Isabelle Carpentier

A20: central gatekeeper in inflammation and immunity.

Inappropriate functioning of the immune system is linked to immune deficiency, autoimmune disease, and cancer. It is therefore not surprising that intracellular immune signaling pathways are tightly controlled. One of the best studied transcription factors in immune signaling is NF-κB, which is activated by multiple receptors and regulates the expression of a wide variety of proteins that control innate and adaptive immunity. A20 is an early NF-κB-responsive gene that encodes a ubiquitin-editing protein that is involved in the negative feedback regulation of NF-κB signaling. Here, we discuss the mechanism of action of A20 and its role in the regulation of inflammation and immunity.

Are the IKKs and IKK-related kinases TBK1 and IKK-ε similarly activated ?

The IkappaB kinases (IKKs) IKK-alpha and IKK-beta, and the IKK-related kinases TBK1 and IKK-epsilon, have essential roles in innate immunity through signal-induced activation of NF-kappaB, IRF3 and IRF7, respectively. Although the signaling events within these pathways have been extensively studied, the mechanisms of IKK and IKK-related complex assembly and activation remain poorly defined. Recent data provide insight into the requirement for scaffold proteins in complex assembly; NF-kappaB essential modulator coordinates some IKK complexes, whereas TANK, NF-kappaB-activating kinase-associated protein 1 (NAP1) or similar to NAP1 TBK1 adaptor (SINTBAD) assemble TBK1 and IKK-epsilon complexes. The different scaffold proteins undergo similar post-translational modifications, including phosphorylation and non-degradative polyubiquitylation. Moreover, increasing evidence indicates that distinct scaffold proteins assemble IKK, and potentially TBK1 and IKK-epsilon subcomplexes, in a stimulus-specific manner, which might be a mechanism to achieve specificity.

ABINs: A20 binding inhibitors of NF-kappa B and apoptosis signaling.

ABINs have been described as three different proteins (ABIN-1, ABIN-2, ABIN-3) that bind the ubiquitin-editing nuclear factor-kappaB (NF-kappaB) inhibitor protein A20 and which show limited sequence homology. Overexpression of ABINs inhibits NF-kappaB activation by tumor necrosis factor (TNF) and several other stimuli. Similar to A20, ABIN-1 and ABIN-3 expression is NF-kappaB dependent, implicating a potential role for the A20/ABIN complex in the negative feedback regulation of NF-kappaB activation. Adenoviral gene transfer of ABIN-1 has been shown to reduce NF-kappaB activation in mouse liver and lungs. However, ABIN-1 as well as ABIN-2 deficient mice exhibit only slightly increased or normal NF-kappaB activation, respectively, possibly reflecting redundant NF-kappaB inhibitory activities of multiple ABINs. Other functions of ABINs might be non-redundant. For example, ABIN-1 shares with A20 the ability to inhibit TNF-induced apoptosis and as a result ABIN-1 deficient mice die during embryogenesis due to TNF-dependent fetal liver apoptosis. On the other hand, ABIN-2 is required for optimal TPL-2 dependent extracellularly regulated kinase activation in macrophages treated with TNF or Toll-like receptor ligands. ABINs have recently been shown to contain an ubiquitin-binding domain that is essential for their NF-kappaB inhibitory and anti-apoptotic activities. In this context, ABINs were proposed to function as adaptors between ubiquitinated proteins and other regulatory proteins. Alternatively, ABINs might disrupt signaling complexes by competing with other ubiquitin-binding proteins for the binding to specific ubiquitinated targets. Altogether, these findings implicate an important role for ABINs in the regulation of immunity and tissue homeostasis.

Expression, biological activities and mechanisms of action of A20 (TNFAIP3).

A20 (also known as TNFAIP3) is a cytoplasmic protein that plays a key role in the negative regulation of inflammation and immunity. Polymorphisms in the A20 gene locus have been identified as risk alleles for multiple human autoimmune diseases, and A20 has also been proposed to function as a tumor suppressor in several human B-cell lymphomas. A20 expression is strongly induced by multiple stimuli, including the proinflammatory cytokines TNF and IL-1, and microbial products that trigger pathogen recognition receptors, such as Toll-like receptors. A20 functions in a negative feedback loop, which mediates its inhibitory functions by downregulating key proinflammatory signaling pathways, including those controlling NF-κB- and IRF3-dependent gene expression. Activation of these transcription factors is controlled by both K48- and K63- polyubiquitination of upstream signaling proteins, respectively triggering proteasome-mediated degradation or interaction with other signaling proteins. A20 turns off NF-κB and IRF3 activation by modulating both types of ubiquitination. Induction of K48-polyubiquitination by A20 involves its C-terminal zinc-finger ubiquitin-binding domain, which may promote interaction with E3 ligases, such as Itch and RNF11 that are involved in mediating A20 inhibitory functions. A20 is thought to promote de-ubiquitination of K63-polyubiquitin chains either directly, due to its N-terminal deubiquitinase domain, or by disrupting the interaction between E3 and E2 enzymes that catalyze K63-polyubiquitination. A20 is subject to different mechanisms of regulation, including phosphorylation, proteolytic processing, and association with ubiquitin binding proteins. Here we review the expression and biological activities of A20, as well as the underlying molecular mechanisms.

A20 inhibits LUBAC-mediated NF-κB activation by binding linear polyubiquitin chains via its zinc finger 7

Linear polyubiquitination of proteins has recently been implicated in NF‐κB signalling and is mediated by the linear ubiquitin chain assembly complex (LUBAC), consisting of HOIL‐1, HOIP and Sharpin. However, the mechanisms that regulate linear ubiquitination are still unknown. Here, we show that A20 is rapidly recruited to NEMO and LUBAC upon TNF stimulation and that A20 inhibits LUBAC‐induced NF‐κB activation via its C‐terminal zinc‐finger 7 (ZF7) domain. Expression of a polypeptide corresponding to only ZF7 was sufficient to inhibit TNF‐induced NF‐κB activation. Both A20 and ZF7 can form a complex with NEMO and LUBAC, and are able to prevent the TNF‐induced binding of NEMO to LUBAC. Finally, we show that ZF7 preferentially binds linear polyubiquitin chains in vitro, indicating A20–ZF7 as a novel linear ubiquitin‐binding domain (LUBID). We thus propose a model in which A20 inhibits TNF‐ and LUBAC‐induced NF‐κB signalling by binding to linear polyubiquitin chains via its seventh zinc finger, which prevents the TNF‐induced interaction between LUBAC and NEMO.

Function and Regulation of Tumor Necrosis Factor Receptor Type 2

TNF is a major proinflammatory cytokine, which exerts its effects through two different receptors known as TNF-R1 and TNF-R2. Whereas TNF-R1 induced signaling pathways have been very well characterized during the past years, TNF-R2 has been much less well studied. Nevertheless, the function of TNF-R2 should not be underestimated, the more because this receptor shows a much more restricted but inducible expression. Several inflammatory diseases and cancers show upregulated levels of soluble TNF-R2 or are associated with TNF-R2 polymorphisms, implicating an important role for TNF-R2 as a therapeutic target. Here we will review the mechanisms that regulate TNF-R2 expression, as well as discuss TNF-R2 induced signal transduction and cross-talk with TNF-R1.

TRAF1 is a TNF inducible regulator of NF‐κB activation

Tumor necrosis factor receptor (TNFR)‐associated factor TRAF1 was first identified as a component of the TNFR2 signalling complex. Unlike the other members of the TRAF family, TRAF1 lacks the N‐terminal ring finger motif and has a tissue specific expression. Here we demonstrate that expression of TRAF1 is induced by TNF and the protein kinase C (PKC) activator PMA, but not by interleukin‐1 (IL‐1). TNF‐induced upregulation of TRAF1 could be prevented by pretreatment of the cells with the proteasome inhibitor MG‐132, whereas the PKC inhibitor Ro31‐8220 was without effect. Interestingly, overexpression of TRAF1 in HEK293T completely prevented NF‐κB activation induced by TNF, IL‐1, or overexpression of TRAF2 or TRAF6. These data suggest that inducible expression of TRAF1 may serve a negative regulatory function in NF‐κB signalling pathways.

TRAF2 plays a dual role in NF‐κB‐dependent gene activation by mediating the TNF‐induced activation of p38 MAPK and IκB kinase pathways

We previously demonstrated that p38 MAPK is a crucial mediator in the NF‐κB‐dependent gene activation induced by TNF. Here, we have studied the role of several TNF receptor‐associated proteins and caspases in p38 MAPK activation by TNF. The latter appears to be dependent on TRAF2, but independent of FADD or caspases. Remarkably, p38 MAPK activation by TNF proceeds independently of the TRAF2‐associated NF‐κB‐inducing kinase NIK, which is known to bind and activate two recently identified IκB kinases. These results demonstrate that two kinase pathways involved in NF‐κB regulation, viz. NIK and p38 MAPK‐mediated, diverge at the level of TRAF2.

LIND/ABIN-3 Is a Novel Lipopolysaccharide-inducible Inhibitor of NF-κB Activation

Recognition of lipopolysaccharide (LPS) by Toll-like receptor (TLR)4 initiates an intracellular signaling pathway leading to the activation of nuclear factor-κB (NF-κB). Although LPS-induced activation of NF-κB is critical to the induction of an efficient immune response, excessive or prolonged signaling from TLR4 can be harmful to the host. Therefore, the NF-κB signal transduction pathway demands tight regulation. In the present study, we describe the human protein Listeria INDuced (LIND) as a novel A20-binding inhibitor of NF-κB activation (ABIN) that is related to ABIN-1 and -2 and, therefore, is further referred to as ABIN-3. Similar to the other ABINs, ABIN-3 binds to A20 and inhibits NF-κB activation induced by tumor necrosis factor, interleukin-1, and 12-O-tetradecanoylphorbol-13-acetate. However, unlike the other ABINs, constitutive expression of ABIN-3 could not be detected in different human cells. Treatment of human monocytic cells with LPS strongly induced ABIN-3 mRNA and protein expression, suggesting a role for ABIN-3 in the LPS/TLR4 pathway. Indeed, ABIN-3 overexpression was found to inhibit NF-κB-dependent gene expression in response to LPS/TLR4 at a level downstream of TRAF6 and upstream of IKKβ. NF-κB inhibition was mediated by the ABIN-homology domain 2 and was independent of A20 binding. Moreover, in vivo adenoviral gene transfer of ABIN-3 in mice reduced LPS-induced NF-κB activity in the liver, thereby partially protecting mice against LPS/d-(+)-galactosamine-inducedmortality. Taken together, these results implicate ABIN-3 as a novel negative feedback regulator of LPS-induced NF-κB activation.

Enhanced osteoclast development in collagen-induced arthritis in interferon-gamma receptor knock-out mice as related to increased splenic CD11b+ myelopoiesis.

Collagen-induced arthritis (CIA) in mice is accompanied by splenomegaly due to the selective expansion of immature CD11b+ myeloblasts. Both disease manifestations are more pronounced in interferon-γ receptor knock-out (IFN-γR KO) mice. We have taken advantage of this difference to test the hypothesis that the expanding CD11b+ splenic cell population constitutes a source from which osteoclast precursors are recruited to the joint synovia. We found larger numbers of osteoclasts and more severe bone destruction in joints of IFN-γR KO mice than in joints of wild-type mice. Osteoclast-like multinucleated cells appeared in splenocyte cultures established in the presence of macrophage colony-stimulating factor (M-CSF) and stimulated with the osteoclast-differentiating factor receptor activator of NF-κB ligand (RANKL) or with tumour necrosis factor-α (TNF-α). Significantly larger numbers of such cells could be generated from splenocytes of IFN-γR KO mice than from those of wild-type mice. This was not accompanied, as might have been expected, by increased concentrations of the intracellular adaptor protein TRAF6, known to be involved in signalling of RANKL- and TNF-α-induced osteoclast formation. Splenocyte cultures of IFN-γR KO mice also produced more TNF-α and more RANKL than those of wild-type mice. Finally, splenocytes isolated from immunised IFN-γR KO mice contained comparatively low levels of pro-interleukin-1β (pro-IL-1β) and pro-caspase-1, indicating more extensive conversion of pro-IL-1β into secreted active IL-1β. These observations provide evidence that all conditions are fulfilled for the expanding CD11b+ splenocytes to act as a source of osteoclasts and to be indirectly responsible for bone destruction in CIA. They also provide a plausible explanation for the higher susceptibility of IFN-γR KO mice to CIA.