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 NFκB via a pathway involving RIP, FADD, and caspase-8. Remarkably, the enzymatic activity of the latter was dispensable for Fas-induced NFκB signaling pointing to a scaffolding-related function of caspase-8 in nonapoptotic Fas signaling. NFκB 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 NFκB activation. Moreover, down-regulation of both endogenous FLIP isoforms or of endogenous FLIPL alone was sufficient to enhance FasL-induced expression of the NFκB target gene IL8. As NFκB signaling is inhibited during apoptosis, FasL-induced NFκB 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 NFκB-related response.

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-B activation. Accordingly, TRAF1 inhibited CD40-dependent but not TNF-R1-dependent NF-B activation. Moreover, down-regulation of TRAF1 with small interfering RNAs enhanced CD40/ CD40 ligand-induced NF-B 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. CD40 and its ligand CD40L1 /CD154 are members of the tumor necrosis factor (TNF) receptor and TNF ligand family and represent major regulators of lymphocyte function (1). Aside from T-and B-cells, CD40 and CD40L are expressed in a variety of non-lymphocytic cell types including monocytes, dendritic cells, fibroblasts, smooth muscle, and endothelial cells (1). The CD40/CD40L system plays a critical role in the regulation of thymus-dependent humoral immune responses but also contributes to chronic inflammatory processes in autoimmune diseases, neurodegenerative disorders, graft-versus-host disease, cancer, and atherosclerosis (1).Engagement of CD40 results in the recruitment of members of the TNF receptor-associated factor (TRAF) adaptor protein family (1, 2). In addition, triggering of CD40 leads to Janus family kinase 3 (Jak3)-dependent activation of signal transducers and activators of transcription (STAT) proteins and to activation of the Src-related tyrosine kinase Lyn (3-6). TRAF proteins couple TNF receptors and Toll/interleukin-1 receptor family members to pathways leading to the activation of the inhibitor of I-B kinases and kinases of the mitogen-activated protein kinase (MAPK) family (2). All members of the TRAF family share a conserved C-terminal homology domain of ϳ180 amino acids (TRAF domain), which mediates interactions with the above mentioned receptors and the majority of cytosolic factors known for their TRAF binding capacity, including kinases, inhibitor of apoptosis proteins, and death domain adaptor proteins (2). With the exception of TRAF1, the N-terminal domain of all six known mammalian TRAFs comprise a single RING finger followed by several zinc finger motifs (2) that are important for ...

No abstract

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-κB pathway up to 90% and dramatically enhanced apoptosis induction by this receptor. When both TNF-receptors were stimulated simultaneously, TNF-R1-induced NF-κB 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-κB-dependent production of anti-apoptotic factors and by blocking the action of anti-apoptotic factors at the post-transcriptional level.

The effect of a purified extract of the flowering herb of Fagopyrum esculentum (buckwheat) on various protein kinases involved in signal transduction was examined. We observed that buckwheat contains red fluorescent compounds having photosensitizing properties. Spectrophotometric analysis of the extract has indicated structural similarity to hypericin. Dose- and light-dependent inhibition of various protein kinases was observed. The purified buckwheat extract strongly inhibited two receptor-associated protein tyrosine kinases (EGF-R and Ins-R) and a Ser/Thr kinase (PK-C) at an ng/ml concentration range. Selectivity was exhibited as a decreased sensitivity to cytosolic PTKs and protein kinase CK-2. The protein kinases are important components of the signal transduction pathway. Aberration of signal transduction is a hallmark of several proliferative diseases. Our results indicate that photosensitizing compounds in buckwheat are potential antiproliferative agents.