Download NF-kB as a primary regulator of the stress response

Oncogene (1999) 18, 6163 ± 6171
ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00
http://www.stockton-press.co.uk/onc
NF-kB as a primary regulator of the stress response
Frank Mercurio*,1 and Anthony M Manning*,1
1
Signal Pharmaceuticals, Inc., 5555 Oberlin Drive, San Diego, California, CA 92121, USA
A myriad of unrelated exogenous or endogenous agents
that represent a threat to the organism are capable of
inducing NF-kB activity, including viral infection,
bacterial lipids, DNA damage, oxidative stress and
chemotherapuetic agents. Likewise, NF-kB regulates
the expression of an equally diverse array of cellular
genes. These ®ndings are indicative of the widespread
signi®cance of NF-kB as a mediator of cellular stress.
Remarkably, the NF-kB pathway displays the capacity
to activate, in a cell- and stimulus-speci®c manner, only
a subset of the total repertoire of NF-kB-responsive
genes. The seemingly promiscuous nature of NF-kB
activation poses a regulatory quagmire as to how
speci®city is achieved at the level of gene expression.
The review will summarize recent ®ndings and explore
how they further our understanding of the mechanism by
which stimulus-speci®c activation of NF-kB is achieved
in response to cellular stress.
Keywords: ubiquitin; ligase; NF-kB; IKK; oxidative
stress
Survival of an organism is dependent on its ability to
rapidly and e€ectively respond to adverse changes in its
environment. This is often achieved through changes in
the program and rates of gene expression, leading to
the production of proteins that exert a protective e€ect
on the cell. Occasionally, these changes promote
dysregulated cell growth and tumorigenicity. Eukaryotic cells possess a number of distinct signal
transduction pathways that couple environmental
stimuli to speci®c changes of gene expression. One
such pathway, that of the transcription factor NF-kB,
orchestrates the rapid cellular response to a multitude
of seemingly unrelated exogenous and endogenous
stress-induced agents. NF-kB plays a critical role in
cell growth and di€erentiation, apoptosis, and adaptive
responses to changes in cellular redox balance. As
would be predicted of such a central signaling pathway,
abberant regulation of NF-kB activation has been
associated with the pathogenesis of several diseases,
including cancer. For this reason, the pharmaceutical
industry has focused signi®cant attention on this
pathway for the identi®cation of novel therapeutic
agents. Although our understanding of the NF-kB
signal transduction cascade has been greatly enhanced
by the recent discovery of several key regulatory
proteins, much remains unclear as to how this
ubiquitiously expressed transcription factor is able to
di€erentially regulate the expression of a diverse array
of genes in a cell- and stimulus-speci®c manner. This
*Correspondence: AM Manning and F Mercurio
review will summarize recent ®ndings on the mechanisms of NF-kB regulation in response to cellular stress
and the potential role of NF-kB as a therapeutic target
in cancer.
NF-kB is a pleiotropic mediator of stress-induced gene
expression
NF-kB is a dimeric transcription factor that is present
in the cytoplasm of most resting cells but that upon
activation can undergo rapid nuclear translocation
and induce gene expression. A myriad of unrelated
exogenous or endogenous agents that represent a
threat to the organism are capable of inducing NF-kB
activity (reviewed in Baldwin, 1996). Some of these
agents include viral infection, bacterial lipids, parasites, UV irradiation, shear stress, chemotherapeutic
agents, oxidative stress, pro-in¯ammatory cytokines
and DNA damage. This ®nding is indicative of the
widespread signi®cance of NF-kB as a mediator of
cellular stress. A growing number of highly diverse
genes have been demonstrated to be induced by NFkB, including viral genes (HIV-1, CMV), immunoreceptors (IL-2 receptor a-chain, T cell receptor b2),
cell adhesion molecules, cytokines and growth factors,
chemokines, acute phase proteins, oxidative stressrelated enzymes and anti-apoptotic proteins (reviewed
in Baeuerle and Baichwal, 1997; May and Ghosh,
1998). Remarkably, the NF-kB pathway displays the
capacity to activate, in a cell- and stimulus-speci®c
manner, only a subset of the total repertoire of NFkB-responsive genes. The seemingly promiscuous
nature of NF-kB activation poses a regulatory
quagmire as to how speci®city is achieved at the
level of gene expression. Rapid growth in our
understanding of signal transduction in general, and
NF-kB in particular, provide intriguing insights as to
how this may occur.
The prototype NF-kB transcription factor consists
of two subunits, NF-kB1 p50 and RelA (p65). The
initial cloning of these two subunits, and the discovery
of their relatedness to the proto-oncogene c-rel,
provided the ®rst insight into the NF-kB pathway
(reviewed in Baeuerle and Henkel, 1994). The products
of these genes share extensive homology in their Nterminal region called the Rel-homology domain
(RHD), whose subregions mediate DNA-binding,
dimerization and nuclear localization. To date, ®ve
proteins belonging to the NF-kB family have been
identi®ed in mammalian cells: RelA, c-Rel, RelB,
NFKB1 (p50/p105) and NFKB2 (p52/p100). The NFkB family members can be divided into two
functionally distinct classes of proteins; those that are
originally sythesized as inactive precursor proteins,
NFKB1 (p105) and NFKB2 (p100), and require
proteolytic processing to yield proteins capable of
NF-kB as a primary regulator of the stress response
F Mercurio and AM Manning
6164
DNA-binding, and those that are produced as
transcriptionally active forms, including RelA, RelB
and c-Rel. Members of the latter group appear to be
primarily responsible for the transcriptional activation
capacity of the complex. Indeed, stimulus-dependent
phosphorylation of these subunits results in the
potentiation of their transcriptional activity (Zhong et
al., 1997; Wang and Baldwin, 1998). NF-kB family
members can form stable homo- or heterodimeric
complexes, which vary in their DNA-binding specificity and transcription activation potential. Thus, the
existence of a multigene family could, in part, account
for the complex regulatory potential displayed by NFkB.
NF-kB exists in the cytoplasm in an inactive form
by virtue of its association with a class of inhibitory
proteins called IkBs. To date, seven IkBs have been
identi®ed: IkBa, IkBb, IkBe, Bcl3, NFKB1 (p105) and
NFKB2 (p100). The IkB family members, which have
common ankyrin repeat domains, regulate the DNAbinding and subcellular translocation of NF-kB
(reviewed in Baldwin, 1996). The IkBs all have 5-7
ankyrin repeat domains, each about 30-33 amino acids,
which stably interact with the RHD and mask the
nuclear localization signal (NLS) of NF-kB, therein
preventing its nuclear translocation. The IkBs display a
preference for speci®c NF-kB complexes, hence each
IkB has the potential to participate in distinct
regulatory pathways a€ecting activation of di€erent
NF-kB complexes. The regulation of IkBa-mediated
NF-kB activity is the best understood of these
pathways and will be discussed in the following
section. The NFKB1 and NFKB2 precursor proteins
are unique in that they posses both an N-terminal
RHD, which is part of the mature NFKB1-p50/
NFKB2-p52 protein, and C-terminal ankyrin repeat
domains. The precursor proteins form stable heterodimers with RelA, c-Rel, RelB, NFKB1-p50, and
NFKB2-p52 proteins, and by virtue of their Cterminal ankyrin repeat domains, retain these NF-kB
complexes in the cytoplasm (Rice et al., 1992; Mercurio
et al., 1993). The physiological relevance of the
precursor's ability to function in an IkB-like capacity
is not well understood. Mutant mice were generated
lacking the C-terminal ankyrin-repeat domain of either
p105 or p100, but expressing the mature p50 and p52
proteins, respectively (Ishikawa et al., 1997). These
mutant mice displayed a variety of pathologies
including gastric lymphoid hyperplasia, splenomegaly
and marked cell type speci®c alteration in NF-kB
activity in response to a variety of stimuli. Thus it
appears that the NFKB1 and NFKB2 precursor
proteins are indispensable in the control of NF-kB
regulation and absence of either precursor cannot be
eciently compensated by the other IkB family
members. Cell stimulation with inducers of NF-kB
activity increase the rate of precursor proteolytic
processing, resulting in the loss of the ankyrin repeat
domain, yielding functionally active NF-kB complexes
that undergo nuclear translocation. The protein kinase
TPL-2, which is more than 90% identical to the human
oncogene COT, has recently been found to associate
with NFKB1 p105 resulting in its phosphorylation and
increased proteolytic processing (Belich et al., 1999).
Kinase-inactive TPL-2 blocks TNFa-induced proteolytic processing of NFKB1 p105. However, TPL-2 does
not appear to directly phosphorylate p105. The relative
levels of the IkBs in a given cell type will in¯uence the
characteristics of NF-kB activation. The IkBs provide
yet another tier of regulatory complexity to modulate
NF-kB-mediated gene expression.
NF-kB activation pathway
Selective, regulated, protein degradation, like protein
synthesis and protein phosphorylation, is a fundamental mechanism by which the organism controls a
large variety of cellular processes. The ubiquitin/
proteosome pathway mediates the regulated degradation of many proteins that play an essential role in cell
cycle progression, immune response, transcriptional
control, development and apoptosis. Activation of
NF-kB is achieved through the signal-induced proteolytic degradation of IkB which is mediated by the 26S
proteosome (Figure 1; Alkalay et al., 1995). The critical
event that initiates proteolytic degradation of IkB is
the stimulus-dependent phosphorylation of IkB at
speci®c N-terminal serine residues (S32 and S36 for
IkBa, S19 and S23 for IkBb). Mutation of IkBa on
these residues to either alanine (S32/364A) or
threonine (S32/364T) was found to block stimulusdependent phosphorylation, thereby preventing degradation and subsequent activation of NF-kB (Chen et
al., 1995; Brown et al., 1995; DiDonato et al., 1996).
We and others recently identi®ed a high molecular
weight multiprotein complex containing an inducible
IkB kinase activity (Lee et al., 1997; DiDonato et al.,
1997; Mercurio et al., 1997; Zandi et al., 1997). Two
kinases contained in this complex, termed IkB kinase 1
(IKK1, IKKa) and 2 (IKK2, IKKb) were cloned and
demonstrated to play a key role in NF-kB activation
by a wide variety stimuli. The IKKs are stimulated by
inducers of NF-kB with kinetics consistent with the
kinetics for appearance of NF-kB in the nucleus. IKK1
and IKK2 are related members of a new family of
intracellular signal transduction enzymes, containing
an amino-terminal kinase domain and a C-terminal
region with two protein interaction motifs, a leucine
zipper and a helix ± loop ± helix motif. The kinase
domains display 64% identity, whereas the C-terminal
domains exhibit 44% identity. IKK1 and IKK2 can
form homo- and heterodimers in vitro and in vivo,
thereby allowing additional opportunities for achieving
signaling speci®city.
Sequence analysis revealed that both IKK1 and
IKK2 contained a canonical MAP kinase kinase
(MAPKK) activation loop motif. Phosphorylation of
both serine residues is necessary for activation of
kinase activity. We generated IKK2 mutants in which
Ser 177 and Ser 181 were mutated to Ala or Glu (S177/
1814A or S177/1814E) to block or mimic, respectively, the e€ect of P-Ser (Mercurio et al., 1997).
Interestingly, IKK2 (S171/1814E) generated a constitutively active IkB kinase, and was capable of
inducing RelA nuclear translocation and NF-kBdependent gene expression in the absence of cell
stimulation. Indeed, baculoviral expressed recombinant versions of IKK2 demonstrated that the speci®c
activity of IKK2-S177/1814E was at least tenfold
greater than that of the wildtype IKK2 protein
(Mercurio et al., 1999). In contrast, IKK2-S177/
NF-kB as a primary regulator of the stress response
F Mercurio and AM Manning
6165
Figure 1 Schematic representation of components of the NF-kB signal transduction pathway leading from the TNF and IL-1
receptors. A number of signal transduction proteins have been identi®ed as associated with these receptors, including TNF-receptor
associated factors 2 and 6 [TRAF2 and 6], death domain-containing proteins [TRADD and FADD], kinases associated with the IL1 receptor [IRAK1 and 2, and MYD88]. Other proteins, such as ring ®nger interacting protein [RIP] have been identi®ed based on
their ability to interact with several of these proteins. Signals emanating from the TNF and IL-1 receptors activate members of the
MEKK-related family, including NIK and MAKK1. These proteins are involved in activation of IKK1 and IKK2, the IkB kinase
components of the IKK signalsome. These kinases phosphorylate members of the IkB family at speci®c serines within their Ntermini, leading to site-speci®c ubiquitination and degradation by the 26S proteosome
1814A yielded an inactive kinase that was refractory
to stimulus-dependent activation, and was able to
block TNFa-stimulated RelA nuclear localization and
NF-kB-dependent gene expression. The corresponding
mutations for IKK1 displayed only modest changes in
NF-kB-mediated activation, suggesting that IKK2
played a more dominant role in NF-kB activation by
pro-in¯ammatory cytokines. These ®nding are consistent with recent genetic analysis of mouse mutants that
lack either IKK1 or IKK2. Mice that are devoid of the
IKK2 gene had extensive liver damage from apoptosis
and died as embryos (Li et al., 1999a,d). This
phenotype is remarkably similar to that seen preciously for mice devoid of RelA, the transactivating
subunit of NF-kB. Of note, IKK27/7 mice could be
rescued by the inactivation of the TNF receptor 1
consistent with a role for NF-kB in preventing TNFainduced hepatocyte apoptosis. Mouse embryonic
®broblasts that were isolated from IKK27/7 embryos
showed a marked suppression in TNFa- and IL-1induced NF-kB activity and enhanced apoptosis in
response to TNFa, even though normal IKK1 levels
were observed associated with IKKAP-1. In contrast,
mice devoid of the IKK1 gene were born alive but died
within 30 min (Hu et al., 1999; Li et al., 1999b). These
mice exhibit a plethora of developmental defects, the
most striking of which includes shiny, taut and sticky
skin without whiskers. Histological analysis revealed
thicker epidermis which is unable to di€erentiate in
vitro. The limbs and tail are highly rudimentary and
the head is signi®cantly shorter than normal. Despite
this marked morphology, the activation of IKK
activity and NF-kB DNA binding activity in IKK17/
7
cells by TNFa, IL-1 or bacterial lipopolysaccharide
appears normal. Therefore, it appears that IKK1 and
IKK2 serve distinct roles in the activation of NF-kB by
pro-in¯ammatoy cytokines, and they cannot substitute
for the loss of each other, even though they exist
together in a heterodimeric complex in cells. Such a
remarkable ®nding suggests exquisite regulation of NFkB activation in response to distinct stimuli. The exact
stimuli that activates IKK1 have not been de®ned, but
they do appear to be responsible for the regulation of
skin and skeletal development.
The precise mechanism and kinase required for IKK
activation is a topic of considerable controversy,
however, it appears likely that members of the MAP
kinase kinase kinase (MAPKKK) family will play a
role. One such upstream kinase, NF-kB Inducing
Kinase [NIK], was identi®ed by its ability to bind
directly to TRAF2, an adapter protein thought to
couple both TNFa and IL-1 receptors to NF-kB
activation (Malinin et al., 1997). A second
MAPKKK, MEKK-1, was shown to be present in
the IKK signalsome complex (Mercurio et al., 1997).
Several labs have reported that co-expression of either
NF-kB as a primary regulator of the stress response
F Mercurio and AM Manning
6166
Figure 2
Schematic representation of reactive oxygen intermediate generation systems and mechanisms of activation of NF-kB
NIK or MEKK-1 enhances the ability of the IKKs to
phosphorylate IkB and activate the NF-kB pathway
(Nakano et al., 1998; Yin et al., 1998). A recent study
suggests that MEKK-2 and MEKK-3 are also capable
of inducing NF-kB activity (Zhao and Lee, 1999). The
exact role of NIK and the MEKKs in IKK activation
has been the subject of considerable controversy. A
recent report demonstrated via phosphopeptide mapping studies that IKK2 is phosphorylated at Ser 177
and Ser 181 in response to pro-in¯amatory cytokines
(Delhase et al., 1999). These studies strongly suggest
that the IKKs are themselves phosphorylated and
activated by one or more upstream kinases. Moreover,
it was found that NIK and MEKK-1 induce IKK
activity via phosphorylation of IKK2 on Ser 177 and
181, and that IKK1 does not undergo induced
phosphorylation at the corresponding sites nor is it
required for IKK activity. Because NIK and MEKK-1
are activated by discrete stimuli, these studies provide
further evidence supporting the IKK complex as being
a central juncture of the NF-kB activation pathway.
In addition to the MAPKKK family, the atypical
protein kinase C [aPKC] subfamily of isozymes
comprised of gPKC and l/iPKC have been implicated
in the direct activation of IKK2 (Sanz et al., 1999). The
aPKCs, while not activated by classical lipid mediators,
are potently induced by TNFa. It was recently
demonstrated that the aPKCs can phosphorylate and
activate IKK2 in vitro and in vivo (Lallena et al., 1999).
TNFa activates the aPKCs with kinetics that are
compatible with the induced phosphorylation and
subsequent degradation of IkBa. gPKC activates
IKK2, but not IKK1 activity. A dominant negative
version of gPKC blocks TNFa, but not PMA, induced
stimulation of IKK2 activity. It appears that the
activity of the aPKCs is modulated by selective,
stimulus-dependent, protein ± protein interactions with
the putative sca€olding protein, p62, which link aPKCs
to membrane signaling proteins. Sanz et al. (1999)
recently elucidated the nature of this interaction,
®nding that upon cell stimulation with TNFa, the
TNF-R1 recruits the protein TRADD that binds
TRAF2 and RIP, p62 then interacts with RIP and
facilitating recruitment of aPKCs to the TNF receptor
complex. The aPKCs then function to directly activate
IKK2 and consequently NF-kB.
The precise mechanism by which intracellular signals
are relayed from the cell membrane to the nucleus are
complex and varied. One theme involves the integration of key signaling proteins into multiprotein
complexes. This is the situation for NF-kB activation
which requires communication between two higherorder complexes, that of the TNF or IL-1 receptor
complex and the IKK signalsome. Varying combinations of related enzymes in a complex, often in a cell
speci®c manner, enables the propagation of distinct
cellular responses. Elucidation of these subtle regulatory processes can be dicult due to misleading results
obtained by ectopic overexpression of kinase de®cient
versions of closely related family members (i.e. titration
of common binding regulatory proteins, overcoming
evolutionary designed binding coecients). Genetic
analysis via knockout/transgenic animals has provided
enormous insight into regulated signal transduction,
however, issues of redundancy and lethality create
limitations to this approach. The development of
highly speci®c pharmaceutical inhibitors of these
kinases should provide valuable tools to further our
understanding in this area.
The identi®cation of additional components of the
IKK signalsome has revealed potential mechanisms by
which receptor activation is linked to IKK activation.
Yamaoka et al. (1998) described the identi®cation of
NEMO (NF-kB-essential modulator) through genetic
complementation of a ¯at cellular variant of HTLV-1
Tax-transformed rat ®broblasts, known as 5R, which
NF-kB as a primary regulator of the stress response
F Mercurio and AM Manning
is unresponsive to all tested NF-kB inducing stimuli.
NEMO was able to complement the 5R cell line, and
restore NF-kB activation in response to LPS, PMA
(phorbol myristylate acetate) and IL-1. NEMO is a
48 kDa protein, which contains a putative leucine
zipper, and was shown to homo-dimerize and interact
with IKK2. Two other groups working independently
biochemically puri®ed and cloned the same non-kinase
component of the IKK signalsome. In our laboratory,
we demonstrated that the IKK signalsome can exist in
two discrete high molecular weight complexes in vivo,
one containing a heterodimer of IKK1 and IKK2, the
other containing a homodimer of IKK2 (Mercurio et
al., 1999). A novel 48 kDa component common to
both complexes was identi®ed and demonstrated to
directly interact with IKK2. This protein was named
IKK associated-1 (IKKAP-1). Rothwarf et al. (1999)
also puri®ed and cloned the identical non-kinase
component of the IKK complex which they termed
IKKg. Functional analysis revealed that binding of
IKK2 required the N-terminal coiled-coil repeat
domain of IKKAP-1. Mutant versions of IKKAP-1,
which either lack the N-terminal IKK2 binding
domain or contain only the IKK2 binding domain,
potently inhibited both IKK2 activation and RelA
nuclear translocation. These studies suggest that the
N- and C-terminal domains of IKKAP-1 play distinct
and essential roles in IKK activation. Immunocytochemical studies revealed that the transient overexpression of the IKKAP-1 C-terminal domain,
which lacks the IKK2 binding domain, displays
stimulus-dependent subcellular localization to the
plasma membrane. This suggests that IKKAP-1
mediates direct interaction with the TNFa receptor
complex. Indeed, a protein called FIP-3, which is
identical to IKKAP-1/NEMO/IKKg, has been shown
to interact directly with RIP, a component of the
TNFa receptor complex. FIP-3 was originally
identi®ed as a 14.7 kDa E3 interacting protein
(distinct from ubiquitin ligase E3 proteins), which is
a virally encoded protein that functions to inhibit the
cytolytic e€ects of TNFa (Li et al., 1999c). Recent
reports have shown that the tax oncoprotein of the
human T cell leukemia virus type1 constitutively
activates the NF-kB pathway via direct interaction
with IKKAP-1 (Harhaj and Sun, 1999; Jin et al.,
1999). It is possible that Tax links interaction of the
IKK complex with upstream activators in the absence
of cell stimulation.
IkB phosphorylation serves as a molecular tag
leading to rapid ubiquitination and degradation of
IkB by components of the ubiquitin-proteasome system
(Hershko and Ciechanower, 1992). The sites of
ubiquitin conjugation are two adjacent lysine residues
[Lys-20 and Lys-21 in IkBa] located just N-terminal to
the two serines that are targets for phosphorylation by
the IKKs (Orian et al., 1995). Short phosphopeptides
that include the IKK phosphorylation sites (Ser32 and
Ser 36), but not lys-20 and Lys-21, were used as
substrate mimics to establish that IkBa phosphorylation generates a binding site for the speci®c ubiquitin
ligase(s) responsible for conjugation of ubiquitin to
IkBa (Yaron et al., 1997). Microinjection of these IkBa
phosphopeptide into cells abrogated NF-kB activation
in response to cytokine stimulation, and consequent
NF-kB-mediated gene expression. These results provide
insight into a novel mechanism for the modulation of
cellular targets of the ubiquitin ± ligase system.
In eukaryotic cells, ubiquitination of the target
substrate is mediated by a series of ubiquitinconjugating enzymes (Ciechanover, 1998). Ubiquitin
is initially activated via thioester formation by a
ubiquitin-activating enzyme (E1), which then transfers
ubiquitin to a member of a distinct class of ubiquitinconjugating enzymes (UBCs or E2s). Some E2s possess
the capacity to directly transfer ubiquitin to the target
protein, whereas others require the participation of a
third component, termed an E3 ubiquitin protein
ligase. E3s can transfer ubiquitin directly to the target
protein, or in some cases, the E3 simply acts to mediate
interaction of a speci®c protein substrate with an E2
ligase charged with ubiquitin. Recently, the pIkBa-E3
ubiquitin ligase was isolated from HeLa cells by virtue
of its phosporylation-dependent, high anity, association with the pIkBa/NF-kB complex, and found to be
a member of the latter class of E3 ligases (Yaron et al.,
1998). Using nanoelectrospray mass spectrometry, the
speci®c component of the E3 ligase that recognizes the
pIkBa degradation motif, DS(PO3)GLDS(PO3)M, was
identi®ed as an F-box/WD-domain protein belonging
to a recently distinguished family of b-TrCP/Slimb
proteins. This component, termed E3RSIkB (pIkBa-E3
Receptor Subunit), binds speci®cally to pIkBa and
promotes its in vitro ubiquitination in the presence of
E1 and UBC5c, an E2 ubiquitin ligase. Additional E2
ubiquitin ligases have also been demonstrated to work
in concert with E3RSIkB (Tan et al., 1999). F-box and
WD domain containing proteins are known to be
involved in protein ± protein interactions, and bind
Skp1, an adaptor component of E3 complexes, and
the target substrate, respectively. An F-box deletion
mutant of E3RSIkB, which binds tightly to pIkBa but
does not support its ubiquitination, acts in vivo as a
dominate negative protein, inhibiting the degradation
of pIkBa and consequently NF-kB activation. Its also
been suggested that E3RSIkB mediates regulated
degradation of beta-catenin (Latres et al., 1999; Fuchs
et al., 1999). E3RSIkB represents a member of a family
of receptor proteins comprising the core component of
a class of ubiquitin-ligases which speci®cally recognize
phosphorylated proteins and target them for proteasomal degradation.
Post-translational modi®cation of the NF-kB complex, occurring subsequent to IkB degradation can
enhance transcriptional activation of NF-kB-dependent
genes. The transcriptional activity of NF-kB is
stimulated upon phosphorylation of its p65 subunit
on serine 276 by protein kinase A (PKA) (Zhong et al.,
1997). The transcriptional coactivator CBP/p300 was
found to associate with NF-kB RelA through two sites,
an N-terminal domain that interacts with the Cterminal region of unphosphorylated RelA, and a
second domain that interacts with RelA phosphorylated on serine 276. Accessibility to both sites is
blocked in unphosphorylated RelA through an
intramolecular masking of the N-terminus by the Cterminal region of RelA. Phosphorylation by PKA
both weakens the interaction between the N- and Cterminal regions of RelA and creates an additional site
for interaction with CBP/p300 (Zhong et al., 1998).
Because PKA phosphorylates a range of substrates in
vivo, it is unlikely that PKA inhibitors could achieve
6167
NF-kB as a primary regulator of the stress response
F Mercurio and AM Manning
6168
selective modulation of NF-kB-dependent gene transcription. Of note, RelA is phosphorylated by IKK1
and IKK2 in vitro on a single tryptic peptide distinct
from that containing serine 276 (Mercurio et al., 1999).
The exact residues phosphorylated by these enzymes
and their e€ects on NF-kB-dependent transcription in
vivo, however, have yet to be reported.
NF-kB as a primary oxidative stress response pathway
NF-kB is recognized as a redox-sensitive transcription
factor, and has been implicated in the cellular response
to oxidative stress. Molecular oxygen presents a
paradox to aerobic organisms in that it is both
essential and at the same time extremely toxic.
Although unreactive in its ground state, molecular
oxygen is reduced via normal metabolic processes,
yielding a variety of highly reactive oxygen intermediates (ROIs) in the process (Figure 2). These ROIs
include the superoxide radical (O27), hydrogen peroxide (H2O2), and the most potent oxidant, the
hydroxyl radical (OH.). ROIs can readily cause
oxidative damage to various biological macromolecules resulting in DNA damage, DNA strand breaks,
oxidation of key amino acid side chains, formation of
protein-protein cross-links, oxidation of the polypeptide backbone resulting in protein fragmentation, and
lipid peroxidation. These oxidative-induced lesions
have been suggested to contribute to various diseases
and degenerative processes such as aging, carcinogenesis, immunode®ciencies and cardiovascular disease.
As a consequence of normal cellular energy
metabolism the superoxide radical is produced via
reactions mediated by a variety of oxidative enzymes,
including NADPH oxidase, cytochrome p450, lipoxygenase, cyclooxygenase, xanthine oxidase and the
coenzyme Q/ubiqinone complex. ROIs are also
generated during chronic and acute in¯ammatory
diseases, and following exposure to g-rays and UV
light irradiation, air pollutants such as ozone, SO2, acid
rain and herbicides. Oxidative stress results when ROIs
are not adequately removed from the cellular environement. This occurs if antioxidants are depleted and/or if
the formation of ROIs is increased beyond the ability
of the defenses to cope with them. Physiological
defenses against oxidative stress include small molecule antioxidants and antioxidant proteins that
maintain the intracellular redox environment in a
highly regulated and reduced state. Antioxidant
proteins act in several ways, including directly destroying oxidants catalytically via superoxide dismutase,
catalase and glutathione peroxidase, or they sequester
transition metals so as to prevent these metal ions from
catalyzing ROIs via the Fenton or Haber-Weiss
reaction. Small molecule antioxidants either act in a
sacri®cial manner by scavenging oxidants, or they
chelate transition metal ions (ferritin) in a way that
prevents metal ions from catalyzing the generation of
ROIs. Growing evidence has indicated that cellular
redox plays an essential role not only in cell survival,
but also in cellular signaling pathways such as NF-kB.
Similar to protein phosphorylation, ROIs can function
as components of signal transduction cascades, acting
as key regulatory switches in many cellular processes.
The delicate balance between oxidants and antiox-
idants, such as glutathione and thioredoxin, ultimately
determine the activity pro®le for many transcription
factors. The NF-kB pathway is generally thought to be
a primary oxidative stress-response pathway, however,
the precise molecular mechanism by which ROIs
activate NF-kB have yet to be elucidated.
Several laboratories have demonstrated that treatment of cells with H2O2 can activate the NF-kB
pathway (reviewed in Henkel and Bauerle, 1994). The
observation that inducers of NF-kB activity, such as
TNFa, IL-1, LPS, PMA, UV and ionizing radiation,
generate elevated levels of ROIs prompted speculation
that ROIs may function as common mediators of NFkB activation. Further support for this model was
provided by indirect evidence showing that nearly all
pathways leading to NF-kB activation could be
blocked by a variety of antioxidants, including
pyrrolidinedithiocarbamate (PDTC), N-acetyl-L-cysteine (NAC), glutathione (GSH), thioredoxin (Txn),
or by overexpression of antioxidant enzymes, including
superoxide dismutase, glutathione peroxidase, or
thioredoxin peroxidase. Contrary to this model,
several reports show that H2O2-induced NF-kB
activation is cell type speci®c, suggesting H2O2 is not
a general mediator of NF-kB activation. However, the
lack of easy, fast and sensitive methods by which to
measure changes in intracellular levels of ROIs hinders
precise elucidation of the mechanism and species of
ROI responsible for oxidative-stress-induced activation
of NF-kB. Shi et al. (1999) recently demonstrated that
the hydroxyl radical was primarily responsible for
activation of NF-kB in Jurkat cells and macrophages.
A hydroxyl radical generating system that did not
involve exogenous H2O2 was used to demonstrate that
antioxidants which scavenge the OH. radical or its
precursor, inhibit NF-kB activation. In addition, metal
chelators that render metal ions incapable of generating the hydroxyl radical from H2O2 were shown to
block NF-kB activation. Thus, the ability of the cell to
convert H2O2 to OH. rather than another metabolic
by-product could in¯uence activation of NF-kB.
Interestingly, since OH. is extremely reactive and
short-lived, the subcellular localization of its generation could be critical to activation of NF-kB. For
example, it was recently shown in macrophages that a
redox reaction at the plasma membrane itself was the
initial signaling process in NF-kB activation (Kaul et
al., 1998).
The process by which ROIs are regulated is highly
complex and consequently the mechanism by which
NF-kB is activated via ROIs may be equally diverse.
For example, Bonizzi et al. (1999) reported that three
distinct cell-speci®c pathways lead to NF-kB activation
in response to IL-1. In lymphoid cells NF-kB
activation was dependent 5-lipoxigenase-mediated
production of ROIs, a pathway requiring ROI
production by NADPH was identi®ed in monocytic
cells, whereas an ROI- and 5-lypoxygenase-independent pathway was found to be involved in epithelial
cells. Interestingly, introduction of a functional 5lypoxygenase system in the epithelial cells allowed ROI
production and 5-lypoxigenase-dependent NF-kB
activation. Hence, the repertoire of oxidative enzymes
expressed in a given cell type will in¯uence the nature
of NF-kB activation. Likewise, adverse conditions that
alter the expression pro®le of the oxidative enzymes
NF-kB as a primary regulator of the stress response
F Mercurio and AM Manning
could result in aberent NF-kB activation and
associated pathologies.
The step(s) in the NF-kB signaling pathway that are
responsive to redox regulation are not well documented. Addition of H2O2 to speci®c cell types results in
the appearance of the hyperphosphorylated form of
IkBa, which undergoes rapid degradation in the
absence of proteosome inhibitors. This data would
suggest that ROIs act upstream or directly on the IKK
complex. Consistent with this model, treatment with
antioxidants or over-expression of antioxidant enzymes
blocked IkBa phosphorylation and degradation induced by LPS, TNFa and PMA. Note however, that
inhibition of stimulus-dependent IkBa degradation by
antioxidants, such as NAC or PDTC, also occurs in
cells that are refractory to H2O2-induced NF-kB
activation. It seems reasonable to suggest that redox
regulation may a€ect more than one step in the NF-kB
pathway. Indeed, reducing agents such as DTT and bmercaptoethanol enhance DNA-binding activity of
NF-kB itself.
In an e€ort to further explore the mechanism by
which redox balance a€ects NF-kB activation, we
treated HeLa cell with PDTC, an antioxidant
demonstrated to inhibit NF-kB activation in response
to a variety of stimuli, prior to stimulation with TNFa
and examined IKK activation and IkBa degradation
(Figure 3). The result from this study was quite
unexpected and suggests that there may be additional
regulatory steps in the NF-kB pathway which have yet
to be elucidated. For example, addition of PDTC to
HeLa cells, in the absence of TNFa, resulted in
dramatic activation of IKK activity. Surprisingly,
IKK activation did not lead to IkBa phosphorylation
or subsequent degradation. This result would indicate
that while IKK activation is required it may not be
sucient for complete propagation of the signal,
perhaps the IkBa substrate must be actively recruited
to the IKK complex. Indeed, IkBa undergoes a marked
shift from a 400 kDa to greater than 700 kDa complex
upon cell stimulation (Mercurio et al., 1997; Yaron et
al., 1997). Moreover, treatment with PDTC prior to
Figure 3 Activation of IKK activity and IkBa phosphorylation
in Hela cells treated with either PDTC (50 mM) prior to TNFa
stimulation
TNFa induction allowed phosphorylation of IkB,
however, the phosphorylated form of IkB was
refractory to degradation, similar to what we observe
when pre-treating cells with inhibitors of the proteasome (LLF, MG132). PDTC does not interfere with
IKK activation in response to TNFa, in fact, PDTC
potentiates IKK activity at higher concentrations. One
possible explanation is that PDTC inhibits ubiquitination and/or degradation of phosphorylated IkBa. These
data indicate that redox regulation of the NF-kB
signaling pathway may function at multiple points.
NF-kB and cancer
Malignant tumor cells have usually accumulated
mutations that a€ect a variety of processes, including
those that sustain cell growth, that block growth
inhibition and apoptosis, that a€ect DNA repair, and
that allow the tumor to escape immune surveillance.
Several lines of evidence suggest that NF-kB family
members are involved in tumor growth and metastasis. The genes for c-rel, NFKB1 [p105], NFKB2
[p52], RelA and Bcl-3 are located at sites of recurrent
tranlsocation and genomic rearrangements in cancer
(Matthew et al., 1993; Gilmore and Morin, 1993;
Bargou et al., 1997). Several Hodgkin's lymphoma cell
lines contain constitutively nuclear NF-kB complexes,
and inhibition of NF-kB activation by overexpression
of a non-degradable IkBa molecule inhibits proliferation and tumorigenicity of these cells (Sovak et al.,
1997). Many breast cancer cells also contain high
levels of nuclear NF-kB DNA-binding activity, which
is essential for survival of these cells in culture
(Nakshatri et al., 1997). Of note, chemically induced
breast cancers in rats and many primary human breast
cancer samples have high levels of nuclear NF-kB.
High levels of activated NF-kB are also associated
with progression of breast cancer cells to estrogenindependent growth (Bours et al., 1994). High levels
of NF-kB activity have been reported in diverse solid
tumor-derived cell lines (Visconti et al., 1997). Human
thyroid carcinoma cells require nuclear NF-kB activity
for proliferation and malignant transformation in vitro
(Hammarskjold and Simurda, 1992). The Tax protein
from the leukemogenic virus HTLV-1 is a potent
activator of NF-kB (Finco et al., 1997). Oncogenic
forms of ras, the most common defect in human
tumors, can activate NF-kB, and NF-kB inhibition
can block ras-mediated cellular transformation (Fisher, 1994; Mayo et al., 1997). Insights into the
molecular mechanims by which NF-kB promotes
these e€ects are being elucidated. Many cancer
therapies function to kill transformed cells through
apoptotic mechanisms. Resistance to apoptosis provides protection against cell killing by these therapies
and may represent a major mechanism of tumor drug
resistance (Wang et al., 1996). The activation of NFkB by tumor necrosis factor a [TNFa], ionizing
radiation, and chaemotherapeutic agents was found
to protect tumor cells from cell killing (Van Antwerp
et al., 1996; Beg and Baltimore, 1996). Inhibition of
NF-kB activation in this setting enhanced apoptotic
killing by these reagents but not by apoptotic stimuli
that do not activate NF-kB. NF-kB can induce the
expression of anti-apoptotic genes such as IAP
6169
NF-kB as a primary regulator of the stress response
F Mercurio and AM Manning
6170
proteins (cellular inhibitor of apoptosis-1 (Chu et al.,
1997)), superoxide dismutase and A20, or genes that
contribute to proliferation such as c-myc and Bcl-2.
Recently, NF-kB activation was shown to result in
transcriptional activation of the genes encoding a
novel factor IEX-1L (Wu et al., 1998) and TRAF1
and 2, and result in supression of caspase 8 activation
(Wang et al., 1999). In this way, persistent activation
of NF-kB represents a unique mechanism by which
these cells express genes which protect against
apoptotic stimuli. NF-kB also regulates the expression cell adhesion molecules that facilitate cell
adhesion and migration (reviewed in Chen and
Manning, 1995). Inhibition of NF-kB activation,
using either antisense oligonucleotides or transcription factor decoys, results in loss of adhesion in tumor
cells in culture (Sokoloski et al., 1993), and reduction
in tumor formation in nude mice (Higgins et al.,
1993). Inhibition of NF-kB represents a novel
approach to cancer drug development, and when
used as combination therapy with established ther-
apeutics, these agents could provide a more e€ective
treatment of drug-resistant forms of cancer.
Conclusion
The NF-kB transcription factor is activated by a
surprisingly broad array of cellular stress stimuli and
current evidence supports a key role for this gene
regulating pathway in a variety of human diseases, and
in particular cancer. Key biochemical components of
the signal transduction cascade mediating activation of
NF-kB by pro-in¯ammatory cytokines have been
identi®ed. In the near future it is anticipated that
other components of these pathways, and those
activated in response to other stimuli will emerge.
This should provide the molecular tools to address the
fundamental question of the mechanism by which celland stimulus-speci®c activation of NF-kB complexes is
achieved.
References
Alkalay I, Yaron A, Hatzubai A, Orian A, Ciechanover A
and Ben-Neriah Y. (1995). Proc. Natl. Acad. Sci. USA, 92,
10599 ± 10603.
Baeuerle PA and Baltimore D. (1996). Cell, 87, 13 ± 20.
Baeuerle PA and Henkel T. (1994). Annu. Rev. Immunol., 12,
141 ± 179.
Baeuerle PA and Baichwal VR. (1997). Adv. Immunol., 65,
111 ± 136.
Baldwin Jr, AS. (1999). Annu. Rev. Immunol., 14, 649 ± 683.
Bargou RC, Emmerich F, Krappmann D, Bommert K,
Mapara MY, Arnold W, Royer HD, Grinstein E, Greiner
A, Scheidereit C and Dorken B. (1997). J. Clin. Invest.,
100, 2961 ± 2969.
Belich MP, Salmeron A, Johnston LH and Ley SC. (1999).
Nature, 397, 363 ± 368.
Beg AA and Baltimore D. (1996). Science, 274, 782 ± 784.
Bonizzi G, Piette J, Schoonbroodt S, Greimers R, Harvard
L, Merville MP and Bours V. (1999). Mol. Cell. Biol., 3,
1950 ± 1960.
Bours V, Dejardin E, Goujon-Letawe F, Merville MP and
Castronovo V. (1994). Biochem. Pharmacol., 47, 145 ± 149.
Brown K, Gerstberger L, Carlson G, Franzoso G and
Siebenlist U. (1995). Science, 267, 1485 ± 1491.
Chen CC and Manning AM. (1995). Agents Actions, 47,
135 ± 141.
Chen Z, Hagler J, Palombella VJ, Melandri F, Scherer D,
Ballard D and Maniatis T. (1995). Genes Dev., 9, 1586 ±
1597.
Chu ZL, McKinsey TA, Liu L, Gentry JJ, Malim MH and
Ballard DW. (1997). Proc. Natl. Acad. Sci. USA, 94,
10057 ± 10062.
Ciechanover A. (1998). EMBO J., 17, 7151 ± 7160.
Delhase M, Hayakawa M, Chen Y and Karin M. (1999).
Science, 284, 309 ± 313.
DiDonato JA, Hayakawa M, Rothwarf DM, Zandi E and
Karin M. (1997). Nature, 388, 853 ± 862.
DiDonato J, Mercurio F, Rosette C, Wu-Li J, Suyang H,
Ghosh S and Karin M. (1996). Mol. Cell. Biol., 16, 1295 ±
1304.
Finco TS, Westwick JK, Norris JL, Beg AA, Der CJ and
Baldwin AS. (1997). J. Biol. Chem., 272, 24113 ± 24116.
Fisher DE. (1994). Cell, 78, 539 ± 542.
Fuchs SY, Chen A, Xiong Y, Pan ZQ and Ronai Z. (1999).
Oncogene, 18, 2039 ± 2046.
Gilmore TD and Morin PJ. (1993). Trends Genet., 9, 427 ±
433.
Hammarskjold ML and Simurda MC. (1992). J. Virol., 66,
6496 ± 6501.
Harhaj EW and Sun SC. (1999). J. Biol. Chem., 274, 22911 ±
22914.
Hershko A and Ciechanover A. (1992). Annu. Rev. Biochem.,
61, 761 ± 807.
Higgins KA, Perez JR, Coleman TA, Dorshkind K,
McComas WA, Sarmiento UM, Rosen CA and Narayanan R. (1993). Proc. Natl. Acad. Sci. USA, 90, 9901 ± 9905.
Hu Y, Baud V, Delhase M, Zhang P, Deerinck T, Ellisman
M, Johnson R and Karin M. (1999). Science, 284, 316 ±
320.
Ishikawa H, Carrasco D, Claudio E, Ryseck RP and Bravo
R. (1997). F. Exp. Med., 186, 999 ± 1014.
Jin DY, Giordano V, Nakano H and Jeang KT. (1999). J.
Biol. Chem., 274, 17402 ± 17405.
Kaul N, Choi J and Forman HJ. (1998). Free Radic. Biol.
Med., 24, 202 ± 207.
Lallena MJ, Diaz-Meco MT, Bren G, Paya CV and Moscat
J. (1999). Mol. Cell. Biol., 19, 2180 ± 2188.
Latres E, Chiaur DS and Pagano M. (1999). Oncogene, 18,
849 ± 855.
Lee FS, Hagler J, Chen ZJ and Maniatis T. (1997). Cell, 88,
213 ± 222.
Li Q, Van Antwerp D, Mercurio F, Lee KF and Verma IM.
(1999a). Science, 284, 321 ± 325.
Li Q, Lu Q, Hwang JY, Buscher D, Lee KF, IzpisuaBelmonte JC and Verma IM. (1999b). Genes Dev., 13,
1322 ± 1328.
Li Y, Kang J, Friedman J, Tarassishin L, Ye J, Kovalenko A,
Wallach D and Horwitz MS. (1999c). Proc. Natl. Acad.
Sci., 96, 1042 ± 1047.
Li ZW, Chu W, Hu Y, Delhase M, Deerinck T, Ellisman M,
Johnson R and Karin M. (1999d). J. Exp. Med., 189,
1839 ± 1845.
Malinin NL, Boldin MP, Kovalenko AV and Wallach D.
(1997). Nature, 385, 540 ± 544.
Matthew S, Murty, VV, Dalla-Favera R and Chaganti RS.
(1993). Oncogene, 8, 191 ± 193.
May MJ and Ghosh S. (1998). Immunol. Today, 19, 80 ± 88.
NF-kB as a primary regulator of the stress response
F Mercurio and AM Manning
Mayo MW, Wang CY, Cogswell PC, Rogers-Graham KS,
Lowe SW, Der CJ and Baldwin Jr, AS. (1997). Science,
278, 1812 ± 1815.
Mercurio F, DiDonato JA, Rosette C and Karin M. (1993).
Gene Dev., 7, 705 ± 718.
Mercurio F, Zhu H, Murray BW, Shevchenko A, Bennett
BL, Li J, Young D, Barbosa M, Mann M, Manning AM
and Rao A. (1997). Science, 278, 860 ± 866.
Mercurio F, Murray B, Bennett BL, Pascaul G, Shevchenko
A, Zhu H, Young D, Li J, Mann M and Manning AM.
(1999). Mol. Cell. Biol., 19, 1526 ± 1538.
Nakano H, Shindo M, Sakon S, Nishinaka S, Mihara M,
Yagita H and Okumura K. (1998). Proc. Natl. Acad. Sci.
USA, 95, 3537 ± 3542.
Nakshatri H, Bhat-Nakshatri P, Martin D, Goulet R and
Sledge G. (1997). Mol. Cell. Biol., 17, 3629 ± 3639.
Orian A, Whiteside S, Israel A, Stancovski I, Schwartz AL
and Ciechanover A. (1995). J. Biol. Chem., 270, 21707 ±
21714.
Rice NR, MacKichan ML and Israel A. (1992). Cell, 71,
243 ± 253.
Rothwarf DM, Zandi E, Natoli G and Karin M. (1998).
Nature, 395, 297 ± 300.
Sanz L, Sanchez P, Lallena MJ, Diaz-Meco MT and Moscat
J. (1999). EMBO J., 18, 3044 ± 3053.
Shi X, Dong Z, Huang C, Ma W, Liu K, Ye J, Chen F,
Leonard SS, Ding M, Castranova V and Vallyathan V.
(1999). Mol. Cell Biochem., 194, 63 ± 70.
Sokoloski JA, Sartorelli AC, Rosen CA and Narayanan R.
(1993). Blood, 82, 625 ± 632.
Sovak MA, Bellas RE, Kim DW, Zanieski GJ, Rogers AE,
Traish AM and Sonenshein GE. (1997). J. Clin. Invest.,
100, 2952 ± 2960.
Tan P, Fuchs SY, Chen A, Wu K, Gomez C and Ronai Z.
(1999). Mol. Cell, 4, 527 ± 533.
Van Antwerp DJ, Martin SJ, Kafri T, Green DR and Verma
IM. (1996). Science, 274, 787 ± 789.
Visconti R, Cerutti J, Battista S, Fedele M, Trapasso F, Zeki
K, Miano MP, de Nigris F, Casalino L, Curcio F, Santoro
M and Fusco A. (1997). Oncogene, 15, 1987 ± 1994.
Wang C, Mayo M and Baldwin AS. (1996). Science, 274,
784 ± 787.
Wang C-Y, Mayo MW, Korneluk RG, Goeddell DV and
Baldwin AS. Science, 281, 1680 ± 1683.
Wang D and Baldwin Jr, AS. (1998). J. Biol. Chem., 45,
29411 ± 29416.
Wu MX, Ao Z, Prasad KV, Wu R and Schlossman SF.
(1998). Science, 281, 998 ± 1001.
Yamaoka S, Courtois G, Bessia C, Whiteside ST, Weil R,
Agou F, Kirk HE, Kay RJ and IsraeÈl A. (1998). Cell, 93,
1231 ± 1240.
Yaron A, Alkalay I, Hatzubai A, Jung S, Beyth S, Mercurio
F, Manning AM, Gonen H, Ciechanover A and BenNeriah Y. (1997). EMBO J., 16, 101 ± 107.
Yaron A, Hatzubai A, Davis M, Lavon I, Amit S, Manning
AM, Andersen JS, Mann M, Mercurio F and Ben-Neriah
Y. (1998). Nature, 396, 590 ± 594.
Yin M-Y, Christerson LB, Yamamoto Y, Kwak Y-T, Xu S,
Mercurio F, Barbosa M, Cobb MH and Gaynor RB.
(1998). Cell, 93, 875 ± 884.
Zandi E, Rothwarf DM, Delhasse M, Hayakawa M and
Karin M. (1997). Cell, 91, 243 ± 252.
Zhao Q and Lee FS. (1999). J. Biol. Chem., 274, 8355 ± 8358.
Zhong H, Yang HS, Erdjument-Bromage H, Tempst P and
Ghosh S. (1997). Cell, 89, 413 ± 424.
Zhong H, Voll RE and Ghosh S. (1998). Molec. Cell, 1, 661 ±
671.
6171