Activation of the transcription factor NF-κB is a major effector of the inducible resistance to death receptor-mediated apoptosis. Previous evidence indicates that the combined transcriptional activation of TRAF-1, TRAF-2, IAP-1, and IAP-2 is required to suppress cell death by tumor necrosis factor (TNF). Here we show that NF-κB activation upregulates the caspase 8 inhibitor FLIP, resulting in increased resistance to Fas ligand (FasL) or TNF. Restoration of either the full-length 55-kDa long form of FLIP or an alternatively spliced short form of FLIP in NF-κB null cells inhibits TNF- and FasL-induced cell death efficiently, whereas the expression of IAP or TRAF family members only partially rescues cells from death. Resistance to either FasL- or TNF-induced apoptosis is overcome when cells are incubated in the presence of the protein synthesis inhibitor cycloheximide. This treatment leads to the rapid downregulation of FLIP but not to that of TRAF2. Our findings suggest that FLIP is an important mediator of NF-κB-controlled antiapoptotic signals.

Death receptors, such as Fas and tumor necrosis factor-related apoptosis-inducing ligand receptors, recruit Fas-associated death domain and pro-caspase-8 homodimers, which are then autoproteolytically activated. Active caspase-8 is released into the cytoplasm, where it cleaves various proteins including pro-caspase-3, resulting in apoptosis. The cellular Fas-associated death domain-like interleukin-1-β-converting enzyme-inhibitory protein long form (FLIP_L), a structural homologue of caspase-8 lacking caspase activity because of several mutations in the active site, is a potent inhibitor of death receptor-induced apoptosis. FLIP_L is proposed to block caspase-8 activity by forming a proteolytically inactive heterodimer with caspase-8. In contrast, we propose that FLIP_L-bound caspase-8 is an active protease. Upon heterocomplex formation, a limited caspase-8 autoprocessing occurs resulting in the generation of the p43/41 and the p12 subunits. This partially processed form but also the non-cleaved FLIP_L-caspase-8 heterocomplex are proteolytically active because they both bind synthetic substrates efficiently. Moreover, FLIP_L expression favors receptor-interacting kinase (RIP) processing within the Fas-signaling complex. We propose that FLIP_L inhibits caspase-8 release-dependent pro-apoptotic signals, whereas the single, membrane-restricted active site of the FLIP_L-caspase-8 heterocomplex is proteolytically active and acts on local substrates such as RIP.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that induces cancer cell death by apoptosis with some selectivity. TRAIL-induced apoptosis is mediated by the transmembrane receptors death receptor 4 (DR4) (also known as TRAIL-R1) and DR5 (TRAIL-R2). TRAIL can also bind decoy receptor 1 (DcR1) (TRAIL-R3) and DcR2 (TRAIL-R4) that fail to induce apoptosis since they lack and have a truncated cytoplasmic death domain, respectively. In addition, DcR1 and DcR2 inhibit DR4- and DR5-mediated, TRAIL-induced apoptosis and we demonstrate here that this occurs through distinct mechanisms. While DcR1 prevents the assembly of the death-inducing signaling complex (DISC) by titrating TRAIL within lipid rafts, DcR2 is corecruited with DR5 within the DISC, where it inhibits initiator caspase activation. In addition, DcR2 prevents DR4 recruitment within the DR5 DISC. The specificity of DcR1- and DcR2-mediated TRAIL inhibition reveals an additional level of complexity for the regulation of TRAIL signaling.