A myriad of unrelated exogenous or endogenous agents that represent a threat to the organism are capable of inducing NF-κB activity, including viral infection, bacterial lipids, DNA damage, oxidative stress and chemotherapuetic agents. Likewise, NF-κB regulates the expression of an equally diverse array of cellular genes. These findings are indicative of the widespread significance of NF-κB as a mediator of cellular stress. Remarkably, the NF-κB pathway displays the capacity to activate, in a cell- and stimulus-specific manner, only a subset of the total repertoire of NF-κB-responsive genes. The seemingly promiscuous nature of NF-κB activation poses a regulatory quagmire as to how specificity is achieved at the level of gene expression. The review will summarize recent findings and explore how they further our understanding of the mechanism by which stimulus-specific activation of NF-κB is achieved in response to cellular stress.
Survival of an organism is dependent on its ability to rapidly and effectively 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 effect 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 specific changes of gene expression. One such pathway, that of the transcription factor NF-κB, orchestrates the rapid cellular response to a multitude of seemingly unrelated exogenous and endogenous stress-induced agents. NF-κB plays a critical role in cell growth and differentiation, apoptosis, and adaptive responses to changes in cellular redox balance. As would be predicted of such a central signaling pathway, abberant regulation of NF-κB activation has been associated with the pathogenesis of several diseases, including cancer. For this reason, the pharmaceutical industry has focused significant attention on this pathway for the identification of novel therapeutic agents. Although our understanding of the NF-κB 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 differentially regulate the expression of a diverse array of genes in a cell- and stimulus-specific manner. This review will summarize recent findings on the mechanisms of NF-κB regulation in response to cellular stress and the potential role of NF-κB as a therapeutic target in cancer.
NF-κB is a pleiotropic mediator of stress-induced gene expression
NF-κB 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-κB activity (reviewed in Baldwin, 1996). Some of these agents include viral infection, bacterial lipids, parasites, UV irradiation, shear stress, chemotherapeutic agents, oxidative stress, pro-inflammatory cytokines and DNA damage. This finding is indicative of the widespread significance of NF-κB as a mediator of cellular stress. A growing number of highly diverse genes have been demonstrated to be induced by NF-κB, including viral genes (HIV-1, CMV), immunoreceptors (IL-2 receptor a-chain, T cell receptor β2), cell adhesion molecules, cytokines and growth factors, chemokines, acute phase proteins, oxidative stress-related enzymes and anti-apoptotic proteins (reviewed in Baeuerle and Baichwal, 1997; May and Ghosh, 1998). Remarkably, the NF-κB pathway displays the capacity to activate, in a cell- and stimulus-specific manner, only a subset of the total repertoire of NF-κB-responsive genes. The seemingly promiscuous nature of NF-κB activation poses a regulatory quagmire as to how specificity is achieved at the level of gene expression. Rapid growth in our understanding of signal transduction in general, and NF-κB in particular, provide intriguing insights as to how this may occur.
The prototype NF-κB 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 first insight into the NF-κB pathway (reviewed in Baeuerle and Henkel, 1994). The products of these genes share extensive homology in their N-terminal region called the Rel-homology domain (RHD), whose subregions mediate DNA-binding, dimerization and nuclear localization. To date, five proteins belonging to the NF-κB family have been identified in mammalian cells: RelA, c-Rel, RelB, NFKB1 (p50/p105) and NFKB2 (p52/p100). The NF-κB 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 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-κB 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 NF-κB.
NF-κB exists in the cytoplasm in an inactive form by virtue of its association with a class of inhibitory proteins called IκBs. To date, seven IκBs have been identified: IκBα, IκBβ, IκBε, Bcl3, NFKB1 (p105) and NFKB2 (p100). The IκB family members, which have common ankyrin repeat domains, regulate the DNA-binding and subcellular translocation of NF-κB (reviewed in Baldwin, 1996). The IκBs 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-κB, therein preventing its nuclear translocation. The IκBs display a preference for specific NF-κB complexes, hence each IκB has the potential to participate in distinct regulatory pathways affecting activation of different NF-κB complexes. The regulation of IκBα-mediated NF-κB 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 C-terminal ankyrin repeat domains, retain these NF-κB complexes in the cytoplasm (Rice et al., 1992; Mercurio et al., 1993). The physiological relevance of the precursor's ability to function in an IκB-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 specific alteration in NF-κB 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-κB regulation and absence of either precursor cannot be efficiently compensated by the other IκB family members. Cell stimulation with inducers of NF-κB activity increase the rate of precursor proteolytic processing, resulting in the loss of the ankyrin repeat domain, yielding functionally active NF-κB 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 TNFα-induced proteolytic processing of NFKB1 p105. However, TPL-2 does not appear to directly phosphorylate p105. The relative levels of the IκBs in a given cell type will influence the characteristics of NF-κB activation. The IκBs provide yet another tier of regulatory complexity to modulate NF-κB-mediated gene expression.
NF-κB 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-κB is achieved through the signal-induced proteolytic degradation of IκB which is mediated by the 26S proteosome (Figure 1; Alkalay et al., 1995). The critical event that initiates proteolytic degradation of IκB is the stimulus-dependent phosphorylation of IκB at specific N-terminal serine residues (S32 and S36 for IκBα, S19 and S23 for IκBβ). Mutation of IκBα on these residues to either alanine (S32/36>A) or threonine (S32/36>T) was found to block stimulus-dependent phosphorylation, thereby preventing degradation and subsequent activation of NF-κB (Chen et al., 1995; Brown et al., 1995; DiDonato et al., 1996). We and others recently identified a high molecular weight multiprotein complex containing an inducible IκB 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 IκB kinase 1 (IKK1, IKKα) and 2 (IKK2, IKKβ) were cloned and demonstrated to play a key role in NF-κB activation by a wide variety stimuli. The IKKs are stimulated by inducers of NF-κB with kinetics consistent with the kinetics for appearance of NF-κB 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 specificity.
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 (S177/181>A or S177/181>E) to block or mimic, respectively, the effect of P-Ser (Mercurio et al., 1997). Interestingly, IKK2 (S171/181>E) generated a constitutively active IκB kinase, and was capable of inducing RelA nuclear translocation and NF-κB-dependent gene expression in the absence of cell stimulation. Indeed, baculoviral expressed recombinant versions of IKK2 demonstrated that the specific activity of IKK2-S177/181>E was at least tenfold greater than that of the wildtype IKK2 protein (Mercurio et al., 1999). In contrast, IKK2-S177/181>A yielded an inactive kinase that was refractory to stimulus-dependent activation, and was able to block TNFα-stimulated RelA nuclear localization and NF-κB-dependent gene expression. The corresponding mutations for IKK1 displayed only modest changes in NF-κB-mediated activation, suggesting that IKK2 played a more dominant role in NF-κB activation by pro-inflammatory cytokines. These finding 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-κB. Of note, IKK2−/− mice could be rescued by the inactivation of the TNF receptor 1 consistent with a role for NF-κB in preventing TNFα-induced hepatocyte apoptosis. Mouse embryonic fibroblasts that were isolated from IKK2−/− embryos showed a marked suppression in TNFα- and IL-1-induced NF-κB activity and enhanced apoptosis in response to TNFα, 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 differentiate in vitro. The limbs and tail are highly rudimentary and the head is significantly shorter than normal. Despite this marked morphology, the activation of IKK activity and NF-κB DNA binding activity in IKK1−/− cells by TNFα, IL-1 or bacterial lipopolysaccharide appears normal. Therefore, it appears that IKK1 and IKK2 serve distinct roles in the activation of NF-κB by pro-inflammatoy 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 finding suggests exquisite regulation of NF-κB activation in response to distinct stimuli. The exact stimuli that activates IKK1 have not been defined, 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-κB Inducing Kinase [NIK], was identified by its ability to bind directly to TRAF2, an adapter protein thought to couple both TNFα and IL-1 receptors to NF-κB 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 NIK or MEKK-1 enhances the ability of the IKKs to phosphorylate IκB and activate the NF-κB 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-κB 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-inflamatory 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-κB activation pathway.
In addition to the MAPKKK family, the atypical protein kinase C [aPKC] subfamily of isozymes comprised of γPKC 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 TNFα. It was recently demonstrated that the aPKCs can phosphorylate and activate IKK2 in vitro and in vivo (Lallena et al., 1999). TNFα activates the aPKCs with kinetics that are compatible with the induced phosphorylation and subsequent degradation of IκBα. γPKC activates IKK2, but not IKK1 activity. A dominant negative version of γPKC blocks TNFα, 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 scaffolding protein, p62, which link aPKCs to membrane signaling proteins. Sanz et al. (1999) recently elucidated the nature of this interaction, finding that upon cell stimulation with TNFα, 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-κB.
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-κB activation which requires communication between two higher-order 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 specific manner, enables the propagation of distinct cellular responses. Elucidation of these subtle regulatory processes can be difficult due to misleading results obtained by ectopic overexpression of kinase deficient versions of closely related family members (i.e. titration of common binding regulatory proteins, overcoming evolutionary designed binding coefficients). 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 specific pharmaceutical inhibitors of these kinases should provide valuable tools to further our understanding in this area.
The identification 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 identification of NEMO (NF-κB-essential modulator) through genetic complementation of a flat cellular variant of HTLV-1 Tax-transformed rat fibroblasts, known as 5R, which is unresponsive to all tested NF-κB inducing stimuli. NEMO was able to complement the 5R cell line, and restore NF-κB 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 purified 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 identified and demonstrated to directly interact with IKK2. This protein was named IKK associated-1 (IKKAP-1). Rothwarf et al. (1999) also purified and cloned the identical non-kinase component of the IKK complex which they termed IKKγ. 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 over-expression 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 TNFα receptor complex. Indeed, a protein called FIP-3, which is identical to IKKAP-1/NEMO/IKKγ, has been shown to interact directly with RIP, a component of the TNFα receptor complex. FIP-3 was originally identified 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 effects of TNFα (Li et al., 1999c). Recent reports have shown that the tax oncoprotein of the human T cell leukemia virus type1 constitutively activates the NF-κB 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.
IκB phosphorylation serves as a molecular tag leading to rapid ubiquitination and degradation of IκB 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 IκBα] 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 IκBα phosphorylation generates a binding site for the specific ubiquitin ligase(s) responsible for conjugation of ubiquitin to IκBα (Yaron et al., 1997). Microinjection of these IκBα phosphopeptide into cells abrogated NF-κB activation in response to cytokine stimulation, and consequent NF-κB-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 ubiquitin-conjugating 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 ubiquitin-conjugating 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 specific protein substrate with an E2 ligase charged with ubiquitin. Recently, the pIκBα-E3 ubiquitin ligase was isolated from HeLa cells by virtue of its phosporylation-dependent, high affinity, association with the pIκBα/NF-κB complex, and found to be a member of the latter class of E3 ligases (Yaron et al., 1998). Using nanoelectrospray mass spectrometry, the specific component of the E3 ligase that recognizes the pIκBα degradation motif, DS(PO3)GLDS(PO3)M, was identified as an F-box/WD-domain protein belonging to a recently distinguished family of β-TrCP/Slimb proteins. This component, termed E3RSIkB (pIκBα-E3 Receptor Subunit), binds specifically to pIκBα 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 pIκBα but does not support its ubiquitination, acts in vivo as a dominate negative protein, inhibiting the degradation of pIκBα and consequently NF-κB activation. Its also been suggested that E3RSIkB mediates regulated degradation of beta-catenin (Latres et al., 1999; Fuchs et al., 1999). E3RSIκB represents a member of a family of receptor proteins comprising the core component of a class of ubiquitin-ligases which specifically recognize phosphorylated proteins and target them for proteasomal degradation.
Post-translational modification of the NF-κB complex, occurring subsequent to IκB degradation can enhance transcriptional activation of NF-κB-dependent genes. The transcriptional activity of NF-κB 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-κB RelA through two sites, an N-terminal domain that interacts with the C-terminal 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 C-terminal region of RelA. Phosphorylation by PKA both weakens the interaction between the N- and C-terminal 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 selective modulation of NF-κB-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 effects on NF-κB-dependent transcription in vivo, however, have yet to be reported.
NF-κB as a primary oxidative stress response pathway
NF-κB 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 (O2−), 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, immunodeficiencies 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 inflammatory diseases, and following exposure to γ-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 sacrificial 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-κB. 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 antioxidants, such as glutathione and thioredoxin, ultimately determine the activity profile for many transcription factors. The NF-κB pathway is generally thought to be a primary oxidative stress-response pathway, however, the precise molecular mechanism by which ROIs activate NF-κB have yet to be elucidated.
Several laboratories have demonstrated that treatment of cells with H2O2 can activate the NF-κB pathway (reviewed in Henkel and Bauerle, 1994). The observation that inducers of NF-κB activity, such as TNFα, IL-1, LPS, PMA, UV and ionizing radiation, generate elevated levels of ROIs prompted speculation that ROIs may function as common mediators of NF-κB activation. Further support for this model was provided by indirect evidence showing that nearly all pathways leading to NF-κB 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-κB activation is cell type specific, suggesting H2O2 is not a general mediator of NF-κB 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-κB. Shi et al. (1999) recently demonstrated that the hydroxyl radical was primarily responsible for activation of NF-κB 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-κB activation. In addition, metal chelators that render metal ions incapable of generating the hydroxyl radical from H2O2 were shown to block NF-κB activation. Thus, the ability of the cell to convert H2O2 to OH. rather than another metabolic by-product could influence activation of NF-κB. Interestingly, since OH. is extremely reactive and short-lived, the subcellular localization of its generation could be critical to activation of NF-κB. For example, it was recently shown in macrophages that a redox reaction at the plasma membrane itself was the initial signaling process in NF-κB activation (Kaul et al., 1998).
The process by which ROIs are regulated is highly complex and consequently the mechanism by which NF-κB is activated via ROIs may be equally diverse. For example, Bonizzi et al. (1999) reported that three distinct cell-specific pathways lead to NF-κB activation in response to IL-1. In lymphoid cells NF-κB activation was dependent 5-lipoxigenase-mediated production of ROIs, a pathway requiring ROI production by NADPH was identified in monocytic cells, whereas an ROI- and 5-lypoxygenase-independent pathway was found to be involved in epithelial cells. Interestingly, introduction of a functional 5-lypoxygenase system in the epithelial cells allowed ROI production and 5-lypoxigenase-dependent NF-κB activation. Hence, the repertoire of oxidative enzymes expressed in a given cell type will influence the nature of NF-κB activation. Likewise, adverse conditions that alter the expression profile of the oxidative enzymes could result in aberent NF-κB activation and associated pathologies.
The step(s) in the NF-κB signaling pathway that are responsive to redox regulation are not well documented. Addition of H2O2 to specific cell types results in the appearance of the hyperphosphorylated form of IκBα, 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 IκBα phosphorylation and degradation induced by LPS, TNFα and PMA. Note however, that inhibition of stimulus-dependent IκBα degradation by antioxidants, such as NAC or PDTC, also occurs in cells that are refractory to H2O2-induced NF-κB activation. It seems reasonable to suggest that redox regulation may affect more than one step in the NF-κB pathway. Indeed, reducing agents such as DTT and β-mercaptoethanol enhance DNA-binding activity of NF-κB itself.
In an effort to further explore the mechanism by which redox balance affects NF-κB activation, we treated HeLa cell with PDTC, an antioxidant demonstrated to inhibit NF-κB activation in response to a variety of stimuli, prior to stimulation with TNFα and examined IKK activation and IκBα degradation (Figure 3). The result from this study was quite unexpected and suggests that there may be additional regulatory steps in the NF-κB pathway which have yet to be elucidated. For example, addition of PDTC to HeLa cells, in the absence of TNFα, resulted in dramatic activation of IKK activity. Surprisingly, IKK activation did not lead to IκBα phosphorylation or subsequent degradation. This result would indicate that while IKK activation is required it may not be sufficient for complete propagation of the signal, perhaps the IκBα substrate must be actively recruited to the IKK complex. Indeed, IκBα 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 TNFα induction allowed phosphorylation of IκB, however, the phosphorylated form of IκB 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 TNFα, in fact, PDTC potentiates IKK activity at higher concentrations. One possible explanation is that PDTC inhibits ubiquitination and/or degradation of phosphorylated IκBα. These data indicate that redox regulation of the NF-κB signaling pathway may function at multiple points.
NF-κB and cancer
Malignant tumor cells have usually accumulated mutations that affect a variety of processes, including those that sustain cell growth, that block growth inhibition and apoptosis, that affect DNA repair, and that allow the tumor to escape immune surveillance. Several lines of evidence suggest that NF-κB 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-κB complexes, and inhibition of NF-κB activation by overexpression of a non-degradable IκBα molecule inhibits proliferation and tumorigenicity of these cells (Sovak et al., 1997). Many breast cancer cells also contain high levels of nuclear NF-κB 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-κB. High levels of activated NF-κB are also associated with progression of breast cancer cells to estrogen-independent growth (Bours et al., 1994). High levels of NF-κB activity have been reported in diverse solid tumor-derived cell lines (Visconti et al., 1997). Human thyroid carcinoma cells require nuclear NF-κB 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-κB (Finco et al., 1997). Oncogenic forms of ras, the most common defect in human tumors, can activate NF-κB, and NF-κB inhibition can block ras-mediated cellular transformation (Fisher, 1994; Mayo et al., 1997). Insights into the molecular mechanims by which NF-kB promotes these effects 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 NF-κB by tumor necrosis factor α [TNFα], 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-κB activation in this setting enhanced apoptotic killing by these reagents but not by apoptotic stimuli that do not activate NF-κB. NF-κB can induce the expression of anti-apoptotic genes such as IAP 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-κB 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-κB represents a unique mechanism by which these cells express genes which protect against apoptotic stimuli. NF-κB also regulates the expression cell adhesion molecules that facilitate cell adhesion and migration (reviewed in Chen and Manning, 1995). Inhibition of NF-κB 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-κB represents a novel approach to cancer drug development, and when used as combination therapy with established therapeutics, these agents could provide a more effective treatment of drug-resistant forms of cancer.
The NF-κB 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-κB by pro-inflammatory cytokines have been identified. 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 cell- and stimulus-specific activation of NF-κB complexes is achieved.
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Mercurio, F., Manning, A. NF-κB as a primary regulator of the stress response. Oncogene 18, 6163–6171 (1999). https://doi.org/10.1038/sj.onc.1203174
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