Download The ubiquitin-editing enzyme A20 (TNFAIP3) is a central regulator of

Review
The ubiquitin-editing enzyme A20
(TNFAIP3) is a central regulator of
immunopathology
Lars Vereecke1,2, Rudi Beyaert1,2 and Geert van Loo1,2
1
Department for Molecular Biomedical Research, Unit of Molecular Signal Transduction in Inflammation, VIB,
B-9052 Ghent, Belgium
2
Department of Biomedical Molecular Biology, Ghent University, B-9052 Ghent, Belgium
Nuclear factor (NF)-kB has an important role in immunity
and inappropriate NF-kB activity has been linked with
many autoimmune and inflammatory diseases. Multiple
mechanisms normally ensure the proper termination of
NF-kB activation. In this context, the intracellular ubiquitin-editing protein A20 (also known as Tumor Necrosis Factor Alpha-Induced Protein 3 or TNFAIP3) is a key
player in the negative feedback regulation of NF-kB
signaling in response to multiple stimuli. Moreover,
A20 also regulates tumor necrosis factor (TNF)-induced
apoptosis. Recent genetic studies demonstrate a clear
association between several mutations in the human
A20 locus and immunopathologies such as Crohn’s disease, rheumatoid arthritis, systemic lupus erythematosus, psoriasis and type 1 diabetes. These findings
further illustrate the importance of A20 in the resolution
of inflammation and the prevention of human disease.
Properties of NF-kB signaling
Nuclear factor (NF)-kB is a transcription factor that
regulates the expression of an exceptionally large number
of genes in response to infection, inflammation and other
stressful situations requiring rapid reprogramming of
gene expression [1]. NF-kB represents a family of structurally related and evolutionarily conserved proteins
(p100 or NF-kB2, p105 or NF-kB1, p65 or RelA, RelB,
c-Rel), which exist as homo- or heterodimers. In resting
cells, NF-kB is kept inactive by binding to inhibitor of
kB (IkB) proteins, of which IkBa is best known. Upon
stimulation with a wide variety of agonists including
tumor necrosis factor (TNF), interleukin (IL)-1 and Tolllike receptor (TLR) ligands, IkBa is phosphorylated and
subsequently polyubiquitinated and degraded by the proteasome, releasing NF-kB, which then accumulates in
the nucleus and activates transcription of its target
genes. NF-kB activating receptors share many signaling
intermediates and they all employ intracellular adaptor
proteins that provide modularity to NF-kB activation [2].
Many of the protein–protein interactions in NF-kB
signaling are regulated by non-degradative polyubiquitination and deubiquitination (Box 1). A central event in
NF-kB signaling is the activation of the IkB kinase (IKK)
Corresponding authors: Beyaert, R. ([email protected]);
van Loo, G. ([email protected]).
complex, which is comprised of the two catalytic subunits
IKK1 and IKK2 (also known as IKKa and IKKb) and the
regulatory subunit, NEMO (NF-kB essential modulator,
also known as IKKg) (Figure 1). The activated IKK complex mediates phosphorylation of the inhibitory IkB
protein, enabling its proteasome-dependent elimination.
Gene targeting experiments showed that IKK2 and
NEMO, but not IKK1, are required for NF-kB activation
by TNF and TLRs [3]. Some receptors such as the lymphotoxin b receptor and CD40 receptor have the ability to
activate an alternative NF-kB pathway, involving IKK1
and leading to p100 NF-kB processing to p52, which
forms a dimer with RelB. The p52–RelB complex regulates
specific genes involved in B-cell development and lymphoid
organogenesis [1].
NF-kB can be activated in virtually every cell type.
Defects in the regulation of NF-kB-dependent gene
expression and apoptosis contribute to a variety of diseases including inflammatory and autoimmune diseases,
neurological disorders and cancer. As such, controllable
modulation of NF-kB signaling might be valuable in treating pathological conditions, and many companies are
directing efforts into the development of NF-kB inhibitors.
Because NF-kB activation is so crucial to many cellular
processes, it is not surprising that a tight regulation of this
pathway and the genes it induces is an absolute requirement. To achieve this, cells employ a multilayered control
system to keep NF-kB signaling in check, and the combined action of different positive and negative regulators
help to fine-tune the immune response. Recently, substantial progress has been made in our understanding of the
mechanisms that control the dynamics of NF-kB activation and several autoregulatory feedback loops terminating the NF-kB response have been described [4]. In this
context, the ubiquitin-editing protein A20 (also referred to
as TNF a-induced protein 3 or TNFAIP3) has been
described as a key player in the termination of NF-kB
signaling. The molecular and biochemical mechanisms by
which this versatile protein modifier regulates NF-kB
signaling have been reviewed in detail elsewhere. [5].
Here we mainly focus on new findings that reveal a key
role for A20 in controlling NF-kB-dependent inflammation. In particular, we discuss the recent discovery of
A20 as a susceptibility gene for a plethora of different
immune pathologies.
1471-4906/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.it.2009.05.007
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Figure 1. Inhibition of TNF- and TLR4-induced NF-kB activation by A20. Left-hand side: stimulation of TNF-R1 leads to the recruitment of the adaptor proteins TRADD, RIP1
and TRAF2 along with the E3 ubiquitin ligases cIAP1 and cIAP2, which then catalyze the attachment of K63-linked polyubiquitin chains to RIP1. These ubiquitin chains bind
the TAB2 subunit of the TAK1 kinase complex, resulting in TAK1 activation. TAK1 phosphorylates and activates IKK2, which is also recruited through the binding of NEMO
to K63-linked ubiquitinated RIP1. Recently, NEMO was shown to selectively bind linear head-to-tail ubiquitin chains [79], and to become itself modified by linear ubiquitin
chains upon TNF and IL-1 treatment [80], the function of which is still unclear. IKK phosphorylates IkBa, resulting in its K48-linked ubiquitination and subsequent
proteasomal degradation. This liberates NF-kB (here shown as a heterodimer of p65 and p50) to translocate to the nucleus and to promote target gene expression. A20 is a
DUB that dampens TNF-induced NF-kB activation by disassembling K63-linked polyubiquitin chains from RIP1, and replacing them by K48-linked polyubiquitin chains,
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Box 1. Regulation of signaling by ubiquitination
Ubiquitination is a post-translational modification of proteins that is
recognized as a fundamental regulatory mechanism of signal
transduction [20,83]. Ubiquitin is a 76 amino acid conserved protein
that is covalently attached to other proteins in a highly regulated
process involving the stepwise activity of an E1 ubiquitin activating
enzyme, E2 conjugating enzymes and E3 ubiquitin ligases. The latter
confer substrate specificity and enable the attachment of ubiquitin
to a specific lysine in the target substrate. Each of the seven lysines
in ubiquitin can themselves also be coupled to another ubiquitin,
thus leading to a polyubiquitin chain on the original target protein.
Recently, also head-to-tail (= linear) polyubiquitination has been
demonstrated to be involved in NF-kB signaling [79,80]. The type of
polyubiquitin chain determines the further fate of the target
substrate. Polyubiquitin chain formation through K48 of ubiquitin
directs proteasomal degradation of the modified protein. By
contrast, K63 polyubiquitin chain linkages or linear polyubiquitination do not lead to degradation of the substrate but mediate a lowaffinity binding of other proteins that contain specific ubiquitinbinding domains. This can stabilize transient protein–protein
interactions and is widely used in NF-kB signaling. Ubiquitination
is reversed by DUBs which are proteases that cleave ubiquitin
chains from their substrate, making ubiquitination a very dynamic
process. Several DUBs such as CYLD, A20 and Cezanne, have been
demonstrated to remove K63-linked ubiquitin chains from specific
target substrates in the NF-kB signaling pathway, thus negatively
regulating NF-kB activation [81].
Immunoregulatory function of A20
A20 is a cytoplasmic zinc finger protein that was originally
identified as a TNF-inducible protein and which has been
characterized as a dual inhibitor of NF-kB activation and
cell death [5]. In most cell types, basal A20 expression is
very low but its transcription is rapidly induced upon NFkB activation. Once expressed, A20 functions as a negative
feedback regulator of NF-kB activation. The essential role
of A20 in the regulation of NF-kB and apoptotic signaling
was clearly demonstrated with the generation of a complete A20 knockout mouse [6]. Mice deficient for A20 are
hypersensitive to TNF and die prematurely due to severe
multi-organ inflammation and cachexia [6]. A20-deficient
cells fail to terminate TNF-induced NF-kB responses and
are more susceptible to TNF-mediated apoptosis. Besides
its crucial role for the regulation of TNF receptor-dependent pro-inflammatory signals, A20 is also required for
terminating NF-kB signaling in response to microbial
products such as lipopolysaccharide (LPS) and muramyl
dipeptide (MDP) [7], which trigger TLR4 and Nucleotidebinding Oligomerization Domain containing 2 (NOD2)
receptors, respectively. Deregulated TLR signaling in
response to commensal bacteria was shown to be responsible for the multi-organ inflammation and premature
death of A20 knockout mice [8]. Several reports suggest
that A20 also restricts innate immune signaling in
response to virus infection [9–11], but in vivo evidence
for this is still lacking. Finally, A20 expression can also
affect adaptive immunity. Two independent studies
Trends in Immunology
Vol.30 No.8
showed that RNA interference-mediated downregulation
of A20 in dendritic cells (DCs) results in enhanced T-cell
stimulatory capacity [12,13]. More specifically, silencing of
A20 in human DCs results in an increase in their cytokine
production following stimulation with the synthetic TLR3
ligand poly(I:C) and an increased T-cell stimulatory
capacity [12]. An increased immunostimulatory potency
of A20 silenced murine DCs was also demonstrated even in
the absence of an activation stimulus [13]. Moreover, this
latter study also reported that A20 silenced DCs inhibit
regulatory T cells and hyperactivate tumor-infiltrating
cytotoxic T lymphocytes and T-helper-cells that then
become refractory to regulatory T-cell-mediated suppression. A20 silencing might thus provide a strategy to overcome regulatory T-cell-mediated suppression in an
antigen-specific manner and increase the efficiency of
vaccines against cancer and perhaps infectious diseases.
Mechanisms of NF-kB inhibition by A20
The molecular mechanism responsible for the NF-kB
inhibitory function of A20 has been clarified by the elucidation of A20 as a ubiquitin-editing protein with dual
functions: a deubiquitinating enzyme (DUB) activity
mediated by its N-terminal ovarian tumor (OTU) domain
and E3 ubiquitin ligase activity mediated by the C-terminal zinc finger containing domain (Figure 2) [14]. In the
case of TNF signaling, Receptor Interacting Protein-1
(RIP1), an essential NF-kB signaling molecule, is polyubiquitinated by the E3 ubiquitin ligases cIAP-1 and -2 (cellular Inhibitor of Apoptosis Protein-1 and -2) [15]. A20
DUB activity removes K63-linked polyubiquitin chains
from RIP1, preventing its interaction with NEMO.
Furthermore, A20 E3 ubiquitin ligase activity then promotes RIP1 K48-linked polyubiquitination, which triggers
the proteasome-mediated degradation of RIP1 [14,16]
(Figure 1). In addition, A20 is capable of targeting the
associated signaling molecule TRAF2 to the lysosomes for
degradation, an activity that does not require A20’s ubiquitin modifying property [17]. A20 can also dismantle
K63-linked polyubiquitin chains from TRAF6 and RIP2,
similarly turning off NF-kB activation by TLR4 and NOD2,
respectively [7,18] (Figure 1). However, removal of K63linked ubiquitin chains from TRAF6 and RIP2 is not
followed by their A20-mediated K48-linked polyubiquitination and subsequent degradation. Because A20 is also
able to inhibit NF-kB activation induced by TAK1 overexpression, which signals downstream of RIP1, RIP2 and
TRAF6 [19], it is likely that A20 acts at multiple steps in
the NF-kB signaling pathway. In this context, several
other molecules that contribute to the proper activation
of the IKK complex have been shown to undergo K63linked polyubiquitination [20], making them potential
targets for A20. The role of A20-mediated ubiquitin editing
has so far only been studied in the context of its NF-kB
thereby targeting RIP1 for proteasomal degradation (reviewed in Ref. [81]). A20 inhibits TNF receptor signaling by mediating the ubiquitination and proteasomal
degradation of RIP1 which is essential for TNF-induced NF-kB activation. Right-hand side: binding of lipopolysaccharide (LPS) to the TLR4-MD2-CD14 receptor complex
leads to the recruitment of the adaptors Mal, MyD88, TRAF6 and multiple members of the IRAK kinase family. TRAF6 is an E3 ubiquitin ligase that is activated by IRAK2,
potentially by promoting TRAF6 oligomerization, leading to the attachment of K63-linked polyubiquitin chains to TRAF6 and IRAK1 [82]. These K63-linked polyubiquitin
chains are then recognized by TAB2 and NEMO, leading to the TAK1-mediated activation of the IKK complex as described earlier for TNF signaling. A20 inhibits LPSinduced NF-kB activation by disassembling the K63-linked polyubiquitin chains from TRAF6 (reviewed in Ref. [81]). Solid lines indicate positive signaling. Broken lines with
arrows indicate negative regulation by A20.
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Figure 2. Domain structure of human A20. The N-terminal OTU domain is essential for deubiquitinating (DUB) activity. C103 represents the catalytic cysteine residue. The Cterminal region of A20 contains seven zinc fingers (1–7) of which zinc finger 4 has been shown to have E3 ubiquitin ligase activity (E3 ligase). The C-terminal zinc finger
containing domain also mediates A20 dimerization. The linker region between zinc finger 1 and 2 contains a cleavage site (GASRG) for MALT1, which disrupts the NF-kB
inhibitory potential of A20. The F127C mutation (SNP rs 2230926) identified in SLE patients, and other SNPs (source: http://www.ncbi.nlm.nih.gov/projects/SNP/) not yet
identified as being associated with immunopathologies, are also indicated.
inhibitory function. Whether similar ubiquitin-editing
mechanisms of A20 mediate the anti-apoptotic activity
of A20 is still unclear, but it is supported by some very
recent data. Death receptor stimulation was shown to
induce cullin3 E3 ligase-mediated polyubiquitination of
caspase-8 at the death-induced signaling complex, resulting in the interaction of caspase-8 with the ubiquitinbinding protein p62 (also known as sequestome-1). The
latter promotes caspase-8 aggregation, leading to its full
activation and driving commitment to cell death. Interestingly, A20 is recruited to caspase-8 in the death-induced
signaling complex, where it reverses its ubiquitination and
blocks the associated increase in caspase-8 activity,
suggesting the existence of a dynamic balance in the
ubiquitin-mediated activity of caspase-8 [21].
Little is known about the molecular mechanisms that
regulate the ubiquitin-editing and NF-kB inhibitory function of A20. ABINs and TAX1BP1 are A20 binding proteins
that have been proposed to recruit A20 to its ubiquitinated
substrate by directly binding ubiquitin [22–24]. The E3
ubiquitin ligases Itch and RNF11 are two other components of the A20 ubiquitin-editing complex that are
essential for the termination of TNF-mediated NF-kB
and JNK signaling [24,25], but their precise role remains
to be discovered. Finally, in the case of T cell receptor (TCR)
and B cell receptor induced NF-kB activation, A20 is itself
proteolytically processed by the paracapase MALT1, compromising its NF-kB-inhibitory potential [26].
A20 in autoimmune and inflammatory diseases
Given its key function in the fine-tuning of NF-kB signaling
and apoptosis, it can be expected that defects in A20
expression or function could lead to chronic inflammation
and tissue damage. Here, we describe some recent findings
that reveal a crucial link between polymorphisms in the
human A20 locus and a wide spectrum of human autoimmune and inflammatory diseases (Table 1). In addition,
we discuss the potential role of A20-mediated regulation of
NF-kB signaling and/or apoptosis in the immunopathology
of these diseases.
A20 and inflammatory bowel disease
Inflammatory bowel disease (IBD), and in particular
Crohn’s disease (CD), is thought to result from a dysregulated interaction between the host immune system and its
commensal microflora [27,28]. Recognition of bacterial
products by TLRs represents a crucial component of the
symbiosis between the commensal microflora and the host
Table 1. Genetic variants in or near A20 at 6q23 (138.230274–138.246135) that are associated with inflammatory autoimmune
pathology in humans
Disease
Atherosclerosis in mice
in diabetic patients
Crohn’s disease
Coeliac disease
Rheumatoid arthritis
Systemic lupus erythematosus
Type 1 diabetes
Psoriasis
386
Mutation in A20 or SNPs identified
E627A (mouse)
rs 5029930
rs 610604
rs 7753394
rs 2327832
rs 10499194/rs 13207033
rs 6920220
rs 5029937
rs 5029939
rs 10499197
rs 7749323
rs 13192841
F127C (rs 2230926)
rs 6922466
rs 10499194
rs 6920220
rs 610604
remark
C57BL/6J versus FVB/N
within intron
within intron
outside gene
outside gene
outside gene
outside gene
within intron
within intron
outside gene
outside gene
outside gene
exon3
outside gene
outside gene
outside gene
within intron
Position (Mb)
138.232377
138.241100
138.126940
138.014760
138.044330
138.048197
138.236844
138.237416
138.174209
138.272082
138.008907
138.237759
138.486623
138.044330
138.048197
138.241100
Refs
[57]
[69]
[69]
[33]
[36]
[39]
[40]
[41]
[43]
[43]
[43]
[44]
[44]
[44]
[49]
[49]
[55]
Review
immune system and is essential for resistance to epithelial
injury [29]. NF-kB signaling in intestinal epithelial cells
was recently shown to be crucial for controlling intestinal
immune homeostasis and limiting chronic inflammation,
because intestinal epithelial cell-specific inhibition of NFkB spontaneously caused severe chronic intestinal inflammation in mice [30,31]. An important role for the p65
(RelA) subunit of NF-kB in intestinal homeostasis was
highlighted by a recent report that mice with specific
deletion of RelA in intestinal epithelial cells exhibit
increased susceptibility to chemically induced colitis
[32]. An important step in the inflammatory process in
these NF-kB-deficient mice involves the sensitization of
NF-kB-deficient epithelial cells to TNF-induced apoptosis,
compromising epithelial integrity and allowing bacterial
translocation into the mucosa, thus leading to recruitment
of inflammatory immune cells and chronic inflammation
[30,32].
Mice genetically deficient in A20 also develop severe
intestinal inflammation [6], leading to the hypothesis that
expression of A20 is essential for intestinal homeostasis
and suppression of NF-kB-dependent inflammation. In
line with this, a recent genome-wide association study
(GWAS) for seven most prevalent common inflammatory
diseases, undertaken in the British population by the
Wellcome Trust Case Control Consortium, identified
A20 as a CD susceptibility gene [33]. An earlier independent study on 260 IBD-affected pairs from 139 families
also associated a region of human chromosome 6q with
the IBD phenotype [34], with one of the candidate genes in
this locus being A20. Finally, mucosal biopsies from 69 CD
patients were analyzed and confirmed a consistent downregulation of mucosal A20 expression, which might hamper their ability to regulate pathological NF-kB activation
induced by acute inflammatory responses [35]. Interestingly, single nucleotide polymorphisms (SNPs) in the A20
region on 6q23.3 were recently also identified as a disease
risk factor in coeliac disease, an autoimmune disorder of
the small intestine that occurs in genetically predisposed
people and which is caused by intolerance to dietary
gluten protein from wheat and related proteins from
barley and rye [36].
The observation that A20 and MyD88 double-deficient
mice are dramatically less inflamed and survive far longer
than A20 single knockout mice suggests that steady state
TLR signals can be profoundly proinflammatory and lethal
[8]. Moreover, A20-deficient mice display splenomegaly
due to infiltration of activated myeloid cells, which can
be rescued in a MyD88-deficient background. Therefore,
A20 is crucial for restricting intestinal inflammation as it
prevents constitutive homeostatic MyD88 signals from
becoming inflammatory, and commensal enteric bacteria
seem to be the driving force inducing cachexia and myeloid
cell proliferation via MyD88 signals in the absence of A20.
A20 and rheumatoid arthritis
Rheumatoid arthritis (RA) is the most common inflammatory arthritis affecting up to 1% of the adult population.
Although the initiating cause of RA is still not fully understood, inflammatory cytokines such as TNF, IL-1, IL-6 and
RANKL are of central importance for the chronic inflam-
Trends in Immunology
Vol.30 No.8
matory state leading to disease. From these, TNF seems to
have a primary role in RA because it regulates the expression of all other pro-inflammatory cytokines found in
joints, making it an attractive therapeutic target. One
effective treatment of severe RA consists of the inhibition
of TNF through administration of TNF antagonists
[37,38]. To identify susceptibility alleles associated with
RA, a GWAS was carried out to identify genetic regions
with potential DNA variants determining susceptibility to
RA [39,40]. Two SNPs were unequivocally identified at
6q23. Although these variants are not located in a gene,
they are thought to influence A20 as its nearest gene
(150 kb downstream of A20), probably by the presence
of potential regulatory DNA elements in this region.
Further fine-mapping studies also indicated an association of these and other polymorphisms in A20 and the
6q23 intergenic region with RA [41,42]. As A20 is required
for termination of TNF-induced signals, these findings
reveal A20 as a likely susceptibility locus for RA.
A20 and systemic lupus erythematosus
Another autoimmune disease for which the A20 gene was
recently identified as being associated with, is systemic
lupus erythematosus (SLE) [43,44]. This is a multigenic
disease characterized by dysregulated interferon
responses and loss of self-tolerance to cellular antigens.
Autoantibody production results in local and systemic
inflammation and organ failure. To identify genetic variants contributing to SLE, a GWAS was carried out, and an
association between SLE and an A20 variant was identified. Evidence for two other SNPs that are associated with
SLE were also found near A20, including the one previously described for RA [43]. When evaluating whether
the A20 rs5029939 risk allele was associated with SLE
clinical manifestations, heterozygous carriers were shown
to have a twofold increased risk of developing renal or
hematologic manifestations compared to homozygous nonrisk subsets [45]. Based on the available evidence for a role
of A20 variants in RA, a second study used data from the
recently published GWAS [46] to specifically examine the
potential association of A20 with SLE [44]. This study
revealed that three independent SNPs in the A20 region
are associated with SLE, including one coding and two noncoding polymorphisms. The coding SNP results in a F127C
amino acid change in the OTU domain of the A20 protein.
To test the biological impact of this SNP, the corresponding
mutant A20 cDNA was transfected in HEK293T cells and
shown to be modestly, but consistently, less effective at
inhibiting TNF-induced NF-kB activity [44]. The epidemiological and genetic similarities between SLE and
RA suggest that not only the same genes but also identical
variants, might affect risk of each disease.
A20 and autoimmune diabetes
Type I diabetes (T1D) is a chronic autoimmune disease
with a complex pathogenesis involving multiple genetic
and environmental factors. The disease is characterized by
the infiltration of inflammatory cells into the pancreatic
islets of Langerhans, followed by the selective destruction
of insulin-producing b-cells. As a result, insufficient insulin
secretion results in hyperglycemia causing complications,
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Review
such as retinopathy, nephropathy and cardiovascular disease. One of the main mechanisms causing b-cell death is
the intra-islet release of inflammatory mediators by activated immune cells [47], a process in which NF-kB is
involved [48]. A transgenic mouse line in which NF-kB
activation is attenuated specifically in b-cells conferred
nearly complete protection against streptozotocin-induced
T1D, with reduced intra-islet lymphocytic infiltration [48].
Analysis of 17 SNPs that have been reported in several
autoimmune diseases (RA, SLE, CD, multiple sclerosis)
also identified 6q23 containing A20 as a susceptibility locus
for T1D [49], suggesting that the uncontrolled activation of
NF-kB, due to reduced A20 expression, participates in T1D
development. A20 is also a cardinal anti-apoptotic gene in
b-cells [50,51]. Islet expression of A20 is rapidly upregulated in response to cytokine stimulation, and direct plasmid-based transfer of the A20 gene into the pancreas has
been shown to protect mice in streptozotocin-induced T1D
[52]. Adenovirus-mediated overexpression of A20 in rodent
and human islets reduces cytokine-induced apoptosis and
subsequent reactive oxygen species production by islets in
vitro, but A20 overexpression has also been associated with
improved islet survival in a syngeneic marginal islet mass
transplantation model [50,51]. Moreover, loss of A20
expression renders b-cells susceptible to apoptotic death
[53]. These data demonstrate that A20 is an important
cytoprotective protein which could be a candidate for gene
therapy in islet transplantation and therapeutic intervention in T1D.
A20 and psoriasis
Psoriasis is a common immune-mediated disorder with a
multifactorial genetic basis that affects the skin, nails and
joints. The most obvious cellular features of psoriasis are
epidermal hyperplasia, altered keratinocyte differentiation and inflammation [54]. Individuals with psoriasis
show increased risk for other autoimmune disorders, such
as arthritis, atherosclerosis and CD [54]. So far, between 10
and 20 chromosome regions have been proposed to harbour
psoriasis genes but only a few genes have been identified
[54]. To identify new psoriasis susceptibility loci, a GWAS
of 1409 psoriasis patients and 1436 controls was carried
out [55]. Next to SNPs for genes involved in IL-23 signaling, loci including A20 and ABIN-1 (also known as
TNFAIP3-interacting protein 1 or TNIP1) showed strong
association with psoriasis [55]. Interestingly, in the mouse
PL/J strain, carrying a CD18 hypomorphic mutation which
gives rise to a skin disease resembling human psoriasis, a
region of mouse chromosome 10 encompassing A20 was
identified as contributing to the psoriasiform skin disease
[56]. Notably, the same region of the mouse genome has
been associated with atherosclerosis [57], a most prevalent
co-morbidity of psoriasis [58].
A20 and atherosclerosis
Atherosclerosis has emerged as a cause of mortality in
systemic autoimmune diseases such as RA and SLE and
compelling evidence demonstrates that these diseases are
risk factors for the development of atherosclerosis [59]. In
addition, psoriasis and diabetes have been associated with
atherosclerosis and cardiovascular mortality [58,60]. Ath388
Trends in Immunology Vol.30 No.8
erosclerosis was once thought to be solely a disease of lipid
accumulation in the vessel wall, but inflammation also
seems to be an important driving factor, leading to the
now largely accepted concept of atherosclerosis as a chronic
inflammatory disease [61]. Several immune cells and
mediators that take part in an inflammatory reaction
can be identified in the atherosclerotic lesions in which
vascular cells interact with immune cells. Cholesterolcontaining low-density lipoproteins that accumulate in
the intima activate the endothelium and promote recruitment of T cells and monocytes, the latter of which differentiate into macrophages and end up as pro-inflammatory
foam cells. Local antigens trigger Th1 responses with
secretion of proinflammatory cytokines that contribute
to local inflammation and plaque growth. At the endothelial level, TLRs are a potential link between inflammation and atherosclerosis, as they are used not only to
sense pathogens but also host-derived ligands such as
oxidized lipoproteins or their component oxidized lipids
and thereby initiate so-called ‘sterile inflammation’. NF-kB
has been implicated in the pathogenesis of atherosclerosis
as it regulates the expression of many proinflammatory
genes involved in the initiation and progression of atherosclerotic lesions [62]. However, NF-kB-dependent expression of anti-apoptotic genes could be seen as antiatherosclerotic, as cell death becomes an important aspect
at later stages of atherosclerosis in which it determines
plaque stability [62].
An intercross between atherosclerosis susceptible
(C57BL/6J ApoE / ) and resistant (FVB/N ApoE / ) mice
identified an atherosclerosis-susceptibility locus on chromosome 10 of the mouse genome [57,63–65]. One of the candidate genes mapping to this locus is A20. Sequencing of the
mouse A20 gene coding region from both parental strains
revealed a SNP resulting in a single E627A amino acid
change (C57BL/6J versus FVB/N). This mutation introduces a putative casein kinase 2 phosphorylation site in
A20 from C57BL/6J that is not present in FVB/N, leading to
less effective termination of TNF-stimulated NF-kB activation in the atherosclerosis susceptible C57BL/6J mice
[57]. Vascular smooth muscle cells from these mice showed
prolonged TNF-induced expression of the NF-kB target
genes A20 and IkBa [57]. In a recent study, the role of
A20 in atherosclerotic lesion development was further
examined using A20 haplo-insufficient mice [65]. This study
showed an increased aortic root atherosclerotic lesion area
in A20 insufficient mice whereas A20 overexpressing transgenic mice had a decreased lesion area. The protective effect
of A20 on atherosclerosis was associated with reduced
expression of pro-atherosclerotic NF-kB target genes, such
as the adhesion molecules VCAM-1, ICAM-1 and monocyte
colony stimulation factor (M-CSF), and reduced plasma
levels of NF-kB-driven cytokines [65]. The atheroprotective
effect of A20 is most likely caused by its ability to shut down
NF-kB activation that is driven via a MyD88-dependent
pathway, as MyD88-, TLR2- and TLR4-deficient mice, on an
ApoE / background, also show decreased atherosclerosis
[66–68]. A20 SNPs were also shown to be associated with
increased atherosclerosis in diabetic patients [69]. Whereas
the susceptibility effect in mice is due to the E627A substitution [57], the effect in humans is mediated by variants
Review
affecting the expression of the A20 gene, with lower mRNA
levels being associated with increased cardiovascular risk
and higher levels with protection [69]. These variants in the
human A20 gene are part of a larger group of polymorphisms, including mutations in lymphotoxin-a and in an IkB
homologue [70], influencing susceptibility to atherosclerosis
through modulation of NF-kB activity [69].
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Vol.30 No.8
how A20 regulates apoptosis signaling. We predict that the
vast expansion of studies on ubiquitination will reveal novel
targets for A20 in addition to the identification of regulatory
mechanisms. A detailed knowledge of these mechanisms
should eventually lead to the development of novel therapeutic approaches that support the activity of A20.
Acknowledgements
A20 as a tumor suppressor in B-cell lymphomas
Persistent NF-kB activation has a crucial role in cancer
development and progression [71], implicating a potential
role for A20 as a tumor suppressor. Recently, different
genetic studies have revealed that A20 is frequently inactivated by somatic mutations and/or deletions in multiple
B-cell lymphomas, including mucosa-associated tissue
(MALT) lymphoma, Hodgkin’s lymphoma, activated B-cell
like diffuse large B-cell lymphoma and follicular lymphoma [72–78], which are all frequently associated with
constitutive activation of the NF-kB pathway. Most of the
A20 mutations were nonsense or frameshift mutations
that prevent production of full-length protein. When reexpressed in lymphoma-derived cell lines carrying biallelic
inactivation of the gene, wild type A20 induced cell growth
arrest and apoptosis, accompanied by downregulation of
NF-kB activation [75,78]. In addition, A20-deficient cells
stably generated tumors in immunodeficient mice whereas
the tumorigenicity was effectively suppressed by reexpression of A20 [75]. Altogether, these results reveal
the intriguing role of A20 as a novel tumor suppressor
protein whose inactivation might thus represent a common
mechanism leading to constitutive or aberrant NF-kB
activation that mediates cell proliferation and survival
of subsets of B-lineage lymphomas. These studies, along
with the recent identification of A20 as a susceptibility
gene for multiple autoimmune diseases, also suggest that
A20 might be a crucial molecular link between chronic
inflammation and cancer.
Conclusions and perspectives
The list of proteins participating in the negative regulation
of NF-kB activation and apoptosis continuous to accumulate, with new molecular mechanisms emerging frequently.
A20 is a unique protein in the sense that it exerts both NFkB inhibitory and anti-apoptotic activities, and it is now
recognized as a key regulator in inflammation and immunity. The large number of GWAS studies over the past two
years have uncovered A20 as a susceptibility locus for
multiple autoimmune and inflammatory diseases. Changes
in A20 expression or activity can alter the intensity or
duration of immune responses that take part in the development and progression of these diseases. It remains to
be resolved if and how the reported polymorphisms in or
near the A20 locus affect the expression and/or function of
A20. In this context, mice lacking A20 in specific cell types or
expressing mutant versions of A20, combined with mouse
models for human autoimmune and inflammatory diseases
will be valuable tools. Another unresolved issue relates to
the molecular mechanisms employed by A20 to regulate
NF-kB activation and apoptosis. Whereas specific ubiquitinated proteins have been identified as A20 targets in the
NF-kB signaling pathway, it is still completely unknown
L.V. is a PhD fellow with the ‘Institute for the promotion of Innovation by
Science and Technology’ (IWT) and G.v.L. is a postdoctoral researcher
with the ‘Fund for Scientific Research of Flanders’ (FWO). G.v.L. is also
supported by an FWO Odysseus Grant, and a grant from the ‘Charcot
Foundation’. Work in the authors’ laboratory is further supported by
research grants from the ‘Interuniversity Attraction Poles program’
(IAP6/18), the FWO, the ‘Belgian Foundation against Cancer’, the
‘Strategic Basis Research program’ of the IWT, the ‘Centrum voor
Gezwelziekten’ and the ‘Concerted Research Actions’ (GOA) of the Ghent
University.
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