Sometimes size really does not matter: recent papers on the tumour necrosis factor (TNF)-like ligand, TWEAK (tumour necrosis factor-like weak inducer of apoptosis), and its diminutive 129 amino-acid receptor, FN14 (fibroblast growth factor (FGF) -inducible 14 kDa protein), describe an impressive and potent range of biological effects. The TNF superfamily (TNSF) receptor FN14 has a small cytoplasmic domain, and the limited similarity of its extracellular domain to the cysteine-rich domains of the TNFSF receptors was only appreciated retrospectively when it was discovered that it bound TWEAK.1 Recent work has demonstrated that TWEAK knockout animals have an activated immune system that is skewed by elevated numbers of natural killer (NK) cells and responds lethally to bacterial endotoxins, but also shows a powerful ability to prevent tumour growth.2 Other topical papers show that TWEAK and FN14 work to promote liver regeneration following injury and prevent the differentiation of a diverse range of cell types. The role of TWEAK and FN14 in cancer has yet to be conclusively established, but several groups have observed high expression of FN14 in cancerous tissues, and demonstrated that it can promote cellular functions important for tumorigenesis, including cytokine production, cellular proliferation, survival, migration and angiogenesis.3

TWEAK is a type II transmembrane protein (extracellular C-terminus) that can undergo proteolysis to produce an active soluble homotrimer,4, 5 whereas FN14 is a type Ia transmembrane protein.6 In some respects the potent effects of the ligand TWEAK are reminiscent to those of TNFα: both factors appear to play roles in inflammation, and both have the capacity to induce cell death, although usually in conjunction with another stimulus. Responses to TNFSF ligands can be complex because several ligands often bind to the same receptor and vice versa. For example, both the human ligands TNFα and LTα bind the receptors TNFR1 and TNFR2, whereas LTα also binds HVEM. In fact, more than half of the TNSF ligands bind to at least two receptors.7 One of the amazing things about the TWEAK/FN14 system is that it is a simple, single ligand, single receptor system7 in which the intracellular signalling domain of FN14 is only 28 amino acids. It remains possible that another TWEAK receptor exists8 which might help to explain the diverse range of effects that TWEAK produces, but this is yet to be conclusively established.

A Role for TWEAK and FN14 in the Wound Response?

FN14 is highly expressed in most newborn mouse tissues,6 but the role of FN14 and TWEAK in development appears to be minimal, as both FN14 and TWEAK knockout mice are viable and show no significant abnormalities.2, 9 Because FN14 is a serum and growth factor (FGF1, FGF2) inducible immediate-early response gene in fibroblasts,6 and FGF2 is required for rapid wound repair,10 the TWEAK/FN14 ligand receptor pair might be involved in co-ordinating tissue responses to wounding. In this context it makes sense to have a ligand that promotes cellular migration and proliferation, inhibits differentiation, promotes angiogenesis, and influences the innate and adaptive immune response.

The potential biological role of TWEAK and FN14 in the wound response of normal tissues was initially supported by several studies demonstrating that TWEAK is angiogenic1, 11, 12 and that FN14 expression is elevated in the proliferating endothelial and smooth muscle cells of damaged rat arteries.1 More recently it has been shown that TWEAK can associate with another novel secreted angiogenic factor, VG5Q, which is mutated in the vascular disease Klippel–Trenaunay syndrome,13 but, the biological significance of this interaction has yet to be elucidated. TWEAK and FN14 have also been implicated in promoting cellular proliferation and inhibiting differentiation. For example, TWEAK enhances the proliferation and invasiveness of mammary cells, and inhibits their differentiation.14 Similarly, myoblasts in skeletal muscle, that are critical for muscle growth and repair, are stimulated by TWEAK and induced to proliferate, with a concomitant decrease in differentiation into myotubes.15 Hence, FGF2, a known inhibitor of myoblast differentiation and wound response mediator, may work through induction of the TWEAK/FN14 signalling system. In support of this notion it has been observed that TWEAK can synergize with FGF2 in promoting endothelial cell proliferation, migration and microvessel formation.11

In a similar way, it has been shown that TWEAK and FN14 may contribute to liver and nerve regrowth. FN14 gene expression is elevated during murine liver16 and sciatic nerve regeneration,17 and can promote neurite outgrowth. Recently it was shown that oval cells (which can become either hepatocytes or biliary epithelial cells) are induced to proliferate via TWEAK/FN14.9 In addition, transgenic mice overexpressing TWEAK develop periportal oval cell hyperplasia, and FN14 expression is increased in several human liver diseases such as chronic viral hepatitis and alcoholic liver disease.9

The Role of TWEAK and FN14 in Immune Regulation and Tumour Growth

Although TWEAK usually stimulates proliferation, it can also synergize with IFNγ to promote cell death in several cell types (see below). IFNγ inhibits erythroid differentiation, and this requires the action of TWEAK in conjunction with the other TNFSF ligands TRAIL and CD95L.18 Thus, as well as synergizing with IFNγ to induce cell death, in some situations TWEAK can mediate the effects of IFNγ. The relationship is even more complex than this, because TWEAK can regulate production of IFNγ in mice by decreasing NK cell numbers and by decreasing the amount of IFNγ that NK cells secrete once activated.2 Moreover, TWEAK appears to suppress macrophage IL-12 production. Because of this dual action by which TWEAK suppresses innate immune cell IFNγ and IL-12 production, TWEAK knock out mice are hyper-responsive to the bacterial endotoxin LPS, and die at doses that leave their wild-type counterparts unaffected.2

The ability of TWEAK to suppress innate immunity differs strikingly from that of its relative, TNFα, which augments the innate inflammatory response by stimulating the secretion of IFNγ and IL-12. In this regard, TWEAK's activities are more reminiscent of the TNFSF ligand TRAIL, because TRAIL-R knockout mice develop normal lymphocyte populations but possess enhanced innate immune responses. In particular, TRAIL-R knockout mice are able to clear murine cytomegalovirus infections better than wild-type mice, and this correlates with increased levels of IL-12 and IFNγ.19

The cytokines IL-12 and IFNγ also play key roles in influencing the transition from the innate to adaptive immune responses. Both IL-12 and IFNγ direct the development of the T helper 1 subtype (TH1), which is generally more effective at combating tumours than the TH2 subtype, because it induces CD8+ effector T cell responses rather than IgG responses. TWEAK knockout mice show a remarkable resistance to tumours, completely resisting establishment of syngeneic B16.F10 melanomas, and significantly impairing the growth of the more aggressive B16.BL6 tumour line.2 Part of the protection against these tumours is due to the increased NK cell response, but the TWEAK knockout mice also have an increased TH1 profile that probably contributes to the more efficient destruction of these tumours.2

One aspect of the TWEAK knockout mice that was not examined by Maecker et al.2 was the (presumed) absence of TWEPRIL, a hybrid protein that results from mRNA splicing in cis between exons coding for the cytoplasmic and transmembrane domains of TWEAK with the nearby exons that code for the extracellular C-terminal domain of APRIL.20 Although the function of this protein is largely unknown, it has been detected in T-lymphocytes and monocytes and may play a role in stimulating T- and B-cells.20 It will be important to ascertain whether loss of TWEPRIL contributes to the antitumour activity observed in the TWEAK knockout mice. An excellent starting point will be to examine the FN14 null mice for the TWEAK knockout phenotype.

Elevated FN14 or TWEAK levels have been observed in several human cancers, including those of the liver,16 brain,21 breast14 and kidney.22 If signalling from FN14 promotes cancer cell growth, angiogenesis, and confers survival advantages to tumour cells, then inhibition of TWEAK/FN14 signalling may contribute to tumour destruction. For example, overexpression of TWEAK in HEK293 cells substantially increases tumour growth and vascularization in athymic mice,22 and a recent study demonstrated that treatment of glioma cells with TWEAK significantly reduced camptothecin and TRAIL induced apoptosis in an FN14 dependent manner.23 The proposed mechanism was that FN14 signalling mediated the activation of NF-κB, which resulted in the upregulation of the antiapoptotic proteins Bcl-xL and Bcl-w. Similarly, TWEAK treatment promotes endothelial cell survival when grown in limiting media.11 If TWEAK and FN14 are required to promote cell growth, survival and angiogenesis, and suppress innate immune cell IFNγ and IL-12 production, then pharmacological inhibition of TWEAK/FN14 may be therapeutic for these cancers.

It has been suggested that the response to cancer resembles the response to wounding,24 and consistent with this hypothesis, FGF2 is upregulated in many tumour cell lines, and FGF2 knockouts show less metastatic cancer in a mouse prostate cancer model.25 Whether FN14 expression contributes significantly to tumorigenesis remains to be proven, but, because FN14 is an FGF2 responsive gene, it seems likely that expression of FN14 in tumours will be significant.

The Involvement of TWEAK and FN14 in Cell Death

An emerging role of TWEAK is in the growth suppression of various cell types, including erythroblasts,18 kidney mesangial cells,26 neuronal cells,27 NK cells2 and monocytes.28 TWEAK can act in concert with other TNFSF members TRAIL and CD95L, to mediate erythropoietic suppression by IFNγ.18 IFNγ treatment of immature erythroid cells upregulated TWEAK and FN14, in addition to TRAIL and CD95. Although addition of TWEAK alone was sufficient to significantly inhibit erythroblast growth and differentiation, the reversal of IFNγ-mediated erythropoietic suppression was most efficient when all three TNF-ligands were neutralized during IFNγ treatment. This is consistent with the observation that IFNγ induces production of TWEAK by peripheral blood mononuclear monocytes, which allows them to kill TWEAK sensitive tumour cells,28 whereas TWEAK treatment in combination with IFNγ can also initiate kidney mesangial cell apoptosis.26

Activated T-cells or autoreactive T-cells from individuals with systemic lupus erythematosus also show increased levels of TRAIL, TWEAK and Fas, that appear to mediate enhanced monocyte apoptosis.29 Antibody neutralization of any one of these three TNFSF ligands is sufficient to significantly inhibit T-cell-mediated monocyte toxicity, suggesting that all three play a pro-death role in monocyte clearance. Similarly, NK cell death mediated through exposure to TNFα, LPS or IFNγ was largely prevented via antibody-mediated blocking of either TWEAK or FN14.2

Unlike the receptors for TNF, TRAIL and Fas, the cytoplasmic domain of FN14 does not bear a death domain, so how does it activate the cell death mechanism? In Kym-1 cells TWEAK indirectly induces apoptosis through induction of endogenous membrane bound TNF, which mediates killing through TNFR1 activation.5 However, TWEAK-mediated killing of HT29 and HSC3 cancer cells appears to occur via a distinct pathway that results in the activation of caspase-8 and caspase-3, without activation of TNFR1.30 FN14 binds TRAF1, 2, 3 and 5, and TWEAK signalling induces TRAF2-dependent activation of both the canonical and noncanonical NF-κB transcription pathways31, 32, 33 which can regulate cytokine and pro-survival gene transcription. In particular, TWEAK induced NF-κB activation has been directly implicated in mesangial cell chemokine secretion,26 glioma cell survival23 and myoblast differentiation.15 However, as suggested above, it appears that TWEAK can also elicit pro-death signalling in some cell types. In this regard TWEAK induced activation of c-Jun N-terminal kinase signalling in HT29 cells and RAW264.7 macrophages has been observed.8

Conclusions

There is substantial evidence that TWEAK/FN14 signalling is increased as part of the wound response, and many of TWEAK's actions are likely to promote wound healing and tissue regeneration. In addition, TWEAK signalling appears to curtail innate immune responses, and influence the transition from innate to adaptive immunity, which could also be important as part of the wound response. These biological roles of TWEAK/FN14 may yet prove important for tumorigenesis, as suggested by the elevated levels of FN14 in human cancer specimens, and the potent ability of TWEAK knockout mice to resist tumour growth. The variety of effects elicited by the short 28 amino-acid cytoplasmic domain of FN14 is nothing short of amazing, and elucidating which proteins bind to it and determining how they influence TWEAK signalling will be the next interesting instalments.