Tumor necrosis factor alpha (TNF-α) is a pleiotropic cytokine that belongs to the TNF ligand superfamily. Through associations with its receptors on a variety of cell types, TNF-α participates in a broad range of biological activities, including cell differentiation, tissue development, inflammatory responses, lipid metabolism, and cellular cytotoxicity (1
). Programmed cell death (PCD) caused by death receptors includes both apoptosis and nonapoptotic mechanisms (13
). For example, TNF-α can trigger different forms of PCD that are morphologically distinguished as apoptosis and necrosis-like death (3
). By using either the Jurkat human T-cell leukemia line or mouse embryonic fibroblasts that are deficient in the Fas-associated death domain (FADD) protein, receptor-interacting protein kinase 1 (RIP1), and caspase-8, substantial evidence indicates that TNF receptor 1 (TNFR1) occupancy can initiate either FADD/caspase-8-dependent apoptosis or a RIP1-dependent nonapoptotic death program that results in necrosis (3
; L. Zheng and M. Lenardo, unpublished data). How the TNFR1 signal is distributed to these different pathways has not been determined.
There are two TNF-α receptors, TNFR1 and TNFR2, both of which belong to the TNFR superfamily. Similar to other members of the superfamily, TNFR1 and TNFR2 harbor four characteristic cysteine-rich domains (CRDs) in their extracellular portions. We have recently shown that the first extracellular CRD, the most membrane-distal domain of TNFRs, contributes to homotypic receptor self-assembly, presumably a prerequisite for the second and third CRDs to bind directly with TNF-α (4
). However, these two TNFRs have different intracellular domains which can initiate distinctive intracellular signals. TNFR1 has a single intracellular death domain (DD), whereas TNFR2 has a TNFR-associated factor (TRAF)-binding motif (24
). The DD is an 80-amino-acid hexahelical bundle homologous to the DDs that exist in some other members of the TNFR superfamily designated the death receptor subfamily (18
). The DD is an essential protein-binding domain that accounts for the ability of TNFR1 to induce apoptotic and nonapoptotic cell death and/or inflammatory signals, depending on the conditions. In this report, we wished to examine how TNFR1 transmits signals that determine different cellular outcomes.
The DD and its flanking sequences in TNFR1 provide docking sites for intracellular adaptors and proximal signal molecules (for details, see references 1
). In response to TNF-α stimulation, the silencer of DD is displaced from the intracellular DD of TNFR1, which allows TNFR1 to associate with other signaling molecules, including the TNFR-associated DD (TRADD) protein (11
), TRAF2, TRAF1 (28
), and other DD-containing adaptors, such as RIP1 (10
) and the FADD protein (26
). The recruitment of TRAF2 to the TNFR1 signaling complex is important for NF-κB activation (31
) and, more prominently, for c-Jun N-terminal kinase (JNK) activation (19
The interactions of the three DD-containing proteins involved in TNFR1 proximal signals pose interesting regulatory questions. TRADD has been shown to associate with the cytoplasmic tail of TNFR1 through homotypic DD interactions. This recruitment initiates two major TNF-induced responses, namely, programmed death by apoptosis and NF-κB activation (11
). FADD has been found in the TNFR1 signaling complex upon stimulation (26
) and is believed to mediate the TNFR1-induced activation of caspase-8 and/or caspase-10 that causes apoptosis. However, the association of FADD with TNFR1 seems to require TRADD as an adaptor (9
). RIP1, another DD molecule found in the TNFR1 signaling complex, induces NF-κB (33
) and a necrotic program of death involving reactive oxygen species (ROS) (9
). Even though it possesses a DD itself, RIP1 seems to still require TRADD as an adaptor to indirectly associate with TNFR1 (10
). Thus, TRADD has been considered a central signaling adaptor for TNFR1 stimulation (1
). Much evidence suggests that through homotypic DD interactions, TRADD is recruited to TNFR1 as a first step which promotes the subsequent binding of all other signal transducers. The place of formation and the components of the TNFR1 signaling complex during TNF-α stimulation have been controversial, partly due to differences in the experimental approaches used by different investigators (26
). Nevertheless, recent data show that upon TNF-α stimulation, signaling complexes can form at the membrane as part of a secondary internal signaling complex or by internalization of the receptor with a signal complex attached to its cytoplasmic tail. In one model, there is an early recruitment of TRADD/RIP1 to the cytoplasmic tail of TNFR1, and then the complex dissociates from the receptors on the membrane and travels into the cytoplasm, recruiting additional proteins into the complex, including FADD and caspase-8 (26
). Alternatively, TNFR1 recruits adaptors and other signaling molecules residing on the membrane and then internalizes as a whole, forming the stabilized signaling complex (29
). Nevertheless, as a serine/threonine protein kinase, RIP1 can deliver both survival and death signals during TNF-α stimulation. A major question posed by the complex array of signaling proteins is how these specific signaling responses from TNFR1 are triggered.
It has been well established that stimulation of TNFR1 can induce the activation of NF-κB. In view of the known PCD pathways, it is interesting that most of the key pro-PCD components are constitutively expressed, which allows cells to promptly sense and execute death signals. In contrast, many of the anti-PCD molecules, such as the cellular FLICE-like inhibitory protein (cFLIP) (17
), inhibitor of apoptosis proteins (cIAPs), TRAFs (35
), and Bcl-2/Bcl-XL (6
), are regulated by NF-κB induction (for a review, see reference 15
). Thus, NF-κB may play a central role in regulating life and death during death receptor stimulation in an inducible manner.
Since TRADD-knockout animals or deficient cell lines are unavailable, it has been difficult to definitively address the physiological role of TRADD. Previous studies suggested that TRADD serves as a central signaling adaptor for both FADD and RIP1 in association with TNFR1. Here we show that silencing the expression of TRADD with small interfering RNA (siRNA) can prevent TNF-α-induced NF-κB activation and caspase-8-dependent apoptosis but has no effect on TNFR1-induced, RIP1-dependent nonapoptotic PCD. We also address the roles of RIP1, FADD, and caspase-8 in the two PCD pathways induced by TNF-α in T-cell lines.