Browsing by Author "Bryde, Susanne"

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    Characterisation of TNF receptor-2 mediated signal initiation and transduction
    (2004) Bryde, Susanne; Scheurich, Peter, Prof. Dr.
    In this work, silica based micro-particles covalently coated with TNF were generated as a new tool to convert soluble into an equivalent of membrane bound TNF. This tool was employed to investigate the molecular events leading to TNFR2 mediated NFkappaB-signalling. For the covalent coupling of TNF to silica beads, a TNF-mutant with a Cysteine and a Histidine tag (CysHisTNF) fused to its N-terminus was used. The particles were activated with sulfoSMCC, allowing the formation of a covalent C-S-thioether between the maleimide group of sulfoSMCC and the SH-group of the free Cysteine being presented by wild type CysHisTNF. Immortalised mouse fibroblasts lacking wild type TNFR1 and TNFR2 but stably over-expressing a TNFR2/Fas receptor-chimera (composed of the extracellular and membrane spanning domains of TNFR2 and the cytosolic domain of Fas/CD95) were employed as a test system to confirm the membrane bound TNF (memTNF)-like activity of TNF-labelled beads. These cells undergo apoptosis within an hour after stimulation with memTNF but are completely resistant to activation with soluble TNF (sTNF). CysHisTNF coated beads were shown to provide a memTNF-like derivative which was able to fully stimulate wild type TNFR2 or the TNFR2/Fas chimera. To allow TNFR1 or TNFR2 selective stimulation in the presence of both receptors, CysHisTNF-mutants binding selectively to one or the other TNF-receptor were employed. Control experiments with wildtype TNF revealed that a significant amount of the cytokine can bind to the beads by adsorption. Particles labelled with CysHisTNF, however, showed a higher bioactivity as compared to beads which had been treated with wildtype soluble TNF only. Investigations using confocal microscopy revealed that an average 6 TNF-coated beads of 1 µm diameter or one respective particle of 10 µm diameter were sufficient to trigger apoptosis in a single TNFR2-Fas expressing mouse fibroblast. This revealed an about 20 fold higher bioactivity as compared to the best so far available memTNF mimicking agent that was generated by sTNF (derivatives) stabilised at the receptor with 80M2, a non-agonistic TNFR2-specific monoclonal mouse antibody. TNFR1/Fas and TNFR2/Fas receptor-chimera were used to distinguish between the receptor-specific activity of sTNF or memTNF-equivalents on TNFR1 or TNFR2. Both receptor-chimera were demonstrated to activate the Fas-signal transduction pathway upon stimulation with memTNF-analoga by recruitment of FADD and/or Caspase-8 to the cytosolic portion of the receptor constructs, observed by confocal microscopy. However, only TNFR1/Fas could be stimulated with soluble TNF whereas TNFR2/Fas was fully resistant to sTNF action. Similar data were obtained after stimulation of wild type TNFR2 with a TNFR2-selective TNF-mutein being stabilised to the receptor with 80M2 or with a wtCysHisTNF-coated bead. Whereas these memTNF derivatives lead to the recruitment of TRAF2 to the TRAF-binding domain of the receptor, sTNF triggered no TRAF2-recruitment (Krippner-Heidenreich et al., 2002). In a HeLa cell overexpressing a full length wild type TNFR2-GFP construct, which was locally stimulated with a wtCysHisTNF-coated bead of 10 µm diameter, clustering of TNFR2-molecules to the bead-cell contact site as well as lateral diffusion of molecules from the direct vicinity to the bead could be observed by confocal microscopy. However, no indication of a lateral signal progression that would have generated the activation of non-stimulated receptor molecules was found. Selective activation of TNFR2-molecules led to their internalisation via a dynamin-1-dependent mechanism. Internalised TNFR2 was found partially associated with caveolae and in late endosomes. Internalisation of TNF-coated beads of 1 µm diameter could be shown to occur independently of interaction of the beads with TNFR2 and was not inhibited by a dominant negative dynamin-1 mutant, suggesting a mechanism of internalisation different of that operative for TNFR2. A TNFR2-receptor-construct devoid of its cytosolic domain, which was replaced by GFP, served to reveal that internalisation but not the clustering of TNFR2 is dependent of its cytosolic domain. TNFR2 mediated NFkappaB-translocation, activated by the stimulation of TNFR2 with memTNF-analogues, was shown to occur within 30 minutes. Of the three investigated NFkappaB-molecules p65, p50 and c-Rel, p65 was found to be the NFkappaB-protein mainly being translocated into the nucleus after stimulation of both TNFR1 as well as TNFR2. IKKgamma/NEMO could be demonstrated to be as essential for the release of NFkappaB from IkappaBalpha via TNFR2-signalling as is known for TNFR1. RIP, however, played no detectable role in TNFR2-mediated NFkappaB-translocation. Finally, TNFR2-mediated recruitment of TRAF2 was demonstrated to occur independently of TNFR2-internalisation.