Tumor necrosis factor alpha (TNFa) is a proinflammatory cytokine involved in various biological processes including regulation of cell proliferation, differentiation, apoptosis and immune response (1). TNFa is mainly produced by macrophages, also by other tissues including lymphoid cells, mast cells, endothelial cells, fibroblasts and neuronal tissues. TNF was identified as a soluble cytokine produced upon the activation by the immune system and able to exert cytotoxicity on tumor cell lines and cause tumor necrosis in animal models (2). TNF is primarily produced as a type II transmembrane protein arranged as stable homotrimers (2,3,4).

The members of TNFa family exert their cellular effect through two distinct surface receptors of the TNF receptor family, TNFRSF1A (TNF-R1) and TNFRSF1B (TNF-R2). TNF-R1 is ubiquitously expressed, whereas TNF-R2 is found typically on cells of the immune system and is highly regulated. TNF-R1 and TNF-R2 binds membrane-integrated TNF (memTNF) as well as soluble TNF (sTNF) (2,5,6,7) TNF-R1 contains a protein-protein interaction domain, called death domain (DD)(8). This domain interacts with other DD-containing proteins and couples the death receptors to caspase activation and apoptosis (9). TNF-R2 induces gene expression by a TRAF-2 dependent signaling mechanism and also crosstalks with TNF-R1 (2).

The pleiotropic biological effects of TNF can be attributed to its ability to simultaneously activate multiple signaling pathways in cells (10). Binding of TNFa to TNF-R1 on the cell surface triggers trimerization of the receptor and exposes intracellular domain of TNF-R1 following the release of an inhibitory protein. This intracellular domain recruits a death-domain containing adaptor protein, TRADD by homophilic interactions (5,11,12,13,14,15,16,17). TRADD, which acts as a scaffold protein, recruits TRAF2 (2,10,18,19,20) and RIPK1 (2,21) to form a complex , referred to as complex 1 (22,23). Complex 1 is believed to be important in NF-?B activation and JNK activation. Complex 1 eventually dissociates from the receptor and integrates FADD and procaspase8 to form a complex referred to as the complex 2(10,23). In some cases, FADD/CASP8 association depends on high molecular weight complexes containing unubiquitinated RIPK1 as scaffold (24). Activated CASP8 induces CASP3 activity and execution of apoptosis (2).

CASP8 activates apoptotic signal through another mechanism involving BID cleavage to truncated BID (tBID) (10,25). tBID translocates to the mitochondria, increasing its outer membrane permeability. This results in cytochrome c release and activation of other caspases ultimately leading to apoptosis (10,25,26). Reactive oxygen species (ROS) have been found to increase during or after complex 1 and 2 formation to mediate or potentiate apoptosis upon TNF stimulation (10,27,28,29). TRAF-2 in complex 1 also activates the MAP kinase cascade, that leads to the activation of JNK (10,25,30), which on prolonged activation is believed to mediate both apoptosis and necrotic cell death (31,32,33).

On complex 1 formation, NF-?B regulated anti-apoptotic gene products efficiently block initiation of apoptosis by complex 2 (22,23,34). There is evidence of an early attempt to signal for apoptosis, which precedes the activation of NF-?B. The intracellular part of TNF-R1 binds to NSMAF (2,22,35,36) which in turn mediates SMPD2-dependant ceramide production from cell membrane. Ceramide induces membrane permeabilization and apoptosis (22,37). This is observed before TNF-R1 internalization and NF-?B activation. This process is repressed on TNF-R1 internalization. This signal however is enough to initiate apoptosis in some cells (22,34).

Another form of cell death, necrosis, is also mediated through TNF stimulation. On TNF stimulation, deubiquitinated RIPK1 dissociates from complex 1 and recruits RIPK3, FADD and CASP8 (38,39). RIPK3 is autophosphorylated and phosphorylates RIPK1 (22,38,40). Taken together, it has been speculated that RIP1 and RIP3 increase carbohydrate and glutamine metabolism of the cell, leading to increased ROS production and eventual necrosis(22).

Recruitment of CASP8, activation of FADD/RIP1 and apoptosis induction, is blunted when RIPK1 becomes ubiquitinated. IKBKG binds to ubiquitinated RIPK1 to induce the activation of NF-?B, which exerts antiapoptotic effects (22,41). Cellular inhibitor of apoptosis, BIRC2 and BIRC3 has E3-ubiquitin ligase activity and functionally interact with TRAF2 (2,13,22,42) and RIPK1 (22,24) to induce polyubiquitination of RIPK1 upon TNF stimulation (22,43,24,44,45). Loss of these inhibitors attenuates TNF-induced NF-?B activation (44). The adaptor proteins TAB2 and TAB3 bind preferentially to Lys-63 polyubiquitinated RIPK1. This facilitates dimerization of MAP3K7, promoting its phosphorylation and activation (22,46).The IKK complex, consisting of CHUK, IKBKB and IKBKG, is recruited to RIP1 through binding of IKBKG to the ubiquitin chain of RIP1 (22,47,48). Activated TAK1 directly phosphorylates IKBKB within the activation loop, leading to activation of the IKK complex and NF-?B (22).

Certain regulatory proteins have been known to intercept NF-?B activation at the level of ubiquitinated RIP1(22). TNFAIP3, an NF-?B inhibitory protein, removes Lys-63 polyubiquitin chain and promotes Lys-48 linked ubiquitination of RIPK1 leading to its degradation (2,22,47,49) and NF-?B signal termination. IKBKG stabilizes the bound polyubiquitinated RIPK1 by inhibiting its degradation, most probably by impairing its interaction with TNFAIP3 (22,47). OTUD7B is recruited to the activated TNF-R1 and promotes RIP1 deubiquitination, thereby attenuating NF-?B activation (22,50). At internalized TNF-receptosomes, RIPK1 is ubiquitinated by endocytic vesicle associated RFFL, inducing RIPK1 degradation, which terminates NF-?B activation (22,51). When successful, TNF-induced NF-?B activation induces transcription and expression of genes encoding proinflammatory IL-6 (22,52), anti-apoptotic factors BIRC2, BIRC3 (22,38) and BCL-2 homologue BCL2L1(22). This causes the cell to remain inert to apoptotic stimuli (53).

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