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The LTβR Signaling Pathway

and *.

* Corresponding Author: Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, California 92121, U.S.A. Email: cware@liai.org

The lymphotoxin-β receptor (LTβR, TNFRSF3) signaling pathway activates gene transcription programs and cell death important in immune development and host defense. The TNF receptor associated factors (TRAF)-2, 3 and 5 function as adaptors linking LTβR signaling targets. Interestingly, TRAF deficient mice do not phenocopy mice deficient in components of the LTβR pathway, presenting a conundrum. Here, an update of our understanding and models of the LTβR signaling pathway are reviewed, with a focus on this conundrum.

Introduction

The lymphotoxin-β receptor (LTβR, TNFRSF3) signaling pathway activates responses controlling cellular differentiation, growth and death manifested in the formation and organizations of peripheral lymphoid organs, dendritic cell homeostasis, hepatic regeneration, interferon responses to pathogens, and death of mucosal derived carcinomas.1-3 The LTβR signaling activates gene transcription programs controlled in part by nuclear factor κB (NFκB) and others, which help orchestrate these diverse processes. Biochemical and genetic evidence supports a model of signal propagation from the LTβR to kinase complexes that activate distinct forms of nuclear factor κB (NFκB). The TNF receptor associated factor (TRAF)-2 and 5 appear to function as adaptors linking LTβR to transcriptional programs for NFκB, and TRAF3 to cell death, however, this is not always apparent from genetically defined phenotypes, presenting an interesting conundrum.

The LTβ Receptor

Gene

The lymphotoxin β receptor (LTβR) was first identified as a transcript containing a cysteine-rich tumor necrosis factor receptor (TNFR)-like domain in somatic cell hybrids.4 The LT βR gene resides on chromosome 12p13 forming a tripartite locus with TNFRI and CD27. Interestingly, this chromosomal region is thought to have duplicated giving rise to the cluster of TNFR genes located on Chromosome 1p36. The mRNA encodes a 435 amino acid protein sharing 41% and 46% homology with TNFRI and TNFRII, respectively. Mouse LT βR maps to chromosome 6 in a region in conserved synteny with human chromosome 12p135 and the encoded protein is highly homologous to the human version with 68% amino acid sequence identity.

Protein Structure and Expression

The LTβR is a type 1 single transmembrane protein with a theoretical mass of 46.7 kDa, however, the observed mass is 61 kDa suggesting that the two potential N-glycosylation sites are utilized. LTβR has a ligand-binding ectodomain containing four cysteine-rich pseudo repeats characteristic of the TNF motif. The cytoplasmic domain is 175 residues, containing a proline-rich membrane proximal region, grouping it with TNFR family members that bind directly to TRAF proteins, including CD40, CD30, HVEM and CD27. This is in contrast to those TNFR with death domains such as TNFRI, Fas and TRAIL Receptors 1 and 2 that require an intermediate adaptor (e.g., TRADD) to engage TRAF. Like other TNFR, LTβR has no intrinsic kinase or other enzymatic activities encoded by its cytosolic domain.

Expression of the LTβR is constitutive on most cells with a promoter region more like a typical house keeping gene. LTβR is expressed on stromal cells in lymphoid tissue6 but is also expressed on myeloid lineage cells,5,7 blood monocytes, alveolar macrophages,8 mast cells9 and dendritic cells.10,11 Most adherent primary cells and cell lines including normal diploid fibroblasts, bronchial airway epithelial cells, the follicular dendritic cell line FDC-1, U937 promyelomonocytic cell line, HT-29 colon adenocarcinoma line, HeLa cervical carcinoma line and HEK 293 embryonic kidney cells express LTβR.6 A prominent feature of LTβR expression is the conspicuous absence on T and B lymphocytes and NK cells. By contrast, the ligands for LTβR are often expressed by T and B cells. This expression pattern indicates that signaling may be unidirectional, and for LTαβ, which is not cleaved into a soluble form, requires cell to cell contact between the lymphocyte to the LTβR-bearing cell to transmit signals.

Ligands

LTβR binds two members of the TNF superfamily (Fig. 1), the LTαβ heterotrimers, and LIGHT (lymphotoxin-like, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes; TNFSF14). Two distinct lymphotoxin heterotrimers, LTα1β2 and LTα2β1, can be formed between LTα and LTβ, which are membrane bound via the LTβ subunit (TNFSF3).12 LTβR also binds another closely related ligand, LIGHT.13 The binding of LTα1β2 is specific for LTβR, whereas LTα2β1 also binds TNFRI and TNFRII, and LIGHT also engages the herpes virus entry mediator (HVEM, TNFRSF14). The LTβR binds the membrane forms of these ligands, the recombinant soluble forms of LTα1β2 and LIGHT with high affinity, but relatively weakly to soluble LTα2β1. DcR3 is a soluble protein, which can compete with LIGHT for binding HVEM or LTβR.14 A novel inhibitory cosignaling membrane protein, B and T lymphocyte attenuator (BTLA) was identified as a receptor for HVEM.15,16 The importance of this pathway is underscored by the finding that two evolutionarily distinct herpesviruses target this pathway.17,18

Figure 1. The Immediate TNF/LT family.

Figure 1

The Immediate TNF/LT family. The TNF ligands are type II transmembrane proteins that form trimers that interact with their corresponding TNF receptors. The arrowed lines indicate the specific binding interactions between ligands and receptors. TNFR1 contains (more...)

LTβR Signaling Pathway

Binding of LTβR by its trivalent ligands induces an ordered aggregation or “clustering” initiating signal transduction pathways. Receptor signaling can also be initiated by anti-LTβR antibodies that mimic receptor “clustering” or by overexpression of the receptor in cell lines. Ligation of the LTβR activates gene transcription via nuclear factor κB (NFκB) and c-Jun N-terminal kinase (JNK) pathways, and, in some cell lines, can activate apoptosis (Fig. 2). Although it can induce death, LTβR does not contain a death domain. The LTβR interacts directly with members of the TNF receptor-associated factors (TRAF) family of zinc RING finger proteins.19 Ligation of the LTβR by LTα1β2 or LIGHT rapidly recruits TRAF to the cytosolic domain.20 TRAF 2, 3 and 5, but not TRAF1 or 6 have been shown to bind directly to its cytosolic domain.20-22 Interestingly, the hepatitis C virus core protein also directly binds to this region.23 TRAF4 has been reported to bind LTβR in in vitro studies, but this has not been confirmed in vivo.24

Figure 2. The LTβR Signaling Pathway.

Figure 2

The LTβR Signaling Pathway. LTαβ2 or LIGHT both engage the LTβR initiating receptor clustering, and recruitment of TRAF2, 3 or 5. LTβR activation leads to activation of both the RelA/p50 and RelB/p52, which activate (more...)

Studies of LTβR cytoplasmic tail mutants suggest that the regulation of LTβR signaling is complex with discrete regions controlling different aspects of signaling.25 A truncation mutant which removes the TRAF binding region (Δ389) greatly diminished receptor-induced NFκB activation and cell death, however, further truncation revealed an adjacent region (Δ379) that negatively regulated signaling, and a cryptic NFκB-activation region (345-359) that is independent of TRAF binding. Additional regions mediated trafficking and self-association.

LT βR-Mediated NF κB Activation

In mammalian cells, the NFκB family of transcription factors consists of five members: RelA, RelB, c-Rel, p50/NFκB1 and p52/NFκB2.26,27 p50 and p52 are the products of the proteolytic processing of p105 and p100, respectively. Homo and hetero-dimers of NFκB family members are held inactive in the cytosol by inhibitors of κB (IκBs), such as IκBα, masking nuclear localization motifs. p105 and p100 also contain C-terminal IκB-homologous inhibitory regions and retain some NFκB dimers within the cytosol. The phosphorylation and ubiquitin-dependent degradation of the IκBs and subsequent nuclear translocation of NFκB occurs in response to a wide variety of stimuli. These stimuli trigger the activation of the IκB kinase (IKK) complex, which consists of two catalytic subunits (IKKα and IKKβ) and a regulatory subunit IKKγ/NEMO, and is responsible for initiation the degradation of IκB.28

Two separate, yet related mechanisms activate distinct forms of NFκB: RelA/p50 and RelB/ p52.29 The first defined (or classical) pathway is activated by IL-1R and TNFR1, and is characterized by the activation of the NFκB dimer Rel A (p65)-p50 in a process occurring within minutes that is initiated through IKKβ-mediated phosphorylation of IκB, ubiquitination and proteosome-dependent degradation. The more recently identified noncanonical (or alternative) pathway involves degradation of p100 forming the active p52 component, which often associates with RelB. This process of RelB/p52 accumulation in the nucleus is optimal several hours after the stimulus. Activation of p100 is independent of IKKβ and γ, but dependent upon IKKα and the NFκB-inducing kinase (NIK).30 Overexpression of NIK has been shown to trigger the processing of p100 to p52 by site-specific phosphorylation and subsequent ubiquitination and degradation of the IκB-like C-terminus of p100.31

NIK, a critical component in the activation of the p100 processing NFκB pathway by LTβR, was identified as a TRAF2-interacting protein that contained a serine/threonine protein kinase motif resembling MAP3K proteins.32 Overexpression of NIK leads to activation of NFκB, but not JNK,33 and NIK is required for activation of the alternative pathway characterized by p100 processing.30,34-37 More recent studies demonstrate that depletion of NIK by siRNA blocked the activation of both the classical and alternative NFκB pathways by CD27, CD40 and BAFF-R, but not by TNFRI, which is restricted to activating RelA/p50 complex through the classical pathway.38 These findings suggest a role for NIK in facilitating the activation of both NFκB pathways by receptors that harbor that capacity, but not in triggering the classical pathway by single NFκB-inducers like TNFRI.

The binding of NIK with TRAF1, 2, 3, 5 and 6 has been demonstrated in overexpression studies,32,33 and in the case of TRAF2, the interaction requires the carboxy-terminus of NIK (residues 624-947). Residues 735-947 of NIK are also required for interaction with IKKα.39 Mutation of glycine 855 to arginine in the C-terminus of murine NIK (G860R in human) causes alymphoplasia (aly) in mice. NIK in the aly mouse has an intact kinase domain and binds to TRAF2, 3 and 5, however, it fails to bind IKKα.40,41 The N-terminus of NIK contains a basic region (residues 127-146) and a proline rich region (250-317) shown to be a cis-acting negative regulatory domain by interacting with the C-terminal region of NIK and interfering with binding to IKKα.42 Liao et al reported that TRAF3 binds at the N-terminus of NIK within residues 78-84 (ISIIAQA), and targets NIK for degradation in the proteasome resulting in an inhibition of NIK-induced p100 processing.43 Treatment of cells with agonistic anti-CD40 antibody or soluble BAFF, but not TNF, lead to decreased TRAF3 levels, an increase in NIK levels and p100 processing, suggesting that TRAF3 acts as a negative regulator of NIK. Unfortunately, many of these studies have yielded conflicting data on the regions responsible for TRAF-NIK interactions perhaps because of an over-reliance on large deletion mutations and forced overexpression systems. Alternatively, NIK may interact with different TRAF molecules at more than one region.

The kinase domain of human NIK resides between residues 366 and 624, and is required for activation of NFκB.32 Autophosphorylation of NIK on threonine 55939 in a manner dependent upon the active-site lysine and an adjacent lysine (KK429/30AA)32 is required for subsequent phosphorylation of IKKα upon its binding to NIK. NIK is thought to serve as a docking molecule recruiting IKKα to p100. The NIK aly mutant exhibits reduced activity in promoting the IKKα/ p100 association, however recruitment of IKKα to p100 by NIK is not dependent upon kinase activity of IKKα. Similarly, kinase inactive NIK promotes binding of IKKα to p100. After recruitment to p100, IKKα phosphorylates serine residues at the N- and C-terminus of the protein (S99, 108, 115, 123 and 872). This phosphorylation is a prerequisite for ubiquitination and degradation of p100 mediated by the beta-TrCP ubiquitin ligase and 26S proteosome.44

In addition to NIK being essential for LTβR-induced NFκB activation, TRAF2 was recently shown to play a key, nonredundant role in LIGHT-LTβR signaling.45 Murine fibroblast lacking TRAF2 failed to activate both NFκB pathways, as well as JNK, in response to treatment with LIGHT. Defects in NFκB or JNK activation were not observed in cells deficient in TRAF5 or RIP, a death domain kinase known to associate with TNFRI. Moreover, following LIGHT treatment, TRAF2 was recruited to the LTβR complex along with IKKα and IKKβ at early times, while only TRAF2 and IKKα were present at 8 hours post treatment. These observations suggest that TRAF2 may recruit different downstream targets to initiate distinct pathways: IKKβγ for activation of the classical NFκB pathway and IKKα for activation of the noncanonical pathway. That TRAF5 does not appear to be essential for LTβR-mediated NFκB or JNK activation may be explained by tissue-restricted expression of these adaptor molecules since these studies were limited to fibroblasts.

JNK Activation

JNK (c-Jun N-terminal kinase or stress-activated kinase) is activated by many apoptosis-inducing stimuli and is thought to be an important mediator of this process.46,47 JNK activation is involved in pathways needed during embryogenesis, cell proliferation and immunological responses. Most members of the TNF superfamily are strong inducers of JNK activation: TNF treatment dramatically elevates JNK activity, as does overexpression of HVEM48 and related receptors. LTβR-mediated activation of JNK has been reported in 293HEK that overexpress the receptor and in HeLa cells and mouse embryo fibroblasts treated with LIGHT.45,49 LIGHT-initiated JNK activation in HeLa cells and fibroblasts was shown to be dependent on TRAF2 but did not require TRAF5. What role, if any, TRAF3 may play in JNK activation initiated by LTβR has yet to be determined.

Cell Death

Treatment of some human adenocarcinoma cell lines with recombinant LTα1β2 or agonistic antibody against LTβR in combination with interferon γ (IFN-γ) results in cell death.20,50 Cell death and growth arrest of tumor cells is also observed with CD40 and CD30, indicating nondeath domain TNFR can impinge signals on cell survival. Similarly, treatment of the adenocarcinoma line HT29 with LIGHT and IFN-γ induces death in an LTβR-dependent, but HVEM-independent, manner even though the cells express both receptors.51 TRAF3 is important for LTβR-mediated apoptosis, as dominant negative mutants of TRAF3 abrogate cell death induced by treatment with LTα1β225 or LIGHT51 but do not alter NFκB activation. Overexpression of the self-association domain of LTβR (324-377) in HT-29 or HeLa cells results in IFN-γ-independent cell death that also is blocked by dominant negative TRAF3 or by caspase inhibitors.52

The death signaling mediated by death domain-containing receptors, such as TNFRI and Fas, can be inhibited efficiently by caspase inhibitors, whereas, LIGHT-LTβR-induced cell death is only partially affected.53,54 In contrast, free radical scavenger carboxyfullerenes can completely inhibit LIGHT-LTβR-induced death indicating the important role for reactive oxygen species (ROS) in this process. ASK1 (apoptosis signal-regulating kinase 1/ MEKK5) can be activated in response to various stress signals, including ROS, and in response to LTβR signaling and the subsequent production of ROS.55 LTβR-mediated activation of ASK1 is dependent on TRAF 3 and 5, but not TRAF2 as it is in the case of TNFRI-activation of ASK1.

Analysis of endogenous LIGHT-LTβR complexes from U937 and HEK293 cells revealed the association of TRAF2, cIAP1 and Smac, in addition to TRAF3, with the receptor. cIAP1, a cellular member of the inhibitor of apoptosis (IAP) family, was first identified as part of the TNFRI complex via its association with TRAF2.56 Smac, whose function is to antagonize the inhibition of caspases by IAP and thus promote apoptosis, is recruited to LTβR in a cIAP-dependent manner and potentiates receptor-induced apoptosis.57 Nonetheless, the intermediates connecting LTβR signaling to death pathways involving either caspases or radicals remain elusive.

The LT βR-TRAF3 Complex

The cytoplasmic domain of LTβR contains a large proline-rich region (˜60 residues) near the C terminus that is responsible for initiating NF-κB activation and apoptosis.25 A series of deletion mutants localized the binding site for TRAF2, TRAF3 and TRAF5 to a minimal region (389PEEGDPG395) with limited homology to the TRAF-binding motifs (PXQXT/S) similar but not identical to other TNFR family members including CD40, HVEM, CD27 and BAFF-R. Structural studies of TRAF3 in complex with a fragment of the cytoplasmic domain of LTβR refined the TRAF3 binding motif to be 388IPEEGD393 (Fig. 3),58 and demonstrated that the primary intermolecular contacts are made in the same surface binding crevice on TRAF3 that accommodates CD4059 and TANK (TRAF-associated NF-κB activator or I-TRAF),60 as well as BAFF-R (B cell-activating factor belonging to the TNF family receptor).61

Figure 3. Structure of the LTβR/TRAF3 complex.

Figure 3

Structure of the LTβR/TRAF3 complex. Upper left panel) Schematic representation of the TRAF3 trimer is shown as a ribbon diagram with each subunit colored separately. Each subunit binds one LTβR molecule, which is represented as a ball-and-stick (more...)

Mutational analysis of the residues within the binding motif of LTβR and the binding crevice on TRAF3 revealed interesting distinctions between the interactions of LTβR with TRAFs 2, 3 and 5 and those of TRAF3 with LTβR, CD40 and TANK.58 Mutation of Pro387 to alanine led to a loss of TRAF5 binding, but not TRAF2 or TRAF3. Similarly, mutation of the two adjacent glutamates, Glu390-Glu391, resulted in a loss of TRAF5 binding and reduced TRAF3 binding but did not affect TRAF2 binding. Only when Glu390-Glu391 and Asp393 were simultaneously mutated was binding by each of the three TRAF affected. These studies suggest that the molecular contacts of these TRAF with LTβR are not identical. The contact residues with LTβR in the TRAF3 binding crevice were compared to those with CD40 and TANK. Binding of each molecule was disrupted upon mutation of either of the phenylalanines (Phe448 and Phe457) in the hydrophobic pocket of the TRAF3 binding crevice to glutamic acid. Mutation of Tyr395 to alanine in TRAF3 reduces its binding to CD40 and TANK while abolishing binding to LTβR. An arginine to alanine substitution at residue 393 reduced binding to each protein. Other residues in TRAF3 proved to be important for CD40 and TANK binding but not LTβR, most notably some of the serine residues in the serine tong (Ser454-456) whose mutation affects binding to CD40 and TANK but not LTβR.

The LTβR, CD40 and the downstream regulator TANK each bind to the same crevice on TRAF3. This observation stimulates an important question regarding the specificity of binding recognition and the trigger of the LTβR signaling process. These three molecules bind at the same surface pocket, and similarly, peptides bearing the motif PxQxT from a number of TNFRs bind to the homologous crevice on TRAF2. TRAF3 appears to contain ‘hot spots’ corresponding to residues that provide the same principal contacts for each of several different binding partners mediated by adjustments of side chains. ‘Plasticity’ or ‘flexibility’ of residues is apparent in the molecular interactions62,63 and may influence binding affinity. In the case of LTβR, signaling through two different NFκB pathways30 may involve similar adaptations at the interface, with distinct responses from the adaptable TRAF molecule. At least 18 different receptors in the TNFR family can engage TRAFs, and others in the IL-1R family, suggesting that selection of an adaptable binding crevice in TRAF3 may be a parallel evolutionary event to compensate for gene duplicative mechanisms driving expansion of the TNFR and IL-1R family. The flexibility of the TRAF binding site may also represent an advantageous evolutionary adaptation, serving as a defense against more rapidly mutating viral pathogens that target TRAF components of the TNFR pathway such as EBV LMP1 protein.21,37

Genes Induced by LT βR

Through its activation of NFκB and JNK, the LTβR plays critical roles in inflammation and lymphoid organogenesis. LTβR signaling induces IKKα-dependent expression of the chemokines CCL19 (ELC), CCL21 (SLC), CXCL3 (BLC) and CXCL12 (SDF-1a) and the cytokine BAFF.30 ELC and SLC are thought to play important roles in the organization of lymphoid organs, while SDF-1 is important in the early stages of B cell development.64,65 Stimulation of the LTβR also increases expression of CCL4 (MIP-1β) and CXCL2 (MIP-2), inflammatory chemokines, and their expression is enhanced in the absence of IKKα, suggesting that the IKKα-dependent pathway suppresses LTβR-mediated induction of MIP-1β and MIP-2.30 Another in vitro study demonstrated that LTβR activation in HEK293 cells increased IL-8 promoter activity and lead to IL-8 release in a manner requiring NFκB and AP-1 binding sites located in the IL-8 promoter.49 LTβR or TNFR1 signaling can activate gene expression of interferon β in diploid fibroblasts but only in the context of virus infection. Recent studies have focused on genes induced by LTbR in vivo in specific tissue systems. In the follicle-associated epithelium (FAE) that overlies Peyerαs patches, expression of the chemokine CCL20 (6Ckine) is mediated, at least in part, by LTβR signaling in an NFkB-dependent manner.66 Additionally, Huber et al67 recently reported a set of LTαβ -responsive transcripts in FDC-enriched cell clusters. This set included transcripts for the cell adhesion related proteins GlyCAM-1 and MFG-1, the chemokine CXCL13, the ECM component cochlin, the apoptosis related protein clusterin and the proteolysis protein serpin a1a.

Genetic Phenotypes of LT and TRAF Deficient Mice

Deletion of LTα in the mouse was the first identified specific deficiency in lymph node formation. 68,69 Both LTβR and LTβ, but neither TNF nor its two receptors, exhibited this phenotype providing genetic evidence that LTαβ and LTβR are involved in a common signaling pathway. We now appreciate that at least two cell types are necessary for lymph node formation, the LTαβ expressing lymphoid tissue inducer cell (a CD4+ IL7Rα+ non T non B lymphocyte) and embryonic stromal organizer cell expressing the LTβR (reviewed in ref. 70). The lymph node deficient phenotype is found in several other knockout mice delineating the framework of a signaling pathway involved in mammalian organ development. Ikaros, ID2 and RORγt are transcriptional regulators essential for lymphocyte progenitors or lymph node inducer cells to develop.71-75 Mice deficient in certain members of the NFκB activation pathway, including NIK, IKKα and Rel B are missing lymph nodes. Potential target genes activated by the LTαβ-LTβR-NFκB pathway includes the lymphocyte organizing chemokines, such as CXCL13, CCL19 and CCL21, and their receptors CXCR5 and CCR7 whose knockouts also show defective lymphoid organogenesis.

We now appreciate the formation of lymph nodes (lymph node) is a complex process that is illustrative of the multiple components associated with the LT signaling pathway. An effective immune response requires transient interactions between multiple cell types that are facilitated in secondary lymphoid tissue (spleen, lymph nodes and Peyerαs patches) by a specialized microarchitecture that positions populations of cells in discrete regions. The development and homeostasis of secondary lymphoid tissue microenvironments require signaling by the LT/TNF related cytokines (reviewed in refs. 70, 76).

TRAF2 deficient mice display premature death due in part to severe runting.77 In addition, TRAF2 deficient cells are highly sensitive to TNF-induced cell death. TNF-mediated toxicity through TNFRI contributes significantly to the survival defects in TRAF2 deficient mice because mice lacking both TRAF2 and TNFRI have increased survival.78 Restriction of the TRAF2 deletion to B cells revealed that TRAF2 acts as a positive mediator of canonical NFκB activation while also serving as a negative regulator of the noncanonical pathway. While this role for TRAF2 was demonstrated in the context of CD40 signaling in B cells, it may also translate to the function of the adaptor in LTβR signaling in other cell types.

Mice lacking TRAF3 have poor perinatal and neonatal survival,79 and, similar to TRAF2 deficient mice, exhibit severe runting and hypotrophy of the spleen and thymus. TRAF3 deficient mice have normal lymph nodes, and the immune system is compromised in T-cell-dependent antigen responses.79

TRAF5 is a close functional and structural homologue of TRAF2, with a more restricted expression pattern compared to the widely expressed TRAF2. Deletion of TRAF5 in mice did not cause perinatal lethality, rather led to more specific defects in CD40- and CD27-mediated lymphocyte activation. As was seen in TRAF2 deficient animals, TNF-mediated NFκB activation was only modestly affected in mice lacking TRAF5.80 The finding that TRAF2 and 5 double knockout animals did exhibit severe defects in NFκB activation suggests that their roles are partially redundant.81

The LT βR-TRAF Conundrum

An interesting but perplexing discordance between genetic and biochemical evidence arises with the TRAF adaptors. LTβR does not engage TRAF6, yet unexpectedly a lymph node deficiency was found in TRAF6-/- mice.82 Moreover, the TRANCE/RANK Ligand-RANK system, which utilizes TRAF6 as an adaptor, when genetically deleted, also revealed a lymph node-deficient phenotype.83 However, Yoshida and colleagues revealed that the RANK/TRAF6 pathway is required for the induction of LTαβ on the lymphoid tissue inducer cell, thus accounting for the discordance with the biochemical data (LTβR does not bind TRAF6).84

Ligation of LTβR recruits TRAF3 to the receptor under normal cellular physiological conditions,20 which is the earliest step identified in signaling. From these types of biochemical studies, TRAF2, 3 and 5 are implicated in the LTβR signaling mechanism, yet deletion of these TRAF genes in mice (including double KO of TRAF2 and 5) failed to disrupt lymph node development (however other dramatic phenotypes are present).77,79,80 TRAF5-/- mice do not phenocopy LT deficient mice, but do with Ox40 and CD27.85 TRAF2 and 3 deficient mice are both neonatal lethal, which has provided technical limits to resolving this question. TRAF2-/- deficiency results in increased TNF production and liver failure, and is partially restored by crossing onto TNFR1-/- mice; TRAF3-/- is not. Thus, LTβR, NIK, IKKα and RelB are clearly linked phenotypically, but TRAF2 and 3 are not, at least to the most obvious LTβR associated phenotypes. The situation remains an intriguing puzzle.

What then is the role of TRAF2 and 3 in LTβR signaling and how can the lymph node phenotype be TRAF2-independent if TRAF2 is required to activate p100 processing and Rel B? Biochemical and genetic evidence supports a model in which LTβR ligation activates NFκB2 via a stepwise event involving the serine kinases NIK and IKKα, which control the proteolytic processing of p100→p52.30 What remains mysterious is how signaling is propagated to NIK and what role TRAF2 and 3 play in this process, if any. One possibility is that TRAF proteins may not function directly as adaptors that propagate signals but rather as regulators restraining the activity of key enzymes or regulators. TRAF3 and NIK are preassociated in an inactive complex and not with LTβR. In this scenario the ligated LTβR cytoplasmic domain binds to the TRAF3 crevice acting to competitively displace NIK, releasing those molecules to interact with their partners or substrates to propagate the signaling event, e.g., liberating NIK to phosphorylate protein substrates, such as IKKα kinase, setting in motion the conversion of p100→52. Alternatively, TRAF independent mechanisms that activate NFκB may account for these developmental phenotypes, which is supported by LTβR deletion mutants that activate NFκB reporter but fail to bind TRAF2, 3 or 5.25 The phenotypes of the TRAF deficient mice are complex in part because TRAF family is utilized by multiple TNFR and IL1-R and phenotypes controlled by these other pathways may be accumulatively displayed in the TRAF deficient mice.

The cell survival and proliferation phenotype displayed by TRAF2 and 3 deficient mice may have some counterpart in LT deficient mice. In addition to the selective death induced signaling by the LTβR in carcinomas, LTβR has recently been shown to provide positive cellular proliferation signals in the context of adult organs. LTβR is needed to maintain the homeostasis of CD4+ myeloid dendritic cells within lymphoid organs,11 a phenotype also seen in RelB deficient mice. The proliferation of hepatocytes may also require LTαβ-LTβR signaling specifically during hepatic regeneration, but not hepatic organogenesis.86 In addition, LTβR signaling is needed to protect T and B cells from death during herpesvirus infection, but through an indirect mechanism suggested to involve interferon β signaling.87 These cell death and proliferation phenotypes associated with the LTβR present an intriguing correlation, however the link between the LTβR and the TRAF molecules remains elusive.

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