TNF ligands and receptors – a matter of life and death (2002)

It had been known for some time that tumour masses which had become contaminated by a bacterial infection would on occasion regress and disappear. It was thought that the bacteria were releasing a factor which would make the tumour necrotic and whither. This factor was termed tumour necrosis factor (TNF) (Old, 1985). It was not until more recent advances in immunology that it became clear that antigens from the bacterial invader (notably lipopolysaccharide LPS) were causing the release of the patient's own TNF which could cause the tumour regression. The hunt was on to isolate this TNF which could be used as a magic therapy to control cancer cell growth and persistence, plus enhance the academic understanding of the ways by which a cell could die. It was discovered that TNF and lymphotoxin (LT) were products from macrophages and lymphocytes that were capable of lysing many cell types including some tumour cells (Carswell et al., 1975; Granger et al., 1969). TNF was also found to be identical to the protein cachectin, which was known to be involved in the fever and muscle wastage seen in cancer patients (Beutler et al., 1985). Hence, the role TNF played in a range of physiological actions was important and the hunt was on to identify TNF and related molecules such as LT. Modern techniques have since allowed the isolation, characterization and cloning of the genes for TNF which is structurally related to LT plus an expanding family of TNF-like ligands (Table 1). These cytokine molecules include ligands such as Fas, CD40 and RANK which cause wide-ranging long-term cellular activities in cells such as differentiation, proliferation or death. Evolution has created this TNF superfamily of cytokines to control and manipulate the immune system, modulating processes such as haematopoiesis, antibody production, or short- and long-term immunity. It is only through its quirky tumour-killing characteristic that TNF cytokine was first identified and may still hold the key to effective tumour therapy. As the majority of information has been gained about TNF and it is the archetypal cytokine of the superfamily, displaying the greatest range of cellular actions, this review will focus on the molecular aspects and biological role of TNF signalling. Biochemically isolated in 1984, TNF has since been found to be a pleiotropic agent produced mostly by activated macrophages and monocytes, but also by many other cell types including B lymphocytes, T lymphocytes and fibroblasts. It is expressed as a 26 kDa transmembrane protein that can be cleaved by the metalloprotease TNF-α-converting enzyme (TACE) to release a 17 kDa soluble TNF form (Idriss & Naismith, 2000). TNF has also referred to as TNFα, cachectin or differentiation inducing factor (DIF) (Aggarwal, 2000). Its superfamily of nearly 20 different homologues including TNF-β (lymphotoxin-α), lymphotoxin-β, and other more specific ligands such as RANKL and TRAIL which are involved more particularly in bone tissue and lymphocyte processes. All the TNF receptor superfamily of ligands are thought form non-covalently-bound homo-trimers which are capable of becoming a secreted form (Figure 1). These TNF ligand are primarily designed for cell – cell contact transfer of signalling information between neighbouring cells, but cleavage into their soluble forms may allow more dispersed cytokine effects. TNF as well as causing necrotic cell death may also cause apoptotic cell death, cellular proliferation, differentiation, inflammation, tumourigenesis, and viral replication (Figure 2). TNF's primary role is in the regulation of immune cells, but is known to be heavily involved in pathogenic disorders such as rheumatoid arthritis, asthma, septic shock, irritable bowel disorder, haemorrhagic fever and cachexia (Locksley et al., 2001). TNF superfamily ligand-receptor interactions. TNF receptor-mediated cellular responses. Just as modern molecular techniques have identified a superfamily of TNF-like cytokine ligands, their receptor molecules consist of a superfamily of proteins that can be activated by one or more ligands (Table 2). TNF receptors are an family of proteins that consist of, to date, at least 27 members characterized by their repeated cysteine-rich extracellular sequence homology, and include LT receptor, Fas, CD40, the low affinity nerve growth factor receptor, TRAIL receptors, RANK and death or decoy receptors (Darnay & Aggarwal, 1999). Many of these members go by multiple names and are activated by specific ligands, however TNF only has the ability to bind two of these receptors which are also activated by LTα. TNF ligand achieves all its different cellular and pathological effects by its binding to either the TNFR1 or TNFR2 receptor subtype. They are single transmembrane glycoproteins with 28% homology mostly in their extracellular domain with both containing four tandemly repeated cysteine rich motifs. Their intracellular sequences are largely unrelated with almost no homology between each other, and early work suggested delineation of their signalling functions (Grell et al., 1994). They contain several motifs with known functional significance. Both TNFR1 and TNFR2 contain an extracellular pre-ligand-binding assembly domain (PLAD) domain (distinct from ligand binding regions) that pre-complexes receptors and encourages them to trimerize particularly upon activation by TNF ligand (Chan et al., 2000). TNFR1 contains a death domain (DD) motif of approximately 80 amino acids in length towards the carboxyl-end of the receptor and is critical in the death-inducing activity of TNFR1 (Tartaglia et al., 1993a). The death domain is present on a number of associating proteins and related molecules that are primarily involved in signalling for cell death. One such molecule is the silencer of death domain protein SODD, which binds to the DD in TNFR1 and prevents other DD-interacting proteins from taking hold (Jiang et al., 1999). SODD dissociates from the TNFR1 DD upon TNF stimulation allowing other activator proteins to access the DD receptor module. TNFR1 also contains an intracellular sequence that is known to bind the adaptor protein FAN, a key stimulatory element in the activation of neutral sphingomyelinase (Adamklages et al., 1998a; Adam et al., 1996) which catalyses the degradation of sphingolipids into smaller ceramide-containing molecules which are key signalling intermediates (Kolesnick & Kronke, 1998). Moreover, a stress responsive 44 kDa protein expressed highly in brain and reproductive organs, BRE, specifically interacts with the juxtamembrane region of TNFR1 (Gu et al., 1998). BRE, like SODD, acts to inhibit and dampen TNFR1-stimulated signalling. More information is known about TNFR1 than TNFR2. This is due to a number of factors including differences in affinity and efficacy of its ligands. The affinity of TNF ligand for TNFR1 receptor varies depending on the study from approximately 100 pM on native receptor (Tsujimoto et al., 1985; Kull et al., 1985; Vanostade et al., 1993), to estimations on cloned receptors from 100 – 600 pM (Schall et al., 1990; Hohmann et al., 1990; Pennica et al., 1992a; Loetscher et al., 1993; Grell et al., 1993; Moosmayer et al., 1994). Another study on native receptor indicated that soluble TNF, as is used experimentally, rapidly binds to TNFR1 with high affinity (Kd of 19 pM) and a slow dissociation from the receptor once bound (t1/2=33 min), a process which efficiently activates the receptor (Grell et al., 1998b). Such kinetics of ligand association are different from TNFR2 association (see below). Stimulation of TNFR1 leads to its internalization with inhibition of its long-term actions (Higuchi & Aggarwal, 1994) (Figure 3). Phosphorylation of TNFR1 occurs at a consensus MAPK site on its cytoplasmic domain or through tyrosine phosphorylation (Darnay & Aggarwal, 1997; Cottin et al., 1999), although it is not fully understood how these phosphorylations control receptor processing. TNFR1 is expressed on the cell surface but large amounts are found localized at the perinuclear-Golgi complex, as is TRADD, but which only associates with TNFR1 once at the plasmamembrane (Jones et al., 1999; Ledgerwood et al., 1999). Major signalling pathways modulated by TNF receptor subtypes. TNFR2 does not contain a DD motif but still recruits adaptor proteins including TRAF2. TNFR2 is thought to be able to signal apoptosis directly (Heller et al., 1992; Declercq et al., 1995) or through a so-called 'ligand-passing' mechanism by which TNFR2's greater affinity and half-life of TNF binding, holds ligand, increases the local TNF concentration in the vicinity of TNFR1 receptors which accept TNF ligand from TNFR2 and are themselves activated, signalling the TNFR1 apoptotic machinery (Tartaglia et al., 1993b). Others believe that additionally, TNFR2 signals for cell death through its cytoplasmic domain to induce mTNF expression, which then signals apoptosis via TNFR1 (Vercammen et al., 1995; Lazdins et al., 1997; Haas et al., 1999; Grell et al., 1999; Weiss et al., 1998). The kinetics of TNFR2 activation by TNF are different from TNFR1 (Vandenabeele et al., 1995). The dissociation kinetics of TNF from native TNFR2 is approximately 20 – 30 fold faster than from TNFR1 (Grell et al., 1998b), with workers finding the affinity of TNF for TNFR2 significantly greater (Tartaglia et al., 1993b) or less (Grell et al., 1998b) than the ligand's affinity for TNFR1. It is not clear how the binding characteristics of membrane-bound TNF at TNFR1 and TNFR2 compare to soluble TNF. Slight structural changes in the TNF sequence can lead to dramatic changes in its binding characteristics to TNF receptors. For example, murine TNF activates mouse TNFR1 and TNFR2 equally well, whereas human TNF acts on mouse TNFR1 but does not bind mouse TNFR2 (Lewis et al., 1991). Such observations led to studies in which mutant proteins ('muteins') of soluble TNF were developed that displayed reduced affinity towards TNFRs compared to wild-type TNF, however these muteins showed marked selectivity between TNFR1 and TNFR2 (Table 3) helping to uncover the role of each TNFR (Loetscher et al., 1993; Vanostade et al., 1993). TNFR2 was fully cloned after TNFR1 and its structural and functional characterization is less well understood. The main reason for the relative lack of signalling information about TNFR2 is that, generally, it is not efficiently activated in vitro. It was assumed that recombinant 17 kDa soluble TNF (as is provided commercially) was an efficient activator of TNFR2. However, it was uncovered that the membrane-bound 26 kDa form of TNF (mTNF) was greater than soluble TNF is activating TNFR2 (Grell et al., 1995) leading to qualitatively different responses and new insight into TNFR2 function (Decoster et al., 1995). TNFR1 is activated equally well by soluble and mTNF. TNF ligand acts in the immune system whereby it would activate TNFRs through cell – cell interactions. As such, most of the TNF effects in vivo may be mediated by mTNF (TNFR1=TNFR2 activation) rather than soluble TNF (TNFR1>TNFR2 activation). As such, the physiological role of TNFR2 may be underestimated by most TNF research conducted in the laboratory which uses soluble TNF as the stimuli. Soluble TNF acts similar to a partial agonist on TNFR2 in that it binds to the receptor, but is not highly efficacious and efficient in its activation. Such limitations of stimulation are overcome by the use of agonistic antibodies capable of efficiently stimulating TNFRs (Grell et al., 1993; Tartaglia et al., 1991; Leeuwenberg et al., 1995; Paleolog et al., 1994; Wajant et al., 2000; Borset et al., 1996; Haridas et al., 1998; Jupp et al., 2001), although how these functionally relate to natural forms of TNF can only be assumed. Both TNFR1 and TNFR2 possess sequences that are capable of binding intracellular adaptor proteins that link TNF receptor stimulation to activation of many signalling processes. These TNF receptor-associating factors (TRAFs) and adaptors are what transduce the TNF signal from the biochemically inert receptors to dramatic changes of the signalling molecules within target cells (Wajant et al., 2001). TRAF molecules all contain a RING finger and zinc finger motifs in their N-terminal, with their C-terminal regions possessing a TRAF domain sequence. To date six mammalian TRAF proteins have been identified. The first TRAFs to be uncovered, TRAF1 and TRAF2, were discovered by their ability to directly interact with the cytoplasmic domain of TNFR2 (Rothe et al., 1994). Work by the same group also identified the apoptotic adaptor proteins c-IAPI and c-IAP2 that bind to TNF receptor via a TRAF1/TRAF2 heterocomplex (Rothe et al., 1995a). Since then, it is now thought that mostly TRAF2 interacts with TNFR2 directly, with TRAF1 interacting indirectly and TRAF3 also able to associate (Table 2). TRAF2 is recruited to TNFR1 indirectly through a specific interaction with the protein TNF receptor-associated death domain (TRADD), a 34 kDa cytosolic adaptor protein that directly binds to TNFR1 through its own death domain sequence (Hsu et al., 1995). TRADD recruits the downstream signalling adaptor molecules FADD (Fas-associated death domain) and RIP (receptor interacting protein). RIP originally identified as a Fas-associating molecule (Stanger et al., 1995) also interacts with TNF receptors (Hsu et al., 1996a; Liu et al., 1996). RIP contains a kinase sequence, but its role as a kinase enzyme is unclear at present. FADD contains a death effector domain (DED) sequence (Chinnaiyan et al., 1995), that interacts with the DED domain in caspase-8 (also known as FLICE or MACH) and a number of other DED-containing molecules that regulate cell death mechanisms (Muzio et al., 1996). Another DD-containing molecule RAIDD is recruited to TNFR1 and interacts with RIP and caspase-2, to allow its activation. RIP and FADD are also thought capable under certain conditions to be able to indirectly bind to TNFR2 via TRAF2 (Pimentel-Muinos & Seed, 1999). TRAF2 is also capable of interacting with downstream signalling molecules such as NF-κB-inducing kinase (NIK) which is a member of the serine/threonine mitogen-activated protein kinase (MAPK) kinase (MEK) kinase (MEKK) family (Liu et al., 1996; Malinin et al., 1997). NIK phosphorylates its target at serine 176, an enzyme named inhibitor of κB (IκB) kinase (IKK) which is implicated in the activation of NF-κB transcription factor involved in transcriptional responses to stress and anti-apoptotic cellular action (Ling et al., 1998). NIK is also capable of interacting with TRAFs 1, 3, 5 and 6 (Wajant et al., 2001). One of the genes under the transcriptional control of NF-κB is the cellular inhibitor of apoptosis protein 2 (c-IAP2) which binds to TRAF2 and is capable of blocking caspase-8 activation and apoptosis. Similarly, A20 is an 80 kDa inducible protein that binds TRAF2 and is anti-apoptotic (Song et al., 1996). RIP and NIK are not the only kinases to interact with TRAF2, apoptosis-stimulating kinase (ASK1) (Nishitoh et al., 1998), germinal centre kinase (GCK) (Yuasa et al., 1998; Shi & Kehrl, 1997) and MEKK1 (Baud et al., 1999), have all been implicated in the activation of p38MAPK and c-Jun N-terminal kinase (JNK) stress kinases, as well as AP-1 and NF-κB transcriptional activation processes (Figure 3). The activation of the MAPK family of kinase enzymes can be achieved by TNF receptor interaction with the factor associated with neutral sphingomyelinase activation (FAN) adaptor protein (Adam et al., 1996). FAN is responsible for neutral sphingomyelinase-mediated generation of ceramide-containing sphingolipids (Adamklages et al., 1998a) which are capable of activating ceramide-activated protein kinases (CAPK) (Mathias et al., 1991; Dressler et al., 1992) (also known as kinase suppresor of ras (Zhang et al., 1997) which activates Raf kinase (Yao et al., 1995), an upstream activator of the MEK and MAPK serine/threonine kinase family (Mathias et al., 1998). Interestingly, TNF receptors have been found to interact with the Grb2 adaptor and son of sevenless (SOS) exchange factor (Hildt & Oess, 1999; Adam et al., 1996). Activated Grb2 binds through its SH3 domain to a motif within the TNF receptor, allowing this activated complex to stimulate c-Raf-1 kinase. Another recently identified protein that interacts with FADD through a DED domain is the FLICE-like inhibitory protein (FLIP (Irmler et al., 1997; Thome et al., 1997) which blocks caspase-8 recruitment and activation. FLIP interacts with TRAFs and RIP to switch signalling pathways through more anti-apoptotic pathways such as NF-κB and Raf-1 kinase, resulting in marked MAPK activation (Kataoka et al., 2000). The death domain within TNFR1 is also able to bind MADD adaptor protein, which contains a DD sequence. MADD activates MAPK when overexpressed in cultured mammalian cells (Schievella et al., 1997; Kataoka et al., 2000; Brinkman et al., 1999). A 59 kDa TNFR2-associated serine/threonine kinase p80TRAK has been described that is TNF-stimulated (Darnay et al., 1994). p80TRAK binds to a 44 amino acid site in TNFR2 cytoplasmic domain in a similar fashion to casein kinase (Darnay et al., 1997). Some of the earliest TNF signalling research revealed the activation of several lipase activities (Dayer et al., 1985; Marquet et al., 1987; Beyaert et al., 1987; Clark et al., 1987; Godfrey et al., 1987). TNF receptors activated phosphatidylcholine-specific phospholipase C (PC – PLC) (Schutze et al., 1991; Wiegmann et al., 1992). PC – PLC stimulation by TNFR1 activates a downstream acidic membrane-bound sphingomyelinase activity. Stimulation of PC – PLC activity degrades phosphatidylcholine into choline (a molecule with no known signalling function) and diacylglycerol, the physiological activator of most protein kinase C (PKC) isoforms. Other reports have suggested TNF ligand stimulates phospholipase D (PLD) creating phosphatidic acid (Devalck et al., 1998; Kang et al., 1998), that may be converted to DAG by phosphatidic acid phosphohydrolase. Others have also implied TNF-stimulated phosphatidylinositol-specific phospholipase C (PI – PLC, generating inositol-1,4,5-trisphosphate and diacylglycerol second messengers) activity (Beyaert et al., 1993). Much interest has centred on the ability of TNF to stimulate sphingomyelinase of which there are two types, neutral and acidic (Kolesnick & Kronke, 1998). These sphingomyelinases stimulate the breakdown of membrane sphingolipids into ceramide which may then be converted into other ceramide-containing lipids such as sphingosine and sphingosine-1-phosphate (S-1-P) (Hannun, 1994). TNF was the first stimuli found to induce ceramide generation (Okazaki et al., 1989; Mathias et al., 1991; Kim et al., 1991; Dressler et al., 1992). TNFR1 is responsible for the stimulation of membrane-associated neutral sphingomyelinase (Wiegmann et al., 1992, 1994), and via FAN adaptor protein, the stimulation of neutral sphingomyelinase (Adam et al., 1996). Exogenous ceramide is a highly specific yet destructive chemical in most cells with rapid stimulation of apoptotic cell death processes. These apoptosis-signalling processes include the sequential activation of Bad (Basu et al., 1998), then activation of CAPK which stimulates raf kinase (Yao et al., 1995) and MAPK activity (Raines et al., 1993; Bird et al., 1994). TNF-induced ceramide production may also stimulates the kinase et al., 1996). are able to isoforms. For example, human cells with ceramide or TNF the rapid to the of and et al., 1997). Similarly, in ceramide and TNF activity and to the plasmamembrane et al., 2000). TNF-stimulated ceramide generation and apoptosis in cells was found to be on et al., 1999), ceramide has also been found to bind directly to the which can activate MAPK and NF-κB transcription et al., et al., 1994; et al., 1995; et al., 2000). TNF-stimulated sphingolipids are also capable of converted into sphingosine-1-phosphate (S-1-P) which has intracellular actions including from and stimulating MAPK activity & 2000). As well as its intracellular actions, is able to from the cell and in an by stimulating differentiation cell surface receptors – a family of receptors & 2000). Another lipase by TNF receptors is the kDa cytosolic phospholipase et al., 1991). is responsible for the of acid from the of The acid acts in an fashion and within its own cell of to be converted into and to stimulate receptors. Moreover, these are responsible for the generation of lipids and that and in cell death mechanisms indicated et al., 1992). cellular enzymes such as these molecules et al., 1989; & TNF receptor stimulation of MAPK and leads to the phosphorylation and activation of at specific et al., 1993; & serine in the of MAPK et al., resulting in a rapid of receptor activation of through p38MAPK has also been implicated et al., 1996) at serine which leads to activation of the lipase et al., 1998), but which may be a of phosphorylation and activation to A mechanism of TNF-stimulated also leading to the of protein and activity which is to inhibition by protein and et al., 1993). It has been in several cell types that protein and activity is for cell death et al., 1993; et al., et al., 1998; et al., 1991; et al., 1997). The mechanism of stimulation by TNF is thought to be as only this receptor has the ability to activate the kinases involved in its phosphorylation et al., 1998; Jupp et al., et al., with TNFR2 a role in the regulation of 1996). activated has been to be the cleaved by but the relative activity of the cleaved is not clear as this of the reports are et al., 1998; et al., et al., 1998; et al., 1997; et al., 1995). As through activation of PC – PLC, TNF receptors are capable of diacylglycerol generation and activation & 1987; et al., at least forms of with activation characteristics and tissue and some of these have been to be For example, activation by TNF can in through an activation process ceramide production et al., 2000). A of the human cell that are in are to TNF-induced apoptosis which can be by protein et al., 1999). is a for TNF-stimulated cleavage and has also been to cause the serine phosphorylation of TNFR1 protein et al., 2000). The and have also been to be by more complex TNF-stimulated mechanisms that may intermediates et al., 1995; et al., 1999; et al., and as in directly bind ceramide to cause the activation of MAPK and Much work has been with the activation by TNF receptors of the extracellular protein kinase superfamily of kinases, responsible for of a cells to a of stimuli and stress responses et al., 1997). These enzymes are characterized by their activation process of phosphorylation on and tyrosine in their with the motif can be for MAPK for p38MAPK or for the c-Jun N-terminal kinase (JNK) family of kinases et al., 1996). All of these are by TNF. kinases control their which are themselves under the control of MEK kinases They have a range of known activated in an including transcription factors and downstream kinases 1999). The first members of the family to be and are activated by TNF through and phosphorylation et al., 1992; et al., 1994) and one in from both receptor are capable of MAPK stimulation et al., 1996) but more recent work has MAPK activation occurs through the TNFR1 receptor only et al., 2001). of these such as and have a role for and activation in TNF of cell death 2000; et al., 1996). and kinases are termed stress kinases as are activated by a range of stress stimuli including or and kinases responsible for p38MAPK activation include and et al., 1998). The and are specific of p38MAPK and have the role of p38MAPK in many TNF-induced cellular responses et al., 2001). One of the first reports to p38MAPK in TNF signalling showed the role of the kinase in TNF-stimulated phosphorylation of protein and of NF-κB activity and (Beyaert et al., 1996). many of the actions of p38MAPK are although the role of p38MAPK in cell death and is not clear et al., 1995; et al., 1999; et al., 2000). The family of are a highly of TNF receptor signalling is activated by but also by 1999). Both TNFR1 and TNFR2 are of et al., Haridas et al., 1998; Jupp et al., through different but TRAF2. inhibitor but the use of such as or a form of have revealed an important role for in cellular processes including a role in apoptosis et al., et al., 1995; Liu et al., 1996; et al., 1996; et al., 1999; et al., 2001). TNF of and processes are critical in many pathological disorders including rheumatoid These processes under the control of several sequences but particularly the stimulation of NF-κB transcription factor (Aggarwal, 2000). The activation of NF-κB is by its association with degradation is by its phosphorylation on serine by NIK (Ling et al., and which is activated by the TNF et al., 1997). of the TNF receptor-associating including TRAF2 (Hsu et al., and RIP (Stanger et al., 1995; Liu et al., have been implicated as upstream NF-κB TRAF2 was the first of these identified to activate with forms of TRAF2 not TRAF1 or able to TNF-induced NF-κB activity (Rothe et al., TRAF2 associated with NIK and was suggested to be to NF-κB however from were still capable of TNF-induced NF-κB but not activation et al., 1997; et al., 1997). on the other were of TNF-stimulated NF-κB with activation and apoptosis et al., 1998; et al., 1995). It that but not TRAF2, was for TNF-stimulated NF-κB with TRAF2 for recruitment and RIP responsible for activation et al., 2000). a range of cell types, that both TNFR1 and TNFR2 are capable of NF-κB activation et al., 1992; et al., 1994; et al., 1994; Weiss et al., 1997; Haridas et al., 1998; et al., 1999; et al., 2001). RIP has thought to have a it binds to both TNFRs and their or et al., 1998; & Seed, 1999). The role of NIK in NF-κB activation processes has been into as activation of NF-κB by TNF was still present in et al., 2001). a