRAF2-specifc rabbit antiserum. Elution volumes of molecular mass standards are indicated above. (B,C) HeLa-TNF-R2 cells (B) and HeLa cells expressing a deletion mutant of TNF-R2 lacking the entire cytoplasmic domain (C) were stimulated for 6 hours with an agonistic TNF-R2-specific rabbit IgG fraction or remained untreated. Cell lysates (200 µl) derived from these groups were separated by size exclusion chromatography and corresponding fractions of stimulated (+) and unstimulated (-) cells were compared with respect to TRAF2 recovery by immunoblotting. (D,E) HeLa-CD40 (D) and HeLa-CD30 (E) cells were stimulated for 6 hours with crosslinked soluble CD40L (100 ng/ml) and the CD30-specific mAb Ki-1 (3 µg/ml), respectively, or remained untreated. The lysates were then analyzed as described in B and C. (F) Lysates from HeLa-TNF-R2 cells stimulated for the indicated times with TNF-R2-specific IgG (2 µg/ml) were separated by gel filtration and the TRAF2 content of corresponding fractions were compared by immunoblotting. (G) HeLa-TNF-R2 cells were stimulated for the indicated times with TNF-R2-specific IgG (2 µg/ml). Total cellular content, soluble proteins and the detergent-insoluble compartment (DIC) fractions were prepared as described in Materials and Methods and analyzed with respect to TRAF2 by western blotting. (H) To thoroughly quantify the loss in the total TRAF2 content observed in receptor-stimulated cells after 18 hours, six independent experiments were analyzed. Cells were stimulated for 0, 3 and 18 hours, boiled in SDS-PAGE sample buffer and analyzed by western blotting.

View larger version (36K): [in a new window]   Fig. 4. Effect of pre- and costimulation of TNF-R2 on TNF-R1-mediated activation of NF-B and TNF-R1-induced cell death. (A-D) HeLa-TNF-R2 cells were grown in 96-well microtiter plates (15x103 cells/well), cultured overnight at 37°C and were transiently transfected the next day with a plasmid mix containing 3xNF-B-luciferase reporter plasmid (20 ng/well), SV40 promoter-driven ß-galactosidase expression plasmid (10 ng/well) and empty vector (pCR3, 120 ng/well). 6 hours after transfection some cells (A,C) were stimulated overnight with 2 µg/ml anti-TNF-R2 IgG (filled bars) to allow next day analysis of TNF-R1-dependent NF-B activation in a situation where TRAF2 had already been depleted. For activation of TNF-R1, cells were challenged in triplicates with the indicated concentration of TNF (A) or the TNF-R1-specific agonistic mAb Htr1 (C). To analyze NF-B activation under costimulatory conditions (B,D), cells were stimulated with anti-TNF-R2 IgG (filled bars) and either TNF (B) or Htr1 (D). In all experiments, cells subjected to costimulation were compared with cells stimulated only with the respective concentration of TNF or Htr1 (open bars). After TNF-R1 stimulation for 6 hours, cells were lysed and assayed for luciferase and galactosidase activity. Finally, luciferase activities were normalized according to the respective galctosidase activities. (E) Effect of temporal order of TNF-R1 and TNF-R2 stimulation with respect to NF-B activation and apoptosis induction. TNF-R2 was stimulated for the indicated times before or after triggering of TNF-R1. For determination of NF-B activation ([UNK]), cells were transfected as described above and assayed for luciferase and galactosidase activities 6 hours after TNF-R1 triggering (10 ng/ml TNF). For determination of cell viability (), TNF-R1 was triggered overnight with 0.1 ng/ml TNF in the presence of CHX (2.5 µg/ml) (i.e. at a concentration of TNF that induces no cell death in the absence of TNF-R2 costimulation). The effect of TNF/anti-TNF-R2 IgG costimulation was normalized to the effect of TNF alone. (F) HeLa-TNF-R2 cells were seeded in 96-well microtiter plates at a density of 15x103 cell/well and cultured overnight at 37°C in the absence () or presence ([UNK]) of anti-TNF-R2 IgG. The next day the cells were challenged with TNF (,) or TNF and anti-TNF-R2 IgG ([UNK]) in the presence of CHX (2.5 µg/ml). After overnight incubation, viable cells were quantified by crystal violet staining.

View larger version (41K): [in a new window]   Fig. 5. Kinetics of TNF and FasL-induced caspase-8 activation. (A) HeLa-TNF-R2 cells were pretreated with CHX (2.5 µg/ml) for 3 hours. Cells were then stimulated for the indicated times with crosslinked FasL (100 ng/ml), TNF (10 ng/ml) alone or in combination with anti-TNF-R2 IgG (2 µg/ml). In an additional group, cells were stimulated with anti-TNF-R2 IgG for 6 hours before TNF treatment. Cell lysates were prepared and procaspase-8 and -3 processing was analyzed by immunoblotting. (B) Cells were treated as described above with crosslinked FasL (), TNF () or TNF and anti-TNF-R2 IgG (costimulation: ; prestimulation: ). After the indicated times, the cells were lysed and analyzed with respect to caspase-8 and caspase-3 activity with fluorogenic substrates. (C) HeLa cells pretreated with CHX (2.5 µg/ml) for 3 hours (left panel) and Kym1 and SKW cells, respectively, (right panel) were challenged with TNF (10 ng/ml) or crosslinked FasL (100 ng/ml). After the indicated times the pan caspase-inhibitor z-VAD-fmk (20 µM) was added and after overnight incubation cell viability was determined by crystal violet staining (HeLa, Kym1) or by the MTT method (SKW).

View larger version (51K): [in a new window]   Fig. 6. (A) HeLa cells were transiently transfected with TNF-R1-YFP and TRADD-CFP. To inhibit the induction of TRADD-dependent apoptosis, cells were treated with z-VAD-fmk (20 µM) immediately after transfection. One day later, transfected cells were analyzed by confocal microscopy. (B) Expression plasmids (120 ng/well) encoding the indicated proteins were transiently transfected into 293 cells along with a 3xNF-B-luciferase reporter plasmid (20 ng/well) and a SV40 promoter-driven ß-galactosidase expression plasmid (10 ng/well). The next day cells were analyzed for luciferase and galactosidase activity. Luciferase activities were normalized according to the respective galctosidase activities. In all transfections z-VAD-fmk (20 µM) was added to block apoptosis. (C) The indicated GFP-tagged variants of TRAF2 were cotransfected along with empty vector or TRADD and cultured overnight in the presence of z-VAD-fmk (20 µM). The next day, the subcellular distribution of the various GFP fusion proteins was analyzed by confocal microscopy. (D-F) The indicated combinations of expression plasmids of TRADD, TRAF2, IKK1-GFP, cIAP2-GFP and cIAP1-GFP were cotransfected and cultured overnight in the presence of z-VAD-fmk (20 µM). The following day, the subcellular distribution of the various GFP fusion proteins was analyzed by confocal microscopy.

View larger version (58K): [in a new window]   Fig. 7. Ligand-induced alterations of TRAF-2 localization in living cells. (A,D,E) TNF-R2 signaling was induced in living Hela-TNF-R2 cells using an agonistic TNF-R2-specific antiserum (A). The distribution of TRAF2-YFP was analyzed online both prior to stimulation and for a further 30 minutes after stimulation by confocal microscopy. TRAF2 accumulated in small granular structures in response to TNF-R2 activation, examples of which are indicated with arrows. In contrast, a normal rabbit control serum (B) or the CD30-specific Ki-1 antibody (C) had no effect on the distribution of TRAF2. (D) HeLa-TNF-R2 cells were transfected with a TRAF2-GFP expression plasmid along with pECFP-Mem encoding a fusion protein consisting of the N-terminal 20 amino acids of neuromodulin and CFP. The next day, nonstimulated or TNF-R2-stimulated (1 hour, 2 µg/ml TNF-R2 IgG) living cells were analyzed by confocal microscopy. (E) HeLa-TNF-R2 cells were transfected with a TRAF2-GFP expression plasmid. The following day, nonstimulated, TNF-R2-stimulated (1 hour, 2 µg/ml TNF-R2 IgG) and TNF-treated (1 hour, 20 ng/ml TNF) living cells were analyzed by confocal microscopy. Endogenous TNF-R2 expression was detected by immunofluorescence using a TNF-R2-specific polyclonal rabbit antiserum. Examples of colocalized TNF-R2 and TRAF2-GFP are indicated with arrows.

View larger version (34K): [in a new window]   Fig. 9. Model of the apoptotic TNF-R1/TNF-R2 crosstalk. (A) Exclusive stimulation of TNF-R1. (B) Prestimulation of TNF-R2. (C) Costimulation of both TNF receptors. For details see Discussion.

View larger version (96K): [in a new window]   Fig. 8. Recruitment of cIAP1 and cIAP2 into the TNF-R2 complex critically depends on TRAF2 in living cells. HeLa-TNF-R2 cells were transfected with expression plasmids encoding for cIAP1(NT)-GFP (A) or cIAP1(NT)-GFP (B), alone or in combination with an expression construct for full-length TRAF2. The following day, distribution of the GFP chimeras was both analyzed prior to stimulation (2 µg/ml TNF-R2 IgG) and monitored online for 15 minutes after stimulation using confocal microscopy. Examples of ligand-induced alterations in the localization of the GFP fusion proteins are indicated with arrows.

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