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Tumour necrosis factors receptor associated signalling molecules and their role in activation of apoptosis, JNK and NF-κB
  1. Bharat B Aggarwal
  1. Cytokine Research Laboratory, Department of Bioimmunotherapy, The University of Texas M D Anderson Cancer Center, 1515 Holcombe Boulevard, PO Box 143, Houston, Texas 77030, USA
  1. Dr Aggarwal (aggarwal{at}utmdacc.mda.uth.tmc.edu)

Abstract

Tumour necrosis factor (TNF) is a pleiotropic cytokine that mediates apoptosis, cell proliferation, immunomodulation, inflammation, viral replication, allergy, arthritis, septic shock, insulin resistance, autoimmune diseases, and other pathological conditions. TNF transduces these cellular responses through two distinct receptors: type I, which are expressed on all cell types, and type II, which are expressed only on cells of the immune system and endothelial cells. At the cellular level, these receptors activate the pathways leading to the activation of transcription factors NF-κB and AP-1, apoptosis and proliferation, and mitogen activated protein kinases. None of these receptors exhibit any enzymatic activity but the signals are transmitted through the recruitment of more than a dozen different signalling proteins, which together form signalling cascades. Inhibitors of TNF signalling have therapeutic value as indicated by the approval of the soluble TNF receptors and anti-TNF antibodies for rheumatoid arthritis and for inflammatory bowl disease.

  • tumour necrosis factor

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Since its initial isolation in 1984, tumour necrosis factor (TNF) has continued to be a major topic of scientific investigation as indicated by over 27 000 citations published within the past 15 years. These studies have indicated that TNF is a homotrimer with a subunit molecular mass of 17 kDa, produced by a wide variety of cell types in response to various inflammatory stimuli. We now know that it plays a major part in growth regulation, differentiation, inflammation, viral replication, tumorigenesis, autoimmune diseases, and the response to viral, bacterial, fungal, and parasitic infections (for references see monographs by Aggarwal and Vilcek,1 Goeddelet al,2 and Aggarwal and Natarajan3). TNF is also referred to as TNFα, cachectin, or differentiation inducing factor (DIF). Since its initial discovery, almost 20 different homologues of TNF with 15–20% identity to each other have been reported (see table 1). Because we know the most about TNFα, this review primarily deals with it.

Table 1

List of the human TNF superfamily members and their characteristics

Soon after TNF protein and its gene were isolated,4 5 a novel TNF receptor was identified6 and shown by ligand receptor cross linking that it has an approximate molecular mass of 70 kDa.7 In 1989, several groups independently reported the isolation of a TNF binding protein or TNF inhibitor from human urine that turned out to be the soluble form of the TNF receptor.8-13 From the amino acid sequence of this protein the cDNA was isolated and cloned.14-16Simultaneously, the cDNA for a second TNF receptor was isolated and cloned.17 18 It is now clear that TNF binds with almost equal affinity to two distinct receptors referred to as p60 (also called p55 or type I) and p80 (also called p75 or type II), with an approximate molecular mass of 60 kDa and 80 kDa, respectively. The p60 receptor has 426 amino acid residues consisting of an extracellular domain (ECD) of 182 amino acids, a transmembrane domain (TMD) of 21 amino acids and an intracellular domain (ICD) of 221 amino acids. From this the predicted molecular mass of this receptor was about 47.5 kDa. As the apparent molecular mass of the p60 receptor is between 55 and 60 kDa, the difference most probably is attributable to three potential N-linked glycosylation sites present in the ECD of the receptors. The ECD of the p60 receptor has a net charge opposite that of the TNF, suggesting electrostatic interaction. The p80 receptor is a 46 kDa protein, and it consists of 439 amino acid residues with an ECD of 235 amino acids, a TMD of 30 amino acid residues, and an ICD of 174 amino acids. This receptor is also glycosylated.

The two TNF receptors are characterised by the presence in their ECD of four cysteine-rich regions, each consisting of six cysteine residues. These cysteines are conserved between the two receptors. The ICD of both the receptors lack enzymatic activity. The two receptors bind TNF with almost equal affinity. The structure of the ICD of the two receptors is quite distinct, suggesting a difference in the signalling pathways. For instance the ICD of the p60 receptor contains a homophilic interaction region of approximately 80 amino acid residues towards its carboxyl terminal, called the death domain (DD),19 which is absent in the p80 receptor. This region was found to be required for TNF induced apoptosis, antiviral activity, and nitric oxide synthase induction. Within the past decade, major advances have been made in understanding how TNF receptors transduce their signals. A series of signalling molecules have been discovered that play a critical part in the TNF induced cellular responses. Some of the major TNF induced cellular responses are shown in figure 1. This review describes various molecules that are recruited by the TNF receptors either directly or indirectly and mediate TNF signalling.

Figure 1

Major TNF receptor mediated cellular responses.

TNF receptor associated death domain (TRADD)

First identified in 1995, TRADD is a 34 kDa cytoplasmic adapter protein that contains a death domain at its C-terminus. It is recruited to the cytoplasmic domain of the p60 receptor through homophilic interaction of the DD present in both the molecules.20This interaction has been shown to be required for TNF induced activation of various cellular responses including NF-κB activation, JNK activation, and apoptosis. On activation, TRADD recruits the downstream signalling molecules FADD and RIP, which mediate apoptosis.21 22 TRADD also interacts with a RING finger protein, TRAF2, leading to NF-κB activation.23 24

Fas associated death domain (FADD/MORT1)

In 1995, two groups independently reported the isolation of a protein that directly associates with the Fas receptor25 26; another member of the TNF receptor superfamily, FADD was subsequently found to interact with TRADD.22 Besides a DD at the carboxyl terminus, FADD contains a motif called the death effector domain (DED) at its amino terminus. FADD interacts with TRADD through its DD. Overexpression of the DD alone inhibits apoptosis as it occupies DD containing proteins and does not allow association of the critical caspases and possibly other molecules. Because overexpression of FADD induces apoptosis and a dominant negative mutant of FADD blocks TNF induced apoptosis,22 it was suggested that FADD is involved in TNF induced apoptosis. Studies done with embryonic fibroblasts from FADD deficient mice suggested that FADD is the major but not the only pro-apoptotic pathway engaged by the TNF receptor.27 28This is based on the observation that FADD-null cells are still 30% sensitive to TNF induced apoptosis.

FADD-like ICE (FLICE/MACH/Caspase-8/Mch5)

Like FADD, FLICE was also first identified by two groups independently.29 30 FLICE is a protein characterised by the presence of an N-terminal DED and a cysteine protease (also called caspase) domain at the C-terminus. The DED is the critical part of the molecule, as it recruits proteins downstream in the pathway that actually modulate apoptosis. Overexpression of this domain alone is sufficient for apoptosis to occur. The FLICE interacts with FADD through DED present in each protein. Several DED containing proteins have been discovered that function in a manner completely opposite of FADD to inhibit apoptosis. The death effector domain of equine herpesvirus protein E8 interacts with FLICE (also called caspase-8) prodomain, whereas that in molluscum contagiosum virus protein MC159 interacts with FADD. Both of these interactions block p60 TNF receptor induced apoptosis.31 Presumably binding of MC159 with FADD prevents FADD from recruiting the caspase-8 and/or caspase-10 (also called FLICE2). Induction of apoptosis by overexpression and suppression of apoptosis by dominant negative mutant have suggested that FLICE is a critical intermediate in TNF induced apoptosis.29 30 Additionally fibroblasts derived from targeted disruption of the FLICE gene were resistant to TNF receptor induced apoptosis but sensitive to TNF induced JNK and NF-κB activation.32 These results indicate that FLICE is required for TNF induced apoptosis. A total of eight different isoforms of FLICE have been described, but only two isoforms are expressed in most cells.33

FADD-like antiapoptotic molecule (FLAME-1/CASH/CASPER/CLARP/FLIP/I-FLICE/MRIT)

FLAME-1 was simultaneously and independently discovered by several groups and given dfifferent names by each.34-38 FLAME contains two DED domains fused to the C-terminal caspase-like domain. The structure of FLAME is similar to caspase-10 (also called FLICE2) and FLICE but lacks caspase activity because of loss of two important residues required for catalysis.33-37 It interacts with a number of cell signalling proteins including FADD, caspase-10, FLICE, caspase-3, and Bcl-xL (a protein known to suppress apoptosis).While some reports demonstrate that FLAME is an inhibitor of TNF induced apoptosis,34 35 others show that when overexpressed FLAME induces apoptosis.36-38

TNF receptor associated factor (TRAF)

Initially two distinct TRAFs that bind to the cytoplasmic domain of the p80 receptor were identified and were referred to as TRAF1 and TRAF2.39 At present six different TRAFs have been identified, all characterised by the presence of a RING finger and zinc finger motif in the N-terminal and a TRAF-domain in the C-terminal region that seem to be responsible for self association and protein interaction. TRAF2, TRAF3, and TRAF6 are ubiquitously expressed, whereas TRAF1 and TRAF5 are preferentially expressed in the spleen, thymus, and lung. TRAF4 is abundantly expressed in breast cancer cells. TRAF1 and TRAF2 can form homotypic and heterotypic dimers. TRAF2 contacts the p80 receptor directly and TRAF1 interacts with this receptor indirectly. TRAF2 is also recruited to the p60 TNF receptor through TRADD, and this requires a specific motif in the receptor (that is, PXQXT).24 In addition TRAF2 interacts with a wide variety of other receptors of the TNF superfamily including CD27, CD30, CD40, GITR, RANK, HVEM, OX40, and 4–1BB. TRAF2 also interacts with downstream cell signalling proteins including ASK1, NIK, I-TRAF, cIAP-1, cIAP2, A20, GCKR, IL15Ra, RIP, and TRIP (fig 2).

Figure 2

Interaction of TRAF2 with upstream and downstream signalling proteins.

Because overexpression of TRAF2 activated NF-κB and DN-TRAF2 blocked TNF induced NF-κB, it was suggested that TRAF2 plays a part in NF-κB activation.40 A recent report, however, indicates that both TRAF2 and RIP are required for NF-κB activation; TRAF2 recruits IκBα kinase (IKK, needed for NF-κB activation) to the TNF receptor while RIP mediates IKK activation.41 TRAF2 is also known to activate JNK, and it was found that TNF mediated pathways leading to the activation of NF-κB and JNK bifurcates at TRAF2.42 Targeted gene deletion studies, however, showed that TRAF2 was not required for TNF induced NF-κB activation but was required for JNK activation.43 44 There are other studies that suggest that the expression of TRAF2 is dependent on NF-κB activation and its expression negatively regulates TNF induced apoptosis.45

How TRAF2 is linked to JNK activation is not fully understood. Some studies suggest that apoptosis-signal-regulating kinase 1 (ASK1) interacts with TRAF2 and activates JNK.46 ASK1 is a mitogen activated protein kinase kinase kinase (MAPKKK) that activates the SEK1-JNK and MKK6-p38 signalling cascades. Another study indicates that interaction of TRAF2 with germinal centre kinase (GCK) leads to activation of JNK.47 The latter group also showed that the interaction of TRAF2 with receptor interacting protein (RIP) leads to activation of both MKK6 and p38 MAPK. Overexpressed TRAF2 activates JNK, p38, and an IκBα kinase (IKK) in the absence of extracellular stimulation. Oligomerisation of the TRAF2 effector domain results in specific binding to MEKK1, a protein kinase capable of JNK, p38, and IKK activation.48 TNF also increases the binding of native TRAF2 to MEKK1 and stimulates the kinase activity of the latter. Thus, TNF may signal by oligomerisation of TRAF2, leading to activation of effector kinases.

TRAF1 was the first TRAF to be identified that, unlike the p60 receptor, binds directly to the intracellular domain of the p80 receptor.39 While bound to the p80 receptor, TRAF1 also binds to TRAF2. Like TRAF2, TRAF1 has been implicated in suppressing TNF induced apoptosis.45 TRAF1 is also required for the recruitment of members of the cellular inhibitor of apoptosis (c-IAP) family to the p80 receptor.49

NF-κB inducing kinase (NIK)

NIK is a ser/thr (MAPKKK) that associates with TRAF2 (also with TRAF1, 3, 5 and 6) and has been implicated in TNF induced NF-κB activation.50 NIK activates IKKα by phosphorylation of Ser-17651 (fig 3). Besides NF-κB, NIK was also reported to be involved in TNF induced activation of AP-1 but had no role in JNK activation.52 Another report suggested that NIK activity is not required for TNF induced NF-κB activation.53 This evidence is based on alymphoplasia caused by a point mutation in the mouse gene encoding NIK. Although NIK is inactive in cells from these animals, TNF induced NF-κB activation is unaffected.53The role of NIK in TNF induced activation of NF-κB has also been questioned based upon other biochemical and genetic experiments.48

Figure 3

TNF signalling pathways leading to the activation of apoptosis, NF-kB, JNK and AP-1.

IκBα kinase (IKK)

The activation of NF-κB by TNF requires phopshorylaton of IκBα that is mediated by IKK. As of today, three different kinases that play an important part in NF-κB activation have been identified, IKKα (also called IKK1), IKKβ (also called IKK2) and IKKγ (also called NEMO). IKKα and IKKβ have very similar primary structures (52% identity) with protein kinase domain at their N-terminus, a leucine zipper (LZ), and a helix-loop-helix (HLH) motif at their C-terminus. IKKγ does not contain a catalytic domain but is composed of three large α-helical regions, including LZ. IKKα was first isolated as a protein that interacts with NIK54; however, this interaction occurs only when is NIK and IKKα are overexpressed.55 The native IKK complex consists of IKKα, IKKβ, and IKKγ.56 No IKK or NF-κB activity can be induced in IKKγ deficient cells treated with TNF,57 suggesting that IKKγ is required. TNF was shown to induce the phosphorylation of all three IKK.58Experiments with embryonic fibroblasts from IKKα gene deleted animals revealed that TNF induced NF-κB activation is unaffected.59 The cells from IKKβ gene deleted cells, in contrast, were unresponsive to TNF induced NF-κB activation.60-62 Thus IKKβ is absolutely required for NF-κB activation.

Silencer of death domains (SODD)

The DD of the TNF receptor p60 is required for the signalling of TNF induced apoptosis and NF-κB activation. Normally, these signals are generated only after TNF induced receptor aggregation. However, p60 receptor self associates and signals independently of ligand when overexpressed. This apparent paradox may be explained by silencer of death domains (SODD), a widely expressed approximately 60 kDa protein that was found to be associated with the DD of TNF receptor.63 TNF treatment released SODD from the TNF receptor, permitting the recruitment of proteins such as TRADD and TRAF2 to the active TNF receptor signalling complex. SODD association may be representative of a general mechanism for preventing spontaneous signalling by death domain containing receptors.

Receptor interacting protein (RIP)

RIP is another DD containing adapter protein that associates with TRADD.21 Besides a DD, it contains a serine/threonine kinase domain, but its function is not understood.64Because overexpression of RIP activates NF-κB65 and RIP deficient cells fail to activate TNF induced NF-κB,66RIP was considered to be required for NF-κB activation in response to TNF. Recent studies indicate that RIP mediates the activation of IKK.41 Although the kinase activity of RIP can autophosphorylate, the kinase domain is not required for NF-κB activation. It is the intermediate domain that resides between the kinase domain and the DD that mediates NF-κB activation.21

Another member of this family of molecules that has been identified is called RIP2 (also called CARDIAK/RICK), which has a conserved kinase domain.67-69 Instead of having a C-terminal DD, RIP2 contains a caspase activation and recruitment domain (CARD) motif, which like the DD, is a homophilic interaction domain found within the prodomain of caspase-1, caspase-2, caspase-9, Apaf-1, and RAIDD (fig4). The CARD motif in RIP2 binds to the prodomain of caspase-168 and promotes the activation of caspase-1. Recently it was shown that the kinase domain of RIP2 is required for the activation of the extracellular signal regulated kinase (ERK) pathway.70 The kinase defective mutant of RIP2 seems to block TNF induced activation of ERK2.

Figure 4

Interaction of various caspase recruitment domain (CARD) proteins.

RIP3, another member of this family that contains N-terminal kinase domain, shares homology with RIP and RIP2. RIP3 has neither a DD nor a CARD motif but has a unique C-terminal domain that binds to RIP and thus is recruited to the TNF p60 receptor complex.71 RIP3 attenuates TNF and RIP induced NF-κB activation and is a potent inducer of apoptosis.

Grb2

TNF receptor has been found to interact with the adapter protein Grb2 and the exchange factor son of sevenless (SOS) in response to TNF.72 Grb2 binds with its -COOH terminal SH3 domain to a PLAP motif within TNF receptor and with its NH2 terminal SH3 domain to SOS. A PLAP deletion mutant of TNF receptor fails to bind Grb2. The TNF receptor/Grb2 interaction is essential for the TNF dependent activation of c-Raf-1 kinase; activation of c-Raf-1 kinase by TNF can be blocked by coexpression of Grb2 mutants harboring inactivating point mutations in the NH2 or -COOH terminal SH3 domain, cell permeable peptides that disrupt the Grb2/TNFR-I interaction or transdominant negative Ras. Functionality of the TNF receptor/Grb2/SOS/Ras interaction is a prerequisite but not sufficient for TNF dependent activation of c-Raf-1 kinase. Inhibition of the TNF receptor/FAN interaction, which is essential for TNF dependent activation of the neutral sphingomyelinase, either by cell permeable peptides or by deletion of the FAN binding domain, prevents activation of c-Raf-1 kinase. Thus the binding of the Grb2 adapter protein via its -COOH terminal SH3 domain to the non-tyrosine kinase receptor results in activation of a signalling cascade known so far to be initiated, in the case of the tyrosine kinase receptors, by binding of the SH2 domain of Grb2 to phosphotyrosine.

Factor associated with neutral-sphingomyelinase activation (FAN)

A novel protein, FAN, which belongs to a family of WD-repeat proteins.73 FAN directly binds to the cytoplasmic nine-amino-acid-binding motif of p60 TNF receptor. This region of the receptor has been shown to be required for the activation of the neutral sphingomyelinase (N-SMase). Overexpression of FAN enhanced TNF induced N-SMase activity, while an N-terminal truncated mutant of FAN suppressed the activity. These results suggest that FAN regulates TNF induced ceramide production.

Mice lacking a functional FAN gene did not show any overt phenotypic abnormalities; in particular, the architecture and cellular composition of lymphoid organs seemed to be unaltered.74 An essential role of FAN in the TNF induced activation of N-SMase was demonstrated using thymocytes from FAN knockout mice. Activation of extracellular signal regulated kinases in response to TNF treatment, however, was not impaired by the absence of the FAN protein. FAN deficient mice showed delayed kinetics of recovery after cutaneous barrier disruption, suggesting a physiological role of FAN in epidermal barrier repair. Although FAN exhibits striking structural homologies with the CHS/Beige proteins, FAN deficient mice do not reproduce the phenotype of beige mice.74

RAIDD/CRADD

RAIDD is another DD containing protein that is recruited directly to the TNF receptor. It has a DD at its N-terminal, and its C-terminal is related to the prodomain of caspase-2, caspase-9 and to the prodomain of the nematode caspase CED-3.75 Because of the interaction with caspase-2 and RIP, it is called CRADD (caspase and RIP adaptor with death domain) (fig 4). The role of this protein in mediating TNF induced apoptosis through the recruitment of caspase-2 has been demonstrated.76 77 However caspase-2-null cells do not show any loss of sensitivity to TNF induced apoptosis.78 Thus the true significance of this interaction is unclear.

Caspases

Caspases are cysteine proteases that play a key part in induction of most forms of apoptosis. Caspases are synthesised as inactive proenzymes that must be activated by proteolytic cleavage after specific aspartic acid residues.79 In most instances the oligomerisation of the proenzyme is sufficient for autoactivation of the caspases.79 80 As of today, 14 different caspases have been identified. However, the role of these caspases in different cell death pathways in various tissues remains elusive. Besides caspase-8 and caspase-10, the role of caspase-3 (also called CPP32) in TNF induced apoptosis have been implicated81 82 (fig 5). Caspase-8, -9 and -10 are classified as upstream initiator caspases, whereas caspase -3, -6 and -7 are called downstream executioner caspases.79 In mice deficient ingenes for caspase-1, caspase-2, caspase-3, and caspase-9, however, the TNF induced apoptosis pathway was unaffected.78 83-85

Figure 5

Role of various caspases in TNF induced apoptosis.

A20

A20 is a TNF inducible 80 kDa zinc finger protein that interacts with TRAF1/TRAF2 and inhibits TNF induced NF-κB activation.86 87 Agents that activate NF-κB also induce A20, suggesting a feedback loop. Deletion analysis has revealed that the N-terminal of A20 interacts with the C-terminal of TRAF1 and TRAF2. The suppression of NF-κB, however, requires the C-terminal zinc finger domain. A20 also protects various cells from TNF induced apoptosis.88 A20 is constitutively expressed in various lymphoid tissues.89

TRAF interacting protein (TRIP/TANK/I-TRAF)

TRIP contains a RING finger motif and an extended coiled coil domain90 91 that interacts with TRAF2 and blocks TRAF2 mediated NF-κB activation. In contrast, when coexpressed with I-TRAF, TRIP forms a strong complex with TRAF2 and germinal centre kinase (GCK) related kinase (GCKR) leading to JNK activation.92

Phosphatidylinositol-4-phosphate 5 kinase (PIP5K)

The treatment of cells with TNF resulted in an increased PIP5K activity. A novel interaction between the juxtamembrane region of the p60 TNF receptor and a newly discovered 47 kDa isoform of phosphatidylinositol-4-phosphate 5-kinase (PIP5K), a member of the enzyme family that generates the key signalling messenger, phosphatidylinositol 4,5-bisphosphate, has been reported.93 The interaction was found to be specific for the p60 TNF receptor and was not observed with the p80 TNF receptor. In vitro experiments using recombinant fusion proteins verified the interaction between the p60 receptor and PIP5KIIβ, a new isoform of PIP5K, but not the previously identified 53 kDa PIP5KIIα. These results indicate that phosphatidylinositol turnover may be linked to stimulation of the p60 TNF receptor and suggest that a subset of TNF responses may result from the direct association of PIP5KIIβ with the p60 TNF receptor.

Apoptosis protease activation factor (Apaf)

Apaf is a 130 kDa protein that participates in cytochrome c (also called Apaf-2) dependent activation of caspase-3.94 The N-terminal 85 amino acid residues of Apaf-1 show 53% similarity to the prodomain of the Caenorhabditis eleganscaspase, CED-3, followed by 320 amino acids with 48% similarity to CED-4. The C-terminal of Apaf-1 comprises multiple WD repeats that mediate protein-protein interactions. Cytochrome c binds to Apaf-1, an event that may trigger the activation of caspase-3 (fig 5), leading to apoptosis. An APAF-1 and cytochrome c multimeric complex is also a functional apoptosome that activates procaspase-9.95

Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors.96 TNF activated caspase-8 cleaves Bid, and the -COOH terminal part of Bid translocates to mitochondria where it triggers cytochrome c release. Immunodepletion of Bid from cell extracts eliminates the cytochrome c releasing activity. The cytochrome c releasing activity of Bid was antagonised by Bcl2. A mutation at the BH3 domain diminished its cytochrome c releasing activity. Bid, therefore, relays an apoptotic signal from the cell surface to mitochondria. Gene deletion experiments have indicated that Apaf1 is required for mitochondria mediated apoptosis.97

Inhibitor of caspase activated DNase (ICAD)

In most cells TNF induced apoptosis is associated with DNA fragmentation, which is induced by activation of DNAse. In the cells, the DNase is kept in its inactive state by association with ICAD. A caspase activated DNase (CAD) and ICAD have been identified in the cytoplasm.98 CAD is a 343 amino acid protein that carries nuclear localisation signals. ICAD exists in a long and short form and serves as a chaperon. When ICAD is degraded by caspase-3, DNase is activated, causing DNA fragmentation.

Inhibitor of cellular apoptosis (cIAP)

Two different inhibitors of cellular apoptosis, (cIAP)-1 and -2, were first identified as proteins recruited by the p80 form of the TNF receptor through TRAF1.49 Now it seems that IAP constitute a family of proteins that have an evolutionarily conserved role in regulating apoptosis. Ectopic expression of cIAP can suppress apoptosis in a variety of cells, but the mechanism of this inhibition is not understood. Human X-chromosome linked IAP directly inhibits caspase-3 and caspase-7.99 Through TRAF2, c-IAP1 is recruited to the p60 form of the TNF receptor.24

MADD

When the DD was used as a bait in the yeast two hybrid system, MADD, was identified by its association with the DD of the p60 TNF receptor through its own C-terminal DD.100 MADD interacts with TNF receptor residues that are critical for signal generation and coimmunoprecipitate with p60 TNF receptor, implicating MADD as a component of the TNF receptor signalling complex. Overexpression of MADD activated the mitogen activated protein (MAP) kinase extracellular signal regulated kinase (ERK) and expression of the MADD stimulates both the ERK and JNK MAP kinases and induces the phosphorylation of cytosolic phospholipase A2. These data indicate that MADD links TNF receptor with MAP kinase activation and arachidonic acid release.

Sentrin

The yeast two hybrid system was also used to identify, a novel protein, sentrin, which interacts with TNF receptor p60 but not with FADD/MORT1 or CD40.101 Sentrin is a novel protein of 101 amino acids with homology to ubiquitin, Nedd8, and aSaccharomyces cerevisiae protein, Smt3. Two hybrid interaction assays revealed that sentrin associates only with the signal competent forms of TNF receptor DD. When overexpressed, sentrin provided protection against TNF induced cell death.

BRE

BRE is a novel stress responsive gene highly expressed in brain and reproductive organs (BRE) and is down regulated after exposure to ultraviolet irradiation, DNA damaging agents, or retinoic acid. The human BRE gene encodes an mRNA of 1.9 kb, which gives rise to a protein of 383 amino acids with a molecular size of 44 kDa.102 In a yeast two hybrid screen, using the juxtamembrane domain of the p60 TNF receptor as a bait, interaction with BRE was identified. The interaction between the p60 receptor and BRE was verified by an in vitro biochemical assay by using recombinant fusion proteins and by coimmunoprecipitation of transfected mammalian cells. In the yeast two hybrid assay, BRE specifically interacted with p60 TNF receptor but not with other TNF family members such as the Fas receptor, the p80 TNF receptor, and p75 neurotrophin receptor. Overexpression of BRE inhibited TNF induced NF-κB activation, indicating that the interaction of BRE protein with the cytoplasmic region of p60 TNF receptor may modulate signal transduction by TNF.

LESSONS LEARNED ABOUT TNF SIGNALLING FROM KNOCKOUT MICE

The deletion of genes for p60 or p80 receptor have produced quite distinct effects in mice. Mice deficient in p60 receptor are resistant to endotoxin induced shock but are susceptible to infection byListeria monocytogenes.103 The p60 receptor was also found to control early graft versus host disease104 and to play an important part in septic shock and in protection from bacterial infection. Another study indicated p60 deficient mice are also resistant to TNF mediated toxicity.105 Interestingly, however, deletion of the gene for the p80 receptor in mice also decreased sensitivity to TNF.106 In addition, p80 receptor deleted mice had normal T-cell development though they exhibited depressed Langerhans cell migration and reduced contact hypersensitivity.107 In addition, a critical role of the p80 TNF receptor in organ inflammation independent of TNF, lymphotoxin α, or the p60 receptor was reported.108

Genes for the signalling proteins through which the p60 receptor mediates its effects have been deleted from mice. The deletion of TRAF2, FADD, FLICE, caspase-9, and the caspase-3 gene was lethal in mice,32 43 44 59-62 65 109 110 whereas deletion of Apaf-1 was not.97 Embryonic lethality suggests a critical role of FADD and FLICE in development. This role must be independent of TNF receptor as deletion of either p60 or p80 had no effect on the survival of the animals. Cells derived from FLICE or FADD knockout animals could be activated for NF-κB and JNK but not apoptosis, suggesting a critical role of FADD and FLICE in TNF induced apoptosis.

Conclusion

It is clear that a number of signalling molecules physically interact with the ICD of the TNF receptor either directly or indirectly (fig 6). Most of these signalling molecules were initially identified by using the yeast two hybrid interaction system. Their physical interaction was also confirmed by overexpression in the mammalian cell system. In most cases, the role of these molecules in TNF signalling was identified by activation of a signal on overexpression of the protein in the cell or by suppression of activity by a dominant negative form of the gene product. In a few cases a role for these signalling molecules in ligand induced signal transduction has been demonstrated. The cells derived from animals targeted with specific genes thought to be involved in the TNF signal transduction were found to be not entirely in agreement with the scheme shown (fig 6). Although deletion of the genes for FADD and FLICE produced the expected results, deletion/mutation of TRAF2, NIK, IKKα, and caspases did not, suggesting there are alternate pathways yet to be identified through which TNF mediates its signals.

Figure 6

A network of TNF signalling proteins leading to various cellular responses.

A series of proteins have been identified that bind to the DD of the p60 receptor, leading to various cellular responses including NF-κB and JNK activation and apoptosis. How p80 receptor that lacks the death domain and thus does not bind to most of these signalling proteins, activates JNK, NF-κB and apoptosis,111 remains to be investigated. In addition, there are indications that TNF receptor with TNF can mediate reverse signalling.112 How the TNF receptor activates TNF also remains to be understood. Nevertheless, within the past decade a great deal has been discovered about TNF signal transduction. This knowledge should assist us in designing better TNF antagonists, which may prove useful in treatment of cancer and inflammatory diseases including rheumatoid arthritis.

Acknowledgments

These studies were supported by The Clayton Foundation. I would like to thank Walter Pagel and Linda Ford for carefully reviewing the manuscript.

References

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