TNFAIP3 (Tumor Necrosis Factor, Alpha-Induced Protein 3)
The intracellular ubiquitin-editing protein tumor necrosis factor alpha-induced protein 3 (TNFAIP3) has been characterized as a dual inhibitor in NF-κB signaling and cell death (Wertz et al. 2004). It is a primary response gene located on chromosome 6q23, originally identified as a gene in human umbilical vein endothelial cells whose expression is rapidly induced by tumor necrosis factor (TNF). The 80 kDa protein encoded by TNFAIP3 is the zinc finger protein A20, which has both ubiquitin ligase and deubiquitinase activities. A20, originally discovered in 1990, acts as a central and inducible endogenous negative feedback regulator of the NF-κB signaling pathway, the inappropriate activity of which has been linked to a host of autoimmune and inflammatory diseases (Dixit et al. 1990). The N-terminal ovarian tumor (OTU) domain (residues 1–370) of A20 comprises the deubiquitinase activity, while the C-terminal zinc finger (ZnF) region (consisting of seven Cys2-Cys2 zinc fingers) acts as an E3 ligase (Yamaguchi et al. 2013). Together, the A20 domains orchestrate a two-step strategy to downregulate NF-κB signaling. Studies in murine models have demonstrated that mice deficient in A20 die prematurely from spontaneous overwhelming systemic inflammation, as well as displaying a degree of hypersensitivity toward TNF (Lee et al. 2000). In human tumors, it has been suggested that the expression of A20 is linked to enhanced tumor formation via increased apoptotic resistance. NF-κB lies on the crossroad of a cell’s decision to live or die. The TNFAIP3 gene encoding the A20 protein is one of several of the cell’s own inhibitory molecules, regulating NF-κB by interacting with upstream signaling pathway components (Lademann et al. 2001, da Silva et al. 2014).
The Diverse Functions of A20
A20 controls NF-κB activity, and the expression of A20 is directly controlled by NF-κB activation, indicating its critical role as a negative feedback regulator of this signaling pathway (Kang et al. 2009). NF-κB is a dimeric transcription factor that regulates the expression of an exceptionally large number of genes in response to infection, inflammation, and other stimuli that require rapid changes in gene expression (Coornaert et al. 2009). A20 acts as a dual ubiquitin-editing enzyme that potently suppresses the canonical NF-κB pathway and effectively inhibits TNF- and IL-1β-induced apoptosis (Lademann et al. 2001). The first substrate identified for the deubiquitinase activity of A20 was receptor-interacting protein kinase 1 (RIP1). Upon stimulation of TNF receptor (TNFR), RIP1 becomes polyubiquitinated at lysine 63 (K63) by the antiapoptotic proteins cIAP1 and cIAP2. A20 removes the K63-linked polyubiquitin chains, preventing their interaction with the essential NF-κB modulator (NEMO). A20 then catalyzes the formation of K48-linked chains to RIP1, targeting it for proteosomal degradation (Wertz et al. 2004) and reviewed (Catrysse et al. 2014). Additionally, A20 functions at the level of Toll-like receptor (TLR) activation by similar deubiquitination/ubiquitination of TLR4- and NOD2-mediated TRAF6 activation. After TLR4 ligand engagement and signaling, A20 inactivates TRAF6 by K63 deubiquitination and disruption of TRAF6 ubiquitin ligase association (Boone et al. 2004). A20 then ubiquitinates TRAF6 on K48, again triggering proteosomal degradation (Yamaguchi et al. 2013). Furthermore, A20 interacts with the TRAF6-binding protein TAX1BP1 to negatively regulate NF-κB signaling. Both proteins work together to form a complex that prevents the E3 ligase TRAF6 polyubiquitination and terminates TLR-mediated inflammatory signaling and subsequent translocation (Catrysse et al. 2014) It has also been reported that A20 plays a major role in suppressing inflammasome activity. Studies have shown that A20-deficient macrophages exhibit spontaneous NLRP3 inflammasome activity to LPS alone, unlike normal cells (Duong et al. 2015). In normal cells, A20 constitutively associates with caspase-1 and pro-IL-1β, and NLRP3 activation further promotes A20 accumulation at the site of the inflammasome. Pro-IL-1β also co-immunoprecipitates with RIPK1, RIPK3, caspase-1, and caspase-8 in a complex containing K63-linked and unanchored polyubiquitin. However, A20-deficient macrophages show notably elevated levels of pro-IL-1β-associated ubiquitination, and the addition of ATP to A20-deficient cells (after 4 h LPS stimulation) induces immense IL-1β secretion, compared to that in wild-type cells. It can be concluded from this that A20 limits the magnitude of NLRP3 inflammasomes while simultaneously preventing spontaneous IL-1β secretion (Duong et al. 2015). IL-1 is also an important mediator of cartilage destruction in rheumatic diseases. Using a mouse model of myeloid cell-specific A20 knockdown, Vande Walle et al. confirmed an IL-1/inflammasome-dependent mechanism of A20 in the development of arthritis, a phenotype that could be reversed by additional IL-1R1 knockdown (Vande Walle et al. 2014). A20 is regarded as a potent regulator of ubiquitin-dependent signaling, and while A20-deficient mice suffer severe systemic inflammation, it has been shown that specific deletions in the A20 protein cause diseases in mice reminiscent of those in humans. For instance, MyD88 depletion negates the severity of several of these conditions, suggesting that A20 restricts TLR and IL-1R family signaling (Duong et al. 2015). This, coupled with the genetic links between inflammasomes and A20-associated human inflammatory disease, points to A20 as being critically positioned at the interface of host responses to commensal microbes (Duong et al. 2015).
A20 also plays a significant role in endo-lysosome formation, thereby indicating its important role in antigen processing. Studies have shown that A20 is involved specifically in endosome/lysosome fusion that consequently determines the degradation of the endocytic allergens present in human colon epithelial cell lines, such as Caco-2 and HT-29 (Huang et al. 2012). Caco-2 cells deficient in A20 also displayed significantly lower endo-lysosome formation rates and rapid transportation of ovalbumin (OVA) to the basolateral chambers, effectively maintaining the cells’ antigenicity. Also, in A20-deficient Caco-2 cells, the number of OVA-carrying endosomes was increased, while the number of OVA-carrying endosomes that were fused to lysosomes was decreased (Huang et al. 2012). These findings imply that A20 may contribute to the tethering of the endosome to the lysosome; however, the detailed mechanism for this process remains unclear. Furthermore, it has been reported that mice that are A20 deficient in their intestinal epithelial cells do not display spontaneous intestinal inflammation but show hypersensitivity to TNF-induced apoptosis. As a result, the intestinal barrier disintegrates, and commensal bacteria begin to infiltrate, triggering a systemic inflammatory response, which ultimately culminates in premature cell death (Vereecke et al. 2010).
Similarly, A20 is important in mediating TLR-4-induced autophagy. Activation of TLR-4 recruits MyD88 and Beclin-1 to create a TLR-4 signaling complex. Beclin-1 is a mammalian yeast homolog that promotes the formation of autophagosomes by aiding the localization of other autophagy proteins to the pre-autophagosomal membrane (Shi and Kehrl 2010). A20 is capable of terminating the NF-κB signaling pathway through the deubiquitination of TRAF6. Shi and coworkers performed a study showing that TRAF6 acts as an E3 ligase to ubiquinate Beclin-1. In murine macrophages (RAW 264.7) and human embryonic kidney cells (HEK 293Y), A20 reduces the extent of K63-linked Beclin-1 ubiquitination, thereby limiting the extent of autophagy elicited by TLR-4 signaling (Shi and Kehrl 2010). Conclusively, TRAF6 and A20 appear to regulate Beclin-1 in a fashion corresponding to their defined roles in regulating the NF-κB signaling pathway. The TRAF6-A20 axis functions as a rheostat to either stimulate or inhibit autophagy (Shi and Kehrl 2010).
A20 plays an important role in the promotion of tolerance to (commensal) bacteria. With respect to mechanisms of tolerance to TLR signaling in the gut, several proposed modes of action have been investigated in this area. Several studies have indicated the importance of A20 for epithelial integrity (Wang et al. 2009; Vereecke et al. 2010). LPS-induced expression of A20 is necessary and normally sufficient for the development of hyporesponsiveness to repeated LPS stimulation. A20 predominantly localized to the luminal interface of villus enterocytes in adult rodents, and A20 levels in the intestinal epithelium positively correlate with the bacterial load (Wang et al. 2009). These findings provide multiple lines of evidence for the key role of the zinc finger protein A20 in the development of tolerance to TLR ligands in the small intestine. Although they are still poorly understood, it is not surprising that such a critical concept as tolerance to commensal bacteria is supported by several mechanisms. For example, posttranslational downregulation of IRAK-1 could possibly be dependent on A20 (Wang et al. 2009). IRAK-1 is regulated by K63 polyubiquitination and could therefore be an A20 target. Another possibility is that other negative regulators of inflammatory signaling, for instance, the downregulation of TLR in villus enterocytes, ligand-induced TLR internalization, NF-κB-mediated resynthesis of IκB, or degradation of MD-2 by trypsin, are redundant with A20 (Wang et al. 2009). Because A20 lacks membrane-spanning domains and lipid modification sites, its association with membranes likely depends on interaction with membrane-tethered proteins. One such protein could be the A20 target TRAF6, which is associated with membranes in unstimulated cells (Wang et al. 2009).
Finally, A20 plays a crucial role in apoptosis, a process of programmed cell death in response to a multitude of stressful stimuli. Apoptosis can occur via two different pathways: the intrinsic pathway, which is mediated by intracellular signaling, or the extrinsic pathway, involving death receptor ligand binding. NF-κB activation results in the expression of antiapoptotic genes that inhibit the apoptosis pathway that is activated in parallel (He and Ting 2002). Studies have shown that isolated Jurkat T cell mutants exhibit hypersensitivity to TNF-induced apoptosis as a result of a deficiency in IκB kinase γ (IKKγ/NEMO), an essential component of the IKK complex and the NF-κB pathway (He and Ting 2002). A20 can protect these deficient cells from TNF-induced apoptosis by disrupting the recruitment of the death domain signaling molecules TRADD and RIP to the receptor signaling complex (He and Ting 2002). This analysis indicates that A20 is not induced in IKKγ-deficient cells and that its ectopic expression in the mutant T cells is sufficient to provide protection against TNF-induced apoptosis. It has also been proven that A20 inhibits apoptosis at a very early stage, inhibiting the recruitment of the death domain-containing signaling molecules TRADD and RIP to their receptors (He and Ting 2002). Contrarily, proapoptotic functions of A20 have also been identified, thought to be mainly due to the termination of NF-κB signaling and subsequent loss of antiapoptotic proteins, such as BCL-2 and BCL-X (Catrysse et al. 2014). This strongly implies that the effect of A20 relies on a balance between its instinctive antiapoptotic tendencies and the expression of NF-κB-mediated antiapoptotic proteins that may be hindered through repression by A20.
Regulation of A20
With the exception of thymocytes, peripheral T cells, and epithelial cells, most cell types do not express A20 under normal resting conditions (Coornaert et al. 2009; Wang et al. 2009; Kelly et al. 2013). Instead, the transcription of A20 is rapidly induced by a large number of stimuli that trigger the binding of NF-κB to two specific NF-κB binding sites in the A20 promoter region (Shembade et al. 2007). At the protein level, several A20-binding proteins such as ABIN and TAX1BP1 have been proposed to regulate A20 activity. With the exception of ABIN-3, the ABIN proteins share a novel ubiquitin-binding domain, and mutations that disrupt the ubiquitin-binding potential of ABIN proteins also disrupts their participation in the NF-κB inhibitory effect on A20 (Coornaert et al. 2009). The binding of TAX1BP1 to A20 was originally thought to be essential for the antiapoptotic of A20. The exact mechanism for this interaction remains an enigma; however, it has been shown that TAX1BP1-deficient cells exhibit a sustained NF-κB response to IL-1, LPS, and TNF treatment. Such a response is associated with elevated ubiquitination of RIP1 and TRAF6, thus recruiting A20 to its substrate through the formation of a ternary complex (Coornaert et al. 2009).
Interestingly, the expression of A20 mRNA itself is controlled by NF-κB and other transcription factors, and inflammatory cytokines such as TNF and IL-1β are capable of upregulating its expression. Initial characterization of the A20 promoter revealed that TNFα-induced A20 upregulation was mediated through two κB elements, which serve as recognition sequences (Krikos et al. 1992). To date several other transcription factor binding sites within the A20 promotor have been identified. Lai et al. showed that LPS upregulates the levels of C/EBP mRNA and protein, while p38 inhibition suppresses this effect, leading to decreased A20 expression (Lai et al. 2013). The work suggests a C/EBPβ binding site within the A20 promotor. MAPK p38 upregulates transcription factor c/EBPβ, which works in conjunction with the flanking NF-κB binding sites to regulate A20 (Lai et al. 2013). We have recently shown that those Cystic Fibrosis patients with ‘close to normal’ basasl levels of A20 also express high basal levels of p38 mRNA (McCallum et al. 2016), supporting the notion of A20 induction by p38.
Regulation of A20 Protein Expression and Activity
A20 can also be regulated by posttranslational modification and consequential protein expression. A20 is a putative substrate of IKKβ, which can phosphorylate A20 at serine 381(Hutti et al. 2007). The phosphorylation process increases the inhibiting potential that A20 exerts over the NF-κB pathway, thereby regulating A20 activity and representing part of the novel feedback mechanism that mediates NF-κB stimulation (Hutti et al. 2007). In T and B cells, antigen receptor stimulation results in the site-specific cleavage of A20 by the paracaspase MALT1, leading to the eventual disruption of its NF-κB inhibitory potential (Coornaert et al. 2008; Duwel et al. 2009). Additionally, there are increasing numbers of genetic association studies uncovering a linkage of loss of function mutations and single nucleotide polymorphisms (SNPs) in the human A20 locus with susceptibility to autoimmune and inflammatory diseases (Koumakis et al. 2012; Mele et al. 2014; Zhou et al. 2016).
A20 in Disease
It is well established that polymorphisms in the human TNFAIP3 gene locus are closely related to a variety of inflammatory diseases, perhaps indicating that A20 inhibits the incidence and/or severity of these diseases (Duong et al. 2015). Recent genetic studies demonstrate a clear association between several mutations in the human A20 locus and immunopathologies including Crohn’s disease, rheumatoid arthritis, lupus erythematosus, and cancer (Vereecke et al. 2009; Zhou et al. 2016; Zhu et al. 2016), as well as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (Kelly et al. 2011, Schuijs et al. 2015). SNIPs in the A20 introns that lead to decreased A20 expression have been associated with an increased risk of coronary artery disease in patients with type II diabetes (Coornaert et al. 2009). Similarly, single amino acid mutations, for example, in amino acid E627A, have been linked to increased susceptibility of mice to atherosclerosis (Coornaert et al. 2009). Furthermore, dysregulated A20 protein expressions and activities have been reported for asthma and cystic fibrosis (Kang et al. 2009, Schuijs et al. 2015). Additionally, in a mouse model, the role of A20 in inflammatory bowel disease was shown: although the selective absence of A20 in intestinal epithelial cells (IEC) did not cause spontaneous intestinal inflammation, additional myeloid-specific A20 deletion synergistically drove intestinal pathology characterized by ileitis, severe colitis, and intestinal microbiota dysbiosis (Catrysse et al. 2014). Most recently, Bhattacharyya S. et al. showed impaired A20 expression in fibroblasts, which, together with a direct suppression of A20 by TGF-ß within the fibrotic milieu in systemic fibrosis, might play a significant functional role in persistence of fibrotic responses (Bhattacharyya et al. 2016).
Collectively, these findings illustrate the importance of TNFAIP3 (A20) in controlling acute and chronic inflammatory responses and highlight its important role as a determinant in human inflammatory disease. A20 makes a critical contribution to immune tolerance, antigen processing, inflammation, and apoptosis, ultimately maintaining immune homeostasis. Therefore, A20 may play an important role as a drug target to normalize excessive inflammatory responses (Schuijs et al. 2015; Bhattacharyya et al. 2016; Malcomson et al. 2016).
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