confers resistance to lung inflammation in mice (Meylan et al., 2008). Meylan et al. (2008) proposed that the interaction of TL1A with DR3 provides an early signal for Th2 cytokine production in lung inflammation and that DR3 could be a valuable target in the treatment of asthma.

4. THE DR6 SYSTEM

Although death receptor 6 (DR6) was identified already in 1998 on the basis of its similarity to TNF-R2, it is the by far least studied DD-containing receptor to date. This is most likely due to the fact that until very recently, a cellular ligand for DR6 has been elusive. In addition, unlike all other members of the DD-containing subfamily of receptors in which the DD is found at or close to the carboxy-terminus of the protein, the DD of DR6 almost immediately follows the transmembrane domain, and then there is an additional domain of approximately 150 amino acids and unknown function at the carboxyterminus of the protein. Thus the sequence of the two intracellular portions is inversed in DR6 as compared with the other five DD-containing receptors. This difference with respect to intramolecular positioning of the DD may be the reason why DR6 seems to recruit adaptor proteins in a manner different from that of the other DD-containing receptors. DR6 has been shown to be capable of engaging a signal transduction pathway that leads to the activation of NF-κB and JNK. However, overexpression of DR6 has been shown to induce apoptosis in a manner dependent on its DD. There are indications that this effect may be mediated via recruitment of TRADD and not FADD. However, the interaction with TRADD is a low-affinity interaction, and possibly DR6 needs a TRADD-related molecule or an additional adaptor protein to engage the cell death machinery. Taken together, it seems that the molecular interactions at the onset of DR6-induced signal transduction have largely remained in the dark, at least thus far.

Despite considerable efforts, to date no TNFSF member that binds to DR6 has been identified. However, in a recent study (Nikolaev et al., 2009), a non-TNFSF protein with an interesting etiology was described as a ligand of DR6. This protein is a specific proteolytically processed form of APP. APP is a transmembrane glycoprotein that undergoes shedding. APP is thought to be causally implicated in Alzheimer’s disease (AD). Trophic factor deprivation in neurons leads to cleavage of APP by β-secretase, followed by further cleavage of the released fragment by an unknown protease, thereby generating the N-terminal 35-kDa fragment of APP (N-APP), which is capable of binding to DR6. The capacity of N-APP to bind to DR6 was identified in COS cells and by an enzyme-linked immunosorbent assay–

like binding assay. Specificity of the interaction between N-APP and DR6 has been tested in pull-down assays showing that N-APP does not interact with any of the other DD-containing receptors. Binding of N-APP to DR6 triggers a caspase-dependent limited cellular destruction process. It has been shown that after trophic factor deprivation, N-APP release triggers DR6-mediated death of the neuronal cell body, which involves activation of caspase-3, whereas, interestingly, axon degeneration is mediated by caspase-6 in a Bax-dependent manner. The mechanism of this differential control of caspase activation between cell body and axons is at present completely unclear. It will be particularly interesting to understand how caspase-6 can be activated without involvement of caspase-3 and also in the absence of any apparent activation of caspases-8 and -10. Because DR6 is widely expressed in neurons as they differentiate and enter a proapoptotic state and APP is highly expressed on axons, and because AD is marked by neuronal and axonal degeneration, the study proposed an involvement of DR6 in loss of neuronal cell mass in AD.

In summary, although this study (Nikolaev et al., 2009) partially illuminates our understanding of DR6mediated processes, it also poses many basic questions regarding the mechanism of DR-mediated signaling behind. Thereby it exemplifies how little we still know about this thus far most elusive death receptor-ligand system, the biochemistry of its signaling pathways, and the physiologic role it may play. It will be exciting to follow the developments of this field as it may hold the key to the treatment of one of the worst neurodegenerative diseases we are faced with today. And who knows, maybe the TNF history will be repeated, and DR6 may even play a role in other neurodegenerative diseases.

5. FUNCTIONAL SPECIALIZATION BY SEQUENTIAL

SIGNALING COMPLEX FORMATION IN DEATH RECEPTOR

SIGNAL TRANSDUCTION

The receptor-associated signaling complexes described in the earlier sections of this chapter form at the plasma membrane. However, they are not the only signaling complexes that form in the cell when TNFSF ligands activate DD-containing TNFRSF receptors. After formation of the receptor-associated protein complex, referred to as complex I, biochemical changes within the complex that are not yet understood induce loss of affinity of the adaptor proteins FADD and TRADD for their respective receptors. Together with at least some of the factors they recruited to the respective receptors, they then form a secondary, cytoplasmic signaling complex, complex II, which can recruit further proteins to the liberated DDs of FADD or TRADD, respectively. Intriguingly, in both cases

complex II is capable of inducing the very signal that was not induced by the respective complex I; that is, complex II, derived from TRADD-binding receptors, induces signals that can lead to apoptosis, and complex II of FADDbinding receptors induces gene activation, resulting in proinflammatory signaling. However, when the primary signals from the respective complex I prevails, the outcome of secondary signaling from complex II is often neutralized by the very effects triggered by the primary complex (Figure 3-4).

This new concept was first introduced for TNF-R1 in a landmark study by Micheau and Tschopp (2003). They found that signaling by this receptor involves the formation of two sequential signaling complexes, leading to activation of transcriptional programs and induction of apoptosis, respectively. The first complex that forms at the plasma membrane when TNF cross-links TNFR1 induces biochemical reactions that ultimately result in the activation of transcriptional events, whereas the cytoplasmic complex II – although derived from complex I – is capable of inducing apoptosis, at least when signaling events induced by complex I do not impede this (Figure 3-4).

More specifically, release of TRADD from the receptor, together with the majority of the signaling proteins that it either directly or indirectly recruited to this complex, leads to the formation of complex II. Complex II then recruits FADD, presumably to the DD of TRADD, which is freed because it left the DD of the receptor behind. Then the initiator caspase-8 and -10 are recruited to FADD, and, initiated by this intracellular secondary DISC, the cell can now undergo apoptosis. However, the signaling outcome of complex II depends on the result of complex I signaling; the gene-inducing events triggered by complex I of the TRADD-binding receptors in most cases lead to an increase in the expression of cFLIP. This then interferes with activation of caspase-8 and-10 at complex II, the cytoplasmic DISC (Figure 3-4), with the result that the cell does not die. It is very likely that the same events are true for DR3 signaling; however, this has not yet been studied.

For the FADD-recruiting receptors, it has in turn been shown that on release of FADD from the TRAIL DISC (i.e., the complex I in this system), complex II recruits TRAF2, cIAP1/2, RIP, NEMO, and possibly a number of other proteins – including TRADD – required to induce the activation of NF-κB, as well as the JNK and p38 MAP kinase pathways (Varfolomeev et al., 2005). Obviously, this pathway would only be induced in a productive manner in cells in which proper execution of the apoptotic cell death program, which is usually quite rapid, would be blocked. Thus this pathway is not the primary reaction of the cell to the stimulus provided by CD95L or

TRAIL but must be regarded as the secondary, alternative outcome of activation of the direct apoptosis inducers.

The spatial and temporal separation of different biochemical tasks into discrete signaling complexes that act in different cellular compartments (i.e., at the plasma membrane versus in the cytoplasm) and are activated sequentially in a hierarchical manner is striking and makes biological sense. In case the first signal prevails, you do not need the second one, and in fact it should probably be minimized. However, if the primary signal is not achieved, then the second, deferred signal kicks in and opens new avenues to achieve a very different, seemingly opposing outcome.

One may ask why the system does not try to achieve the same outcome in its second attempts. Perhaps it does, but in an unexpected manner. When a certain outcome of signaling is not achieved, then this means there is a problem in its execution. In such situations, biology often follows a new path to achieve the same physiologic end point. If a cell that should die does not do so, this means trouble. Therefore, the activation of proinflammatory signaling to attract other cells of the innate immune system (and, possibly later, also adaptive immune cells, which may be able to handle the situation around the cell that did not die) seems like a very sensible thing to do. With respect to the TRADD binders, if proper immunostimulatory signaling, as supervised by induction of cFLIP, cannot be achieved, then the induction of the cell death program kicks in. This cell death is apoptotic. Apoptotic cell death can either be immunogenic or nonimmunogenic. It is not clear which type of cell death is induced by TNF when the geneinductive path does not prevail. However, our prediction would be that it is the immunogenic one and that thereby the same biological outcome (i.e., the creation of an immunostimulatory, pro-inflammatory environment) could be achieved, yet via a path very different from the originally intended apoptosis. Thus in the end it appears that cellular suicide and inflammation may be linked closer to each other than it first seemed.

6. CONCLUDING REMARKS AND OUTLOOK

Since the discovery of TNF, many truly exciting developments have characterized the research into the function of TNFR-like receptors that are capable of inducing cell death. The level to which the study of the different receptor-ligand systems has pushed our understanding of the biochemical processes that are at the heart of the induction of the specific cellular responses associated with both their physiologic and pathological consequences is astonishing. However, these studies have also made it very clear that we will

have to understand them in even more detail. This means that we have to be able to study them in a timeresolved manner, and we have to get close-up pictures of parts of the system to unravel the molecular simplicity behind the processes that today still seem so incredibly complex. By determining the biochemical interactions at the molecular, in some cases submolecular, atomic level, we will ultimately be able to unravel their mysteries and interpret them correctly.

Set apart from the beauty of solving the mysteries of molecular interactions and their connection to biology, the most important achievement of the research into the function of the receptors and ligands discussed in this chapter is the translation of knowledge on the basic biochemical mechanisms into clinical practice. Astonishing achievements in the TNF field have been made to date. However, a number of additional new avenues into clinical application are currently being followed within the TNF and TNFR superfamilies, and some of them have been touched on in this chapter. It seems we are far from having appreciated the full potential of the death receptor-ligand systems as targets for the treatment of diseases. So this fascinating and hopefully rewarding journey continues.

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