NFkappaB activation by Fas is mediated through FADD, caspase-8, and RIP and is inhibited by FLIP.

Fas (APO-1/CD95) is the prototypic death receptor, and the molecular mechanisms of Fas-induced apoptosis are comparably well understood. Here, we show that Fas activates NFkappaB via a pathway involving RIP, FADD, and caspase-8. Remarkably, the enzymatic activity of the latter was dispensable for Fas-induced NFkappaB signaling pointing to a scaffolding-related function of caspase-8 in nonapoptotic Fas signaling. NFkappaB was activated by overexpressed FLIPL and FLIPS in a cell type-specific manner. However, in the context of Fas signaling both isoforms blocked FasL-induced NFkappaB activation. Moreover, down-regulation of both endogenous FLIP isoforms or of endogenous FLIPL alone was sufficient to enhance FasL-induced expression of the NFkappaB target gene IL8. As NFkappaB signaling is inhibited during apoptosis, FasL-induced NFkappaB activation was most prominent in cells that were protected by Bcl2 expression or caspase inhibitors and expressed no or minute amounts of FLIP. Thus, protection against Fas-induced apoptosis in a FLIP-independent manner converted a proapoptotic Fas signal into an inflammatory NFkappaB-related response.

Generation of a FasL-based proapoptotic fusion protein devoid of systemic toxicity due to cell-surface antigen-restricted Activation.

We describe the construction of a FasL fusion protein devoid of systemic toxicity, inducing apoptosis only on cell-surface antigen-positive cells. The fusion protein consists carboxyl-terminally of the extracellular domain of FasL and amino-terminally of a fibroblast activation protein (FAP)-specific single chain antibody fragment (sc40-FasL). The latter allows immobilization-dependent conversion of the inactive soluble FasL fusion protein into an entity with membrane FasL-like activity. Thus, sc40-FasL efficiently induced apoptosis only in FAP-expressing cells. In accordance with a strict target-selective activity of sc40-FasL, the intravenous application of this reagent in mice revealed no signs of systemic toxicity and prevented growth of xenotransplanted FAP-positive (but not FAP-negative) tumor cells. The principle described here for the first time, in which cell-surface antigen-mediated activation of Fas permits local activation of Fas in vivo, opens novel avenues for the use of Fas signaling in cancer therapy.

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

We have recently shown that stimulation of TNF-R2 selectively enhances apoptosis induction by the death receptor TNF-R1. Here, we demonstrate that stimulation of CD30 or CD40 also leads to selective enhancement of TNF-R1-induced cell death. Enhancement of apoptosis was correlated with the depletion of endogenous TRAF2 within 1 to 6 hours. Selective prestimulation of TNF-R2 for several hours inhibited TNF-R2-induced activation of the anti-apoptotic NF-kappaB pathway up to 90% and dramatically enhanced apoptosis induction by this receptor. When both TNF-receptors were stimulated simultaneously, TNF-R1-induced NF-kappaB activation remained unaffected but TNF-R1-induced apoptosis was still significantly enhanced. Compared with FasL-induced cell death TNF-R1-induced activation of caspase-8 was significantly weaker and delayed. Costimulation or prestimulation of TNF-R2 enhanced caspase-8 processing. Life cell imaging and confocal microscopy revealed that both TNF-R1 and TNF-R2 recruited the anti-apoptotic factor cIAP1 in a TRAF2-dependent manner. Thus, TNF-R2 may compete with TNF-R1 for the recruitment of newly synthesized TRAF2-bound anti-apoptotic factors, thereby promoting the formation of a caspase-8-activating TNF-R1 complex. Hence, TNF-R2 triggering can interfere with TNF-R1-induced apoptosis by inhibition of NF-kappaB-dependent production of anti-apoptotic factors and by blocking the action of anti-apoptotic factors at the post-transcriptional level.

Tumor necrosis factor receptor-associated factor (TRAF) 1 regulates CD40-induced TRAF2-mediated NF-kappaB activation.

To investigate CD40 signaling complex formation in living cells, we used green fluorescent protein (GFP)-tagged CD40 signaling intermediates and confocal life imaging. The majority of cytoplasmic TRAF2-GFP and, to a lesser extent, TRAF3-GFP, but not TRAF1-GFP or TRAF4-GFP, translocated into CD40 signaling complexes within a few minutes after CD40 triggering with the CD40 ligand. The inhibitor of apoptosis proteins cIAP1 and cIAP2 were also recruited by TRAF2 to sites of CD40 signaling. An excess of TRAF2 allowed recruitment of TRAF1-GFP to sites of CD40 signaling, whereas an excess of TRAF1 abrogated the interaction of TRAF2 and CD40. Overexpression of TRAF1, however, had no effect on the interaction of TRADD and TRAF2, known to be important for tumor necrosis factor receptor 1 (TNF-R1)-mediated NF-kappaB activation. Accordingly, TRAF1 inhibited CD40-dependent but not TNF-R1-dependent NF-kappaB activation. Moreover, down-regulation of TRAF1 with small interfering RNAs enhanced CD40/CD40 ligand-induced NF-kappaB activation but showed no effect on TNF signaling. Because of the trimeric organization of TRAF proteins, we propose that the stoichiometry of TRAF1-TRAF2 heteromeric complexes ((TRAF2)2-TRAF1 versus TRAF2-(TRAF1)2) determines their capability to mediate CD40 signaling but has no major effect on TNF signaling.