TNFR1 contains a cytoplasmic region designed death domain (DD), which initiates a signal of cytotoxicity with homology to the intracellular domain of Fas antigen [33]. TNFR1 activates different signalling pathways, including neutrophil migration, complement pathway, regulation of other cytokines and chemokines, adhesion molecules and their receptors, generally promoting inflammatory responses [34]. Several molecules were described as essential for NF-κB activation through a TNFR1-dependent pathway, for example, TRADD (TNFR1-associated death domain), RIPK1 (receptor-interacting protein kinase 1, also known as RIP1), TRAF2 (TNFR-associated factor 2) and FADD (Fas-associated death domain) [35]. The TNF/tmTNFR1 complex induces activation signalling pathways leading to opposite effects such as cell survival and cell death. Once TNF binds tmTNFR1, TRADD, RIPK1, TRAF2 and TAK1 molecules are recruited near DD and complex I. This complex I mediates the activation of MAPK and NF-κB, promoting cell survival. To activate this pathway, the phosphorylation of Jak-(Janus kinase)1 and Jak2, STAT-(signal transducer and activator of transcription)3 and STAT5 are required [36,37]. RIPK1 (RIPK1^U) is ubiquitinated to recruit Ikappa B kinase (IKK), and then there is a binding between RIPK1^U/NEMO (regulatory subunit IKKγ) to finally activate NF-κB [38] (Figure 2, left). However, if TRADD, RIPK1 and TRAF2 are dissociated from DD, they can interact with FADD, assembling the complex II that activates Caspase-8, inducing cell death. It has been suggested that to induce a full caspase 8-activation, ROS generation is required upstream and downstream of complex II [37,39] (Figure 2, left).

The interactions of solTNF or tmTNF with tmTNFR1 are also crucial to trigger apoptotic signals. It has been recently shown that tmTNF induces the binding of STAT1 to a region spanning amino acids 319–337 of tmTNFR1. STAT1-phosphorylation (at the serine residue in position 727) favors its binding to TRADD and FADD, promoting apoptosis but not NF-κB activation [40]. Thus, the regulatory balance between survival versus death by the TNFR1 pathway is crucial to maintain homeostasis, and several authors suggested that TNFR1-mediated-signal transduction includes a checkpoint resulting in cell death when the signal activating NF-κB fails. Although it is not yet clarified how this checkpoint is controlled, experimental evidence suggested that RIPK1 is a crucial target to regulate this balance. It was reported that IKKα/IKKβ mediates direct phosphorylation of RIPK1 as the last step regulating cell death [41].

Toso, also known as FAIM3 (Fas apoptosis inhibitory molecule 3) or FcμR, is a transmembrane protein with a negative regulatory function, which promotes the ubiquitination of RIP1 inducing NF-κB activation and consequently cell survival [42], affecting caspase 8 activation [43]. Another molecule that has been involved in cell death is MAPKAP kinase-2 (MK2) [44]. If the effector MK2 induces phosphorylation of the kinase RIPK1, the binding with FADD/Caspase 8 is inhibited, thus complex-II-dependent cell death is blocked [44,45]. TNFR1-mediated cell survival may depend on ubiquitination and phosphorylation of RIPK1 (Figure 2, left).

Additionally, TNF/tmTNFR1 pathway can induce cell death called necroptosis as the assembly of FADD, RIPK1, and RIPK3 form the complex called necrosome [46]. Roca and collaborators [47], have shown in zebrafish or in human macrophages (infected with Mycobacterium marinum or Mycobacterium tuberculosis) that an excess of TNF triggers necrosis through TNF-RIPK1-RIPK3 interactions increasing the production of ROS, cyclophilin D (mitochondrial matrix protein), BID and BAX (pro-apoptotic proteins) [48]. The same group has recently reported that TNF triggers the production of ROS activating cyclophilin D (mitochondrial matrix protein) and leading to BID and BAX (pro-apoptotic proteins) activation, which results in mitochondrial Ca²⁺ overload through ER ryanodine receptor and necrosis [47].

The Ubiquitin-binding protein ABIN-1 is critical for the activation of RIPK1 [49]. This protein is recruited into the complex I and plays a critical role in the control of ubiquitylation and deubiquitylation. It has been proposed that ABIN-1 deficiency reduces the recruitment of A20 molecule (negative regulator of NFκB), and consequently, there are ubiquitylation and activation of RIPK1 to mediate necroptosis [49] (Figure 2, left).

Although the tmTNFR2 signalling pathway has been mainly implicated in cell proliferation, activation and survival, data have also reported to transduce apoptotic signal under specific models and potentiate TNFR1-induced cell death [50,51].

Two molecules with the ability to interact with the TNFR2 cytoplasmic domain and to induce signalling were reported and called TNF receptor-associated factor 1 (TRAF1) and TRAF2 [52]. TNFR2 signalling was first simplified as a pathway where TRAF2 induces c-Jun-N-terminal kinase (JNK) activation, using the apoptosis signal-regulating kinase 1 (ASK1) as a mediator to activate NF-κB and facilitating anti-apoptotic signals [53].

JNK plays a fundamental role to determine the outcome of the TNFR2 pathway. It has been shown that after TNF/tmTNFR2 engagement, TRAF2 and the inhibitor of apoptosis molecules called cIAP1 (molecule to mediate the TRAF2 ubiquitination) are recruited to the TNFR2 cytoplasmic domain, and later it is translocated to an ER-associated compartment, where TRAF2 ubiquitination occurs [54,55] (Figure 2, right). The ER is sensitive to homeostasis alterations favoring the accumulation of misfolded proteins, which trigger ER stress and promote apoptotic cell death [56]. Thus, after ER-stress, TRAF2 forms a complex with the ER-stress sensor called IRE1 and interacts with procaspase-12 promoting its activation [57]. It has also been proposed that ER-stress induces expression of TNF in an IRE1- and NF-κB-dependent manner, and TRAF2 is decreased; these together inhibit the JNK activation and makes cells susceptible to cell death [58]. Those data support evidence for a relevant role of the ER in an additional apoptotic control point of the TNFR2 pathway.

It has also been proposed that following tmTNFR2 activation, the E3 ubiquitin ligase Smurf2 forms a ternary complex with tmTNFR2 and TRAF2, inducing relocalization of TNFR2 to the insoluble membrane/cytoskeletal fraction promoting the JNK activation [59]. Mixed lineage kinase 3 (MLK3), a mitogen-activated protein kinase kinase kinase (MAP3K) required for optimal activation of JNK signalling, has been shown to associate with TRAF2, TRAF5 and TRAF6. However, only TRAF2 induces the kinase activity of MLK3 by conjugating with polyubiquitin chains to activate JNK [60,61].

Finally, there is controversial evidence about the role of TNF on mitochondrial integrity. Reports have shown that TNF causes mitochondrial fragmentation (fission), but other authors suggested that TNF stimulates mitochondrial biogenesis [62,63]. In human airway smooth muscle (hASM) non-asthmatic cells, fission was associated with an increased level of Drp1 (Dynamin related protein (1) and the decreased level of Mfn2 (GTPase Mitofusin (2), and TNF was involved in Mfn2 reduction [64]. Recently, it was reported that hASM cells exposed to TNF showed fission and mitochondrial biogenesis, with increased organelle volume density, cell proliferation, reduction of Ca²⁺ influx, and decrease of O2 consumption per mitochondrion [65]. In this regard, the TNFR2-activation mediates interactions between Stat3 (signal transducer and activator of transcription (3) and Re1A (Stat-Re1A protein) as the Stat3/Re1A complex interacts with two lysine residues within Stat3 that is acetylated by p300 and OPA1 (optic atrophy 1) expression is increased, suggesting an enhanced mitochondrial fusion [66].

In summary, the tmTNFR2 pathway is tightly regulated depending on different cellular organelles, which are alternative for specific cellular systems when TNF is produced in inappropriate concentration. These mechanisms can help to develop novel therapeutic targets for treating or preventing diseases where TNF expression is dysregulated.