NFκB-dependent signaling pathways

Experimental Hematology 30 (2002) 285–296

NFB-dependent signaling pathways Xiaoxia Lia and George R. Starkb Departments of aImmunology and bMolecular Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio, USA

The transcription factor NFB is activated by numerous stimuli. Once NFB is fully activated, it participates in the regulation of various target genes in different cells to exert its biological functions. NFB has often been referred to as a central mediator of the immune response, since a large variety of bacteria and viruses can lead to the activation of NFB, which in turn controls the expression of many inflammatory cytokines, chemokines, immune receptors, and cell surface adhesion molecules. Recent studies have shown that NFB may function more generally as a central regulator of stress responses, since different stressful conditions, including physical stress, oxidative stress, and exposure to certain chemicals, also lead to NFB activation. Furthermore, NFB blocks cell apoptosis in several cell types. Taken together, these findings make it clear that NFB plays an important role in cell proliferation and differentiation. It is the intention of this review to cover the various NFB-dependent signaling pathways, thereby to achieve a better understanding of the mechanisms of NFB activation and the physiological functions of activated NFB. © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.

Fifteen years following the first publication describing NFB [1], its study has attracted many researchers from different fields, including molecular biology, cell biology, biochemistry, genetics, immunology, and developmental biology. From this collective effort we have gained extensive knowledge regarding the molecular basis of the physiological functions of NFB. The family of NFB transcription factors includes a collection of proteins, conserved from Drosophila to humans and related through a highly conserved DNA-binding and dimerization region, the Rel homology (RH) domain [2]. However, the NFB family can be divided into two groups, based on differences in their structures, functions, and modes of synthesis [3,4]. Members of one group (p105, p100, and Drosophila Relish) have long C-terminal domains that contain multiple copies of ankyrin repeats, which act to inhibit these molecules. Members of this group give rise to active, shorter proteins that contain the Rel homology domain (p50 from p105, p52 from p100) either by limited proteolysis [5–9] or arrested translation [10,11]. Members of this group do not function as transcription activators, except when they form dimers with members of the second group, which includes p65 (RelA), Rel (c-Rel), RelB, and the Drosophila Rel proteins dorsal and Dif [12]. These pro-

Offprint requests to: Xiaoxia Li, Ph.D., Department of Immunology/ NB30, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195; E-mail: [email protected]

teins are not synthesized as precursors, and in addition to the N-terminal Rel homology domain, they possess one or more C-terminal transcriptional activation domains. Members of both groups of NFB proteins can form homodimers or heterodimers. NFB was the original name for the p50p65 heterodimer. In unstimulated cells, NFB-family proteins exist as heterodimers or homodimers that are sequestered in the cytoplasm by virtue of their association with a member of the IB family of inhibitory proteins [5,12]. From biochemical studies and, more recently, direct structural determinations, it is clear that IB makes multiple contacts with NFB. These interactions mask the nuclear localization sequence of NFB and interfere with sequences important for DNA binding [13–15]. More than 150 extracellular signals can lead to activation through the dissociation of NFB from the IB proteins. These signal transduction pathways lead to the activation of the IB kinase (IKK), which in turn phosphorylates two specific serine residuals on IB proteins (S32 and S36) [5,16]. Phospho-IB is then recognized by the -TrCP–containing SCF ubiquitin ligase complex, leading to its ubiquitination and degradation by the proteasome. The destruction of IB unmasks the nuclear localization signal of NFB, leading to its nuclear translocation and binding to the promoters of target genes [5,17]. Recently, more and more evidence suggests that the phosphorylation and degradation of IB and the consequent liberation of NFB are not sufficient to activate NFB-dependent transcription. A second level of regulation of NFB activity re-

0301-472X/02 $–see front matter. Copyright © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(02)0 0 7 7 7 - 4

286

X. Li and G.R. Stark/Experimental Hematology 30 (2002) 285–296

lies on phosphorylation of members of the second group of NFB proteins (p65/RelA, RelB, and c-Rel), resulting in the activation of transcriptional activity of NFB [18–25]. A large variety of bacteria and viruses can lead to the activation of NFB; which in turn controls the expression of many inflammatory cytokines, chemokines, immune receptors, and cell surface adhesion molecules (Fig. 1) [26]. Therefore, historically, NFB has been considered as a central mediator of the innate immune responses. However, recent studies have shown that NFB may function more generally as a central regulator of stress responses, since different stressful conditions, including physical stress, oxidative stress, and exposure to certain chemicals, also lead to NFB activation (Fig. 1) [26]. In addition, mitogens, growth factors, and hormones activate NFB. Furthermore, NFB blocks cell apoptosis in several cell types. This review will discuss the mechanisms of NFB activation and then outline the functions of activated NFB in the immune system, stress responses, and cell survival/development. Mechanisms of NFB activation The role of IB kinases Many of the extracellular signals that lead to the activation of NFB converge on a high-molecular-weight oligomeric protein, the serine-specific IB kinase (IKK) [27–32]. IKK is composed of at least three subunits: the catalytic subunits IKK and IKK, and the regulatory subunit, IKK. IKK and IKK have very similar primary structures (52% identity), with protein kinase domains near their N-termini and leucine zipper (LZ) and helix-loop-helix (HLH) motifs near their C-termini. IKK lacks a kinase domain but contains long stretches of coiled-coil sequence, which function in protein-protein binding, including a C-terminal LZ motif. The IKK and IKK subunits preferentially form heterodimers, and both can directly phosphorylate the critical

Figure 1. NFB-dependent signaling pathways. NFB is activated by many extracellular stimuli, including many different pathogens, cytokines, stress signals, and growth factors. Once NFB is fully activated, it helps to regulate various genes in different target cells that have important roles in immune responses, stress responses, cell survival, and development.

S32 and S36 residues of IB. IKK serves a structural and regulatory role and is thought to mediate interactions with upstream activators of IKK in response to cellular activation signals. However, the identity of the immediately upstream IKK activators remains to be identified. Activation of the IKK complex involves the phosphorylation of two serine residues located in the activation loop within the kinase domain of IKK (S176 and S180) or IKK (S177 and S181). Certain MAP3 kinases (MEKK1, MEKK2, MEKK3, and NIK) are capable of phosphorylating these serine residues in vitro and of activating NFB when overexpressed [5]. Furthermore, dominant negative mutants of MEKK1 or NIK can inhibit NFB activation induced by certain stimuli. However, none of these kinases has been definitively proven to be an IKK kinase in vivo. MEKK1null mouse embryonic fibroblasts (MEFs) display normal NFB activation in response to stimulation [33]. Interestingly, although NIKnull cells also display normal NFB DNA-binding activity in response to many stimuli [34], they exhibit weak activation of NFB-dependent genes specifically in response to lymphotoxin- receptor (LTR) signaling. Recently, it has been shown that NIK and IKK kinase are required specifically for signal-dependent p100 processing, which helps to explain the phenotype of NIKnull cells [35,36]. Curiously, MEKK3, which can phosphorylate IKK in vitro, is required for tumor necrosis factor (TNF)–induced NFB activation. MEKK3null MEFs exhibit a greatly decreased level of IKK activation, IB degradation, and NFB activation in response to TNF- [37]. Although it is still not clear how IKK is activated, the IKK// complex is surely required for NFB activation in response to numerous stimuli. IKKnull MEFs have a greatly reduced ability to degrade IB or activate NFB in response to various stimuli, including TNF- and LPS [38– 40]. However, IKKnull cells still have some residual IKK activity, suggesting that IKK also plays some role in NFB activation. In supporting this, IKK/ double knockout cells have no NFB response [41]. However, IKKnull cells retain nearly normal IB degradation and NFB DNA binding activity in response to stimulation [42–44]. Instead, IKKnull MEFs are deficient in inducing several NFBdependent mRNAs in response to interleukin (IL)-1 and TNF [43]. These findings raise the possibility that IKK may play a more important role in transcriptional activation driven by NFB than in IB degradation, which will be further discussed below. IKKnull cells fail to degrade IB or activate NFB in response to TNF and LPS, indicating that IKK is required for these processes [32,45–48]. In addition to IKK//, two related kinases, IKK/IKKi and TBK/NAK/T2K, were identified recently and found to be involved in NFB activation [49–53]. Both IKK/IKKi and TBK1/NAK/T2K are activated by a specific subset of physiological inducers of NFB. Both have been implicated in PMA-stimulated activation of NFB. In addition, IKK/ IKKi is involved in the activation of T cells through CD3

X. Li and G.R. Stark/Experimental Hematology 30 (2002) 285–296

and CD28 costimulation, and TBK1/NAK/T2K may be involved in activating NFB in response to PDGF. While both IKK/IKKi and TBK1/NAK/T2K are capable of phosphorylating S36 of IB, it has also been proposed that they may actually serve as IKK kinases, activating IKK, and/or an unknown S32/S36 kinase. However, neither IKK/IKKi nor TBK1/NAK/T2K appear to associate tightly with the IKK// complex [49]. Recently, deletion of the TBK1/NAK/T2K gene in mice suggests a function in TNF signaling. TBK1/NAK/T2Knull MEFs exhibit decreased activation of some, but not all, NFB-responsive genes in response to TNF- or IL-1. However, TBK1/NAK/T2Knull MEFs display normal induction of IKK activity, IB degradation, and NFB DNA binding activity [54]. These observations suggest that TBK1/NAK/T2K functions after IB degradation. For example, TBK1/NAK/T2K could function to directly or indirectly activate the transcriptional activity of the p65 subunit of NFB. Other possible explanations are that TBK1/NAK/ T2K is required for a separate TNF-inducible pathway, or that TBK1/NAK/T2K is required for the activation of a certain subset of NFB dimers. More studies are required to further elucidate the function of these IKK-related kinases. Regulation of NFB transcriptional activity In addition to the signal-induced liberation of NFB from IB and the consequent nuclear localization of NFB, the transcriptional activity of NFB is also regulated in response to stimulation [18–25]. The NFB p65 protein has a site for phosphorylation by protein kinase A on serine 276, and phosphorylation of this residue is required for efficient binding to the transcriptional activator protein CBP. The catalytic subunit of protein kinase A was shown to be bound to inactive NFB complexes and, upon IB degradation, to phosphorylate p65, resulting in a conformational change of p65 and consequent interaction with CBP [19,20]. Moreover, TNF- treatment of cells results in phosphorylation of Ser529 in the transactivation domain of p65, resulting in activation of its transcriptional activity. Recently, casein kinase II was implicated in the TNF-–dependent phosphorylation of Ser529 [21,22]. IB protects p65 from phosphorylation by constitutively active CKII, but signal-dependent degradation of IB exposes the p65 phosphorylation site to CKII activity. Thus, once released from IB, at least two kinases, PKA and CKII, phosphorylate p65 at different serine residues, to increase its transcriptional activity. On the other hand, PI3K and Akt have been shown to be required for IL-1– and TNF-–induced NFB activity. IL-1 and TNF induce the activation of PI3K and Akt, which lead to the phosphorylation and activation of p65 [23]. Akt has also been implicated in Ras-induced NFB activation, through the activation of IKK and phosphorylation of p65 at serines 529 and 536 [24,25]. The same serine residues were shown to be required for the activation of the p65 fusion proteins by activated Akt or treatment with IL-1. Re-

287

cently, IL-1–stimulated PI3K/Akt activation has also been implicated in the phosphorylation of the p50 NFB subunit, which increases the DNA-binding capacity of the NFB complex [55]. As mentioned above, in TBK1/NAK/T2Knull and IKKnull MEFs, IB phosphorylation and degradation occur in response to stimulation, but NFB-regulated genes are not activated, suggesting that TBK1/NAK/T2K and IKK function downstream of IB degradation and may play important roles in the phosphorylation and activation of NFB [43,54]. Recently, Sizemore et al. [56] found that IKK complexes from IKKnull and IKKnull MEFs are both deficient in PI3K-mediated phosphorylation of the transactivation domain of the p65 subunit of NFB in response to IL-1 and TNF. These results indicate that both IKK and IKK are activated by the IL-1– and TNF-induced PI3K/Akt, leading to the phosphorylation and activation of the p65 NFB subunit [56].

Role of NFB in innate immunity Innate immunity Innate immunity is the first line of defense against infectious microorganisms. The Toll-like receptors (TLRs) play important roles in innate immunity, functioning as the pattern recognition receptors (PRRs) [57–61]. These receptors recognize conserved molecular patterns (pathogen-associated molecular patterns [PAMPs]) expressed by large groups of microorganisms. Infection leads to activation of the TLRs, which in turn initiate intracellular signaling cascades that culminate in the activation of NFB/Rel family transcription factors. Activated NFB controls the expression of many genes, including those encoding inflammatory cytokines, chemokines, immune receptors, and cell surface adhesion molecules. These inflammatory mediators initiate a second wave of signaling cascades that also activates NFB, leading to the upregulation of a variety of antimicrobial effector molecules that attack microorganisms at many different levels. In this section, we discuss recent advances in understanding NFB-dependent signaling pathways involved in innate immunity. IL-1 and TNF receptor–mediated NFB activation Recent studies have begun to reveal the signaling events from the receptor to the activation of IKK for different innate immune NFB-dependent pathways. Among them, TNF-– and IL-1–mediated NFB activation are characterized best (Fig. 2) TNF- signals by trimerizing the type 1 TNF- receptor (TNFR1), which contains an intracellular death domain [62]. The TNF-–TNFR1 complex then recruits the death domain–containing molecule TRADD [63], which then interacts with the serine-threonine kinase RIP and TRAF2 to activate downstream components that eventually lead to NFB activation [64,65]. RIP is the prototypi-

288

X. Li and G.R. Stark/Experimental Hematology 30 (2002) 285–296

cal member of an emerging family of related molecules (including RIP, RIP2, and RIP3) [66,67]. The function of the other RIP members is not well characterized and will be further discussed below. The IL-1 receptor uses parallel but distinct proximal components to activate NFB. IL-1 induces the formation of a complex involving the type 1 receptor (IL-1R) and the receptor accessory protein (IL1RAcP) [68–71]. The death domain–containing protein MyD88 is then recruited to the activated receptor complex [72,73]. Similarly to TRADD, MyD88 functions as an adaptor to recruit the serine-threonine IL-1 receptor-associated kinase IRAK to the receptor through its death domain [73,74]. Another adaptor, Tollip, has also been implicated in the recruitment of IRAK to the IL-1 receptor [75]. The detailed mechanism by which Tollip and MyD88 recruit IRAK to the receptor is still not clear. IRAK then leaves the receptor complex and interacts with TRAF6, which is required for the activation of NFB [76]. Just like the RIP family, more IRAK-like molecules (including IRAK, IRAK2, IRAKM) have been identified [77,78]. Similarly, a family of TRAF proteins (a total of six) has been described [79]. The different TRAFs are used by various NFB-dependent pathways, as will be discussed further below. The three proteins TAK1/TAB1/TAB2 have been implicated in linking TRAF6 to IKK in IL-1–mediated signaling [80–85]. TAK1 is a previously identified MAP3K; TAB1 and TAB2 are TAK1 binding proteins. Both TRAF6 and TAB2 have been shown to be membrane bound before stimulation. The IRAK that is phosphorylated in response to IL1 is also membrane bound, where it forms a complex with TRAF6 and TAB2. Consequently, IRAK mediates the translocation of TRAF6 and TAB2 from the membrane to the cytosol, leading to the activation of TAK1. Chen and colleagues, using an in vitro system to study TRAF6-mediated IKK activation [86,87], showed that TRAF6-

Figure 2. TNF and IL-1 signaling pathways. While both TNF and IL-1 induce the activation of NFB and JNK, TNF also induces apoptosis. NFB activation protects cells from TNF-induced apoptosis by upregulating antiapoptotic genes, including TRAF1, TRAF2, IAP1, IAP2, and FLIP.

mediated IKK activation requires nonclassical ubiquitination catalyzed by the ubiquitination proteins Ubc13 and Uev1A, which plays a regulatory role and does not lead to proteasome-mediated degradation. TRAF6 functions as part of a unique E3 complex, with Ubc13 and Uev1A, and TRAF6 itself is the target of ubiquitination. Furthermore, it was shown that the TAK1 complex (TAK1/TAB1/TAB2) is activated by association with ubiquitinated TRAF6. Once activated, TAK1 can directly phosphorylate IKK and MKK6, leading to the activation of both the JNK and NFB signaling pathways. TAK1 is also activated upon stimulation by TNF or LPS. A kinase-negative mutant of TAK1 inhibited IL-1–, TNF-, and LPS-mediated NFB activation [88,89]. Therefore, TAK1 is probably a common component shared by several different signaling pathways. Since TAB2 seems to be specific for the IL-1 signaling pathway, TAB2-like molecules might exist for the other pathways to mediate signal-induced TAK1 activation. Other possible components of the IL-1/TNF signaling pathways include the atypical PKCs,  and , and their associated protein p62 [90]. IL-1 induces an interaction between endogenous TRAF6 and p62, whereas TNF- has been shown to induce the association of atypical PKCs and p62 with RIP. Inhibition of these PKCs or downregulation of p62 severely abrogates NFB activation by IL-1. Activation of NFB mediated by the IL-1/Toll receptor superfamily The IL-1/Toll receptors play essential roles in inflammation and innate immunity. The defining feature of a member of the superfamily is a Toll-like domain on the cytoplasmic side of the receptor (Fig. 1) [58,91]. The first members of this superfamily are in the IL-1R family. These receptors contain three Ig domains in their extracellular regions. The second group of IL-1/Toll receptors is the recently identi-

Figure 3. Ligand specificities of Toll-like receptors.

X. Li and G.R. Stark/Experimental Hematology 30 (2002) 285–296

fied pattern recognition receptors, the Toll-like receptors (TLRs, 9 members so far, Fig. 3), which consist of two major domains characterized by extracellular leucine rich repeats (LRRs) and an intracellular Toll-like domain [57–61]. TLR4 has been genetically identified as a signaling molecule essential for the responses to LPS [92]. Mice with targeted disruption of the TLR4 gene are LPS unresponsive. Unlike TLR4, TLR2 responds to mycobacteria, yeast, and gram-positive bacteria [93–96]. TLR9 has been shown to recognize bacterial DNA [61], while TLR5 mediates the induction of the immune response by bacterial flagellins [97]. Recent studies show that TLR3 recognizes dsRNA, and that activation of this receptor induces the activation of NFB and the production of type 1 interferons [98]. The last group includes only one receptor so far, single Ig IL-1R-related molecule (SIGIRR), which has only a single extracellular Ig domain [99]. Since the receptors in this superfamily are similar in their cytoplasmic domains, they share some signaling components that function with the IL-1 receptor, including MyD88, TRAF6, and IRAK, leading to the activation of NFB. Previous studies with MyD88null mice showed that this molecule is indispensable for the responses to IL-1, the IL-1–related cytokine IL-18, LPS, and MALP-2 (macrophage-activating lipopeptide-2 kD), demonstrating that MyD88 functions as a general adaptor for the IL-1/TLR receptor superfamily [94–101]. However, the difference between TLR2- and TLR4-mediated signaling has been shown [101]. In MyD88null macrophages, the production of IL-1b, TNF-, and IL-6 in response to LPS and MALP-2 was completely defective. However, LPS stimulation of MyD88null macrophages activates NFB and JNK, although this activation is delayed when compared with that of wild-type cells [101]. In contrast, NFB activation in response to MALP-2 is completely abolished in MyD88null macrophages [94]. These results suggest that MyD88-independent pathways lead to NFB and JNK activation in TLR4-dependent signaling. Recently, a novel MyD88adaptor-like protein (Mal/TIRAP) has indeed been identified. It controls the activation of MyD88-independent signaling pathways downstream of TLR4 [102,103]. Activation of NFB by Mal/TIRAP requires IRAK2, but not IRAK, whereas MyD88 requires both IRAKs. A dominant negative form of Mal/TIRAP inhibits NFB, activated by TLR-4 or lipopolysaccharide, but not NFB activated by IL-1R1 or IL-18R. NFB activation by the mediated TNF receptor superfamily The TNF receptor superfamily is another growing group of NFB-activating receptors (Table 1) [104]. These receptors have a characteristic cysteine-rich pseudorepeat in their extracellular regions. The corresponding ligands isolated so far also share significant sequence homology and belong to the TNF family. The cytoplasmic domains of TNF receptor superfamily members are relatively short and contain no known catalytic motifs. They also lack significant sequence

289

homology among themselves, except for the death domains in TNFR1, DR4, and DR5. Therefore, unlike the IL-1/Toll receptor superfamily, the TNF receptor superfamily uses quite distinct downstream signaling components, such as different TRAF proteins. The formation of homodimers and/or heterodimers among the six members of the TRAF family generates useful complexity. Other NFB-dependent pathways The receptor for IL-17 (IL-17) is a type I transmembrane protein that does not share sequence homology with the receptors for TNF, IL-1, or other cytokines. IL-17R mediates the activation of NFB by utilizing TRAF6 [105]. The Nod genes encode cytosolic proteins that contain an LRR domain similar to that found in the extracellular domains of TLRs, an ATPase domain related to APAF-1/CED4, and one or more CARD domains. At least 20 Nod-like genes have been identified in the human genome sequence [18]. Through the CARD domain, Nod1 and Nod2 can interact with RIP2/RICK and activate NFB. Recently, LPS has also been shown to induce TLR4-independent NFB activation in 293 cells ex-

Table 1. Members of the TNF receptor superfamily and their ligands

Receptor

Standard Name TNFRSF16 TNFRSF19

Other Names

NGFR Troy EDAR XEDAR CD40 DcR3 FAS OX40 AITR CD30 HveA 4-1BB TNFR2

p75 Taj

TNFRSF5 TNFRSF6B TNFRSF6 TNFRSF4 TNFRSF18 TNFRSF8 TNFRSF14 TNFRSF9 TNFRSF1B

DR3

TNFRSF12

CD27 TNFR1

TNFRSF7 TNFRSF1A

LTR

TNFRSF3

RANK TACI

TNFRSF11A

TRANCE-R CAML interactor

BCMA

TNFRSF17

BCM

DR6 OPG DR4 DR5

TNFRSF11B TNFRSF10A TNFRSF10B

DcR1 DcR2

TNFRSF10C TNFRSF10D

EDA-A2R p50, Bp50 CD95, APO-1, APT1 CD134, ACT35, TXGP1L G1TR Ki-1, D1S166E HVEM, ATAR, TR2, LIGHTR CD137, ILA CD120b,p75, TNFBR, TNFR80, TNF-R-11 TRAMP, WSL-1, LARD, WSLLR, DDR3, TR3, APO-3 Tp55, S152 CD120a p55-R, TNFAR, TNFR60, TNF-R-1 TNFR2-RP, TNFCR, TNF-R-111

TR7 OCIF, TR1, osteoprotegerin Apo2, TRAILR-1 KILLER, TRICK2A, TRAIL-R2, TRICKB TRAILR3, LIT, TRID TRUNDD, TRAILR4

Ligand NGF EDA1 EDA2 CD40L FASL OX40L AITRL CD30L 4-1BBL TNF DR3L CD27L TNF LT, LT, Light RANKL APRIL, BLYS APRIL, BLYS

TRAIL TRAIL

290

X. Li and G.R. Stark/Experimental Hematology 30 (2002) 285–296

pressing trace amounts of Nod1 and Nod2. It has been suggested that Nod1 and Nod2 might function as intracellular receptors for LPS [106,107]. Thus, the Nod genes may encode a family of PRRs that function within the cell to recognize substances released from intracellular pathogens. Interestingly, mutations in the NOD 2 gene are linked to susceptibility to Crohn’s inflammatory bowel disease [108–110].

Role of NFB in stress responses Many activators of NFB are not bacterial or viral pathogens. NFB activity, for instance, is induced in response to various physiological stresses such as ischemia/reperfusion, liver regeneration, and hemorrhagic shock. Physical stress in the form of irradiation as well as oxidative stress to cells also induce NFB. In addition, NFB is activated by both environmental stresses, such as heavy metals, and various chemotherapeutic agents. Therefore, NFB is activated by and induces responses to many forms of cellular stress and can thus more generally be thought of as a central mediator of stress responses [26]. NFB activation by oxidative stress In respiring cells, a small amount of the consumed oxygen is reduced to highly reactive chemical entities collectively called reactive oxygen species (ROS) or reactive oxidative intermediates (ROIs). A state of moderately increased levels of intracellular ROS is referred to as oxidative stress [111]. Cells respond to these adverse conditions by modulating their antioxidant levels, inducing new gene expression, and modifying proteins. Direct evidence that the level of ROS may regulate NFB was provided by frank exposure of cells to H2O2 [112,113]. The available data indicate that H2O2-induced NFB activation is highly cell-type dependent and therefore that H2O2 is unlikely to be a general mediator of NFB activation [114–117]. Adding H2O2 to HeLa cells induces the appearance of a slowly migrating form of IB in SDS-polyacrylamide gels, which is rapidly degraded unless cells are treated with a proteasome inhibitor [118]. Addition of antioxidants (PDTC or NAC) or overexpression of peroxidases blocked the IB phosphorylation and degradation induced by TNF, PMA, and LPS [115,119]. Therefore, IB phosphorylation and degradation might be a step that is responsive to oxidative stress [113]. However, the IKK activity induced by TNF was not affected by these antioxidants. One possibility is that antioxidants affect the recognition of IB by the IKK complex to slow down the phosphorylation of IB upon TNF stimulation. Alternatively, antioxidants may directly inhibit the ubiquitination or degradation of IB. Stress-induced NFB activation through IKK-independent pathways Short-wavelength ultraviolet (UV-C) light activates NFB in certain cell types concomitantly with IB degradation.

Pretreatment of cells with proteasome inhibitors blocked IB degradation and NFB activation induced by UV, indicating that IB degradation is required. However, neither IKK activation nor the phosphorylation of IB on Ser32 and Ser36 was observed after UV-C [120,121]. Furthermore, even the IB mutant that contains alanines at position 32 and 36 was still susceptible to UV-C–induced degradation. Similar to UV-C, treatment of cells with amino acid analogs also activates NFB through proteasome-mediated IB degradation without apparent phosphorylation at Ser32 or Ser36 [118]. It is likely that exposure to amino acid analogs activates a stress response similar to the one triggered by UV-C. Another pathway leading to NFB activation was activated when cells were treated with tyrosine phosphatase inhibitors (e.g., pervanadate) or upon reoxygenation of hypoxic cells [122,123]. In these cases, NFB was activated through the tyrosine phosphorylation of IB without degradation. The phosphorylation site was identified as Tyr-42, a site that is present only in IB and not IKK. The tyrosine phosphorylation of IB led to its dissociation from NFB [122].

Role of NFB in cell survival and development NFB as a survival factor NFB provides a survival signal in B lymphocytes [124– 126]. In WEHI 231 immature B-lymphoma cells, engagement of surface IgM by anti-IgM antibodies leads to apoptosis, and overexpression of c-Rel protects these cells from IgM-mediated apoptosis. C-myc is thought to be an essential target gene for the NFB-mediated block to apoptosis in these cells [125,127]. As with anti-IgM antibodies, treatment of WEHI 231 cells with TGF-1 also induces apoptosis and causes a decrease in c-myc expression [128], resulting from a TGF-1–mediated increase in IB expression, which decreases NFB DNA-binding activity. Engagement of the CD40 receptor can protect these cells from TGF-1–induced apoptosis by maintaining high levels of NFB activity and c-myc expression. B lymphocytes lacking the adaptor protein B-cell linker (BLNK) do not proliferate in response to B-cell antigen receptor (BCR) engagement [129]. Examination of the various BCR-activated signaling pathways in mouse BLNKnull B cells reveals intact activation of Akt and mitogen-activated protein kinases but impaired activation of NFB. Recently, phospholipase C (PLC)-2 and Bruton’s tyrosine kinase (Btk) have also been demonstrated to be essential for NFB activation upon BCR engagement [130,131]. Since BLNK interacts with Btk and PLC-2, BLNK might be involved in mediating the formation of a Btk-PLC-2 signaling axis that regulates NFB activation. The NFB activation defect may be sufficient to explain similar defects in BCR-induced B-cell proliferation in BLNKnull [132–134], Btknull [135,136], and PLC-2null mice [137].

X. Li and G.R. Stark/Experimental Hematology 30 (2002) 285–296

bcl-10, a CARD-containing protein identified from the t(1;14)(p22;q32) breakpoint in MALT lymphomas, has also been implicated in B- and T-cell proliferation through antigen receptor–mediated NFB activation [138–140]. bcl10null–deficient mice are severely immunodeficient and bcl10null lymphocytes are defective in antigen receptor or PMA-induced proliferation. Tyrosine phosphorylation, MAPK and AP-1 activation, and Ca2 -dependent signaling are normal in mutant lymphocytes, but antigen receptor–induced NFB activation is absent. Thus, bcl-10 functions as a positive regulator of lymphocyte proliferation that specifically connects antigen receptor signaling in B and T cells to NFB activation. Activation of NFB and pre–T cell receptor (pre-TCR) expression are tightly correlated during thymocyte development [141,142]. Inhibition of NFB in isolated thymocytes results in spontaneous apoptosis of cells expressing preTCR, whereas inhibition of NFB in transgenic mice through the expression of a mutated, suppressor form of IB leads to a loss of -selected thymocytes. In contrast, the forced activation of NFB through the expression of a dominant-active IB kinase allows differentiation to proceed to the CD4 , CD8 stage in a Rag1null mouse that cannot assemble pre-TCR. Therefore, signals emanating from pre-TCR are mediated at least in part by NFB, which provides a selective survival signal for developing thymocytes with productive -chain rearrangements. A protective role for NFB in signal-induced cell death Some of the inflammatory stimuli that activate NFB also lead to apoptosis, although NFB itself induces responses that suppress apoptosis. One example is the TNFR-1–mediated signaling pathway. While the N-terminal half of TRADD interacts with TRAF2 to activate NFB, TRADD also interacts with FADD through its C-terminal death domain [143–147]. The death effector domain at the N-terminal end of FADD/MORT1 then interacts with a related motif in the prodomain of caspase 8 (also called FLICE, MACH, or Mch5), thereby leading to the activation of apoptotic protease cascades (Fig. 2) [143]. Several members of the death domain–containing TNF receptor superfamily (including TNFR-1, DR4/Trail-R1/Apo-2, and DR5/Trail-R2) can induce apoptosis [148,149]. Although death domains are absent in other members of the TNFR family, some of these receptors, such as TNFR2, CD30, and LTR, are nevertheless capable of inducing cell death under certain circumstances [150,151]. Recently, it has been shown that the activation of TLR2 by bacterial lipoprotein (BLP) can also lead to apoptosis, presumably through the adaptor molecule MyD88 [100,152]. Activation of the putative intracellular LPS receptors, Nod1 and Nod2, has also been shown to lead to apoptosis. It is quite intriguing that many signals that can initiate apoptosis also activate NFB, which suppresses apoptosis. This seemingly contradictory phenomenon has been eluci-

291

dated for the TNF pathway. NFB induces a group of gene products, including TRAF1, TRAF2, and c-IAP1 and cIAP2, that function cooperatively at the earliest checkpoint to suppress TNF-–mediated apoptosis [153]. Recently, it has been shown that NFB activation upregulates the caspase 8 inhibitor FLIP, resulting in increased resistance to Fas ligand (FasL) or TNF-mediated apoptosis [154]. NFB plays an important role in preventing apoptosis in embryonic liver and during liver regeneration. Both RelAnull and IKKnull mice are embryonic lethal due to liver degeneration and hepatocyte apoptosis [38,40]. The apoptosis of embryonic liver cells in RelAnull and IKKnull mice appears to be due to their increased sensitivity to circulating TNF-, since there is no apoptosis of embryonic livers in mice doubly deficient for either RelA and TNF- or IKK and TNFR1 [38,155]. Although it has been clearly shown that inflammatory cytokines and bacterial pathogens induce apoptosis, the implications of this phenomenon remain elusive. The apoptosis induced by inflammatory stimuli may be important for (1) the initiation of inflammation, (2) the resolution of inflammation, or (3) generation of signals necessary for other immune responses [152]. Constitutive activation of NFB in human cancers NFB proteins also participate in cellular growth control and neoplasia. The viral oncoprotein v-Rel was the first member of this family to be identified and is still the only one that is acutely transforming in vitro and in vivo [156]. Several oncogenic viruses, such as human T-cell leukemia virus type 1 and Epstein-Barr virus, activate NFB as part of the transformation process. Moreover, there is growing evidence implicating roles for all vertebrate NFB factors in human cancer [157]. Chromosomal aberrations involving the human c-rel, relA, NFB1 (encoding p105/p50), and NFB2 (encoding p100/p52) genes are found in many hematopoietic and solid tumors. Constitutively high levels of nuclear NFB activity has also been described in many cancer cell types, as a result of the constitutive activation of upstream signaling kinases or mutations inactivating IBs. Constitutive IKK activity was observed in Hodgkin’s disease and childhood acute lymphoblastic leukemia. In addition to its antiapoptotic role, as discussed above, NFB also induces cell proliferation and cell-cycle progression by regulating the expression of target genes including c-myc and cyclinD1 [157,158]. In certain systems the upregulation of NFB is associated with advanced stages of oncogenesis, supporting a role in tumor progression. In this respect, NFB activates the expression of genes important for invasion and metastasis, including those encoding angiogenic factors such as VEGF, proteolytic enzymes such as matrix metalloproteinases, urokinase plasminogen activator (uPA), and cell adhesion molecules such as ICAM-1. uPA, significantly increased in most breast cancer cell lines that contain constitutively active NFB, is required for intrava-

292

X. Li and G.R. Stark/Experimental Hematology 30 (2002) 285–296

sation and is associated with poor prognosis [159], supporting a role for NFB in metastasis. Development In Drosophila melanogaster, NFB participates in both pattern formation and innate immunity. Given the high degree of functional conservation between insect dorsal and mammalian NFB pathways, a developmental function for mammalian NFB is expected [18]. As discussed above, the analysis of IKK- and IKKnull mice showed that IKK plays a critical role in NFB activation in response to most common NFB inducing agents, whereas IKK is not required for the phosphorylation and degradation of IB in response to proinflammatory stimuli. Instead, IKK has been shown to be required for signal-dependent p100 processing and also to play an important role in regulating the transcriptional activity of NFB by mediating signal-dependent p65 phosphorylation. Interestingly, it has also been shown that loss of IKK interferes with multiple morphogenetic events, including limb and skeletal patterning and proliferation and differentiation of epidermal keratinocytes [38,40,43]. Transgenic mice expressing a transdominant form of IB under the control of the keratin 14 promoter exhibited hyperplastic epidermis, a phenotype very similar to that caused by the loss of IKK. Genomic rearrangement in Nemo (IKK) impairs NFB activation and is a cause of incontinentia pigmenti (IP) [32,45–48]. In affected IP females the defective Nemo (IKK) causes highly variable abnormalities of the skin, hair, nails, teeth, eyes, and central nervous system. The individual inactivation of each of four of the five known members of the NFB family results in defects in the immune system of varying severity. The absence of developmental defects in mice lacking individual NFB subunits might be due to redundant functions of NFB isoforms in mammals. Interestingly, mice devoid of both the p105/p50 and the p100/p52 subunits fail to generate mature osteoclasts, causing severe osteopetrosis [160,161]. All these findings suggest that the NFB proteins may regulate genetic programs in mammals that are associated with development as well as immunity.

Conclusions As discussed in this article, NFB proteins play an important role in innate immunity, stress responses, and cell proliferation and differentiation. The identification of IB kinase marks the turning point for elucidating the molecular mechanisms of NFB activation. Signal-induced activation of IB kinase leads to the phosphorylation and degradation of IB, liberating NFB from the IB inhibitory proteins. The identity of the kinase that activates IB kinase remains controversial, although several candidates have been proposed, including MEKK1, MEKK3, and TAK1. Further biochemical and genetic studies are required to unify the identity of the IB kinase kinase in the field, although it is

also quite possible that more than one kinase are capable of activating IB kinase. To fully activate NFB, in addition to its liberation from IB proteins, NFB is also phosphorylated. Many kinases have been implicated directly or indirectly in phosphorylating NFB, including PKA, CKII, PI3K, Akt, TBK1/NAK/T2K, and IKK. Future studies are required to clarify the function of these kinases in the phosphorylation of NFB proteins. While many stimuli lead to activation of NFB, including cytokines, bacterial products, viruses, and environmental insults, the proximal signaling components of the pathways activated by most of these stimuli are still largely unknown. Therefore, the future NFB research also demands identification and characterization of the specific proximal components for these NFB-dependent signaling pathways. The further understanding of the NFB-dependent signaling pathways will lay a solid foundation for the development of small-molecule drugs for controlling inflammation and treatment of cancers.

References 1. Sen R, Baltimore D (1986) Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46:705 2. Hoffmann JA, Kafatos FC, Janeway CA, Ezekowitz RA (1999) Phylogenetic perspectives in innate immunity. Science 284:1313 3. Baeuerle PA, Henkel T (1994) Function and activation of NF- B in the immune system. Annu Rev Immunol 12:141 4. Siebenlist U, Franzoso G, Brown K (1994) Structure, regulation and function of NF- B. Annu Rev Cell Biol 10:405 5. Karin M, Ben Neriah Y (2000) Phosphorylation meets ubiquitination: the control of NF-B activity. Annu Rev Immunol 18:621 6. Lee C, Schwartz MP, Prakash S, Iwakura M, Matouschek A (2001) ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal. Mol Cell 7:627 7. Lin L, Ghosh S (1996) A glycine-rich region in NF-B p105 functions as a processing signal for the generation of the p50 subunit. Mol Cell Biol 16:2248 8. Orian A, Schwartz AL, Israel A, Whiteside S, Kahana C, Ciechanover A (1999) Structural motifs involved in ubiquitin-mediated processing of the NF-B precursor p105: roles of the glycine-rich region and a downstream ubiquitination domain. Mol Cell Biol 19:3664 9. Chen ZJ, Maniatis T (1998) Role of the ubiquitin-proteasome pathway in NF-B activation. In J-M Peters, D Finley (eds): Ubiquitin and the biology of the cell. New York: Plenum, pp 303–322 10. Lin L, DeMartino GN, Greene WC (1998) Cotranslational biogenesis of NF-B p50 by the 26S proteasome. Cell 92:819 11. Lin L, DeMartino GN, Greene WC (2000) Cotranslational dimerization of the Rel homology domain of NF-B1 generates p50-p105 heterodimers and is required for effective p50 production. EMBO J 19:4712 12. Baeuerle PA, Baltimore D (1996) NF- B: ten years after. Cell 87:13 13. Chen FE, Huang DB, Chen YQ, Ghosh G (1998) Crystal structure of p50/p65 heterodimer of transcription factor NF-B bound to DNA. Nature 391:410 14. Cramer P, Larson CJ, Verdine GL, Muller CW (1997) Structure of the human NF-B p52 homodimer-DNA complex at 2.1 A resolution. EMBO J 16:7078 15. Ghosh G, van Duyne G, Ghosh S, Sigler PB (1995) Structure of NF B p50 homodimer bound to a  B site. Nature 373:303 16. Ghosh S, May MJ, Kopp EB (1998) NF- B and Rel proteins: evolu-

X. Li and G.R. Stark/Experimental Hematology 30 (2002) 285–296

17.

18. 19.

20.

21.

22.

23.

24.

25.

26. 27.

28.

29. 30.

31.

32.

33.

34.

35.

36. 37.

tionarily conserved mediators of immune responses. Annu Rev Immunol 16:225 Read MA, Brownell JE, Gladysheva TB, et al. (2000) Nedd8 modification of cul-1 activates SCF((TrCP))-dependent ubiquitination of IB. Mol Cell Biol 20:2326 Silverman N, Maniatis T (2001) NF-B signaling pathways in mammalian and insect innate immunity. Genes Dev 15:2321 Zhong H, SuYang H, Erdjument-Bromage H, Tempst P, Ghosh S (1997) The transcriptional activity of NF-B is regulated by the IBassociated PKAc subunit through a cyclic AMP-independent mechanism. Cell 89:413 Zhong H, Voll RE, Ghosh S (1998) Phosphorylation of NF- B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol Cell 1:661 Wang D, Baldwin AS Jr (1998) Activation of nuclear factor-B–dependent transcription by tumor necrosis factor- is mediated through phosphorylation of RelA/p65 on serine 529. J Biol Chem 273:29411 Wang D, Westerheide SD, Hanson JL, Baldwin AS Jr (2000) Tumor necrosis factor –induced phosphorylation of RelA/p65 on Ser529 is controlled by casein kinase II. J Biol Chem 275:32592 Sizemore N, Leung S, Stark GR (1999) Activation of phosphatidylinositol 3-kinase in response to interleukin-1 leads to phosphorylation and activation of the NF-B p65/RelA subunit. Mol Cell Biol 19:4798 Madrid LV, Wang CY, Guttridge DC, Schottelius AJ, Baldwin AS Jr, Mayo MW (2000) Akt suppresses apoptosis by stimulating the transactivation potential of the RelA/p65 subunit of NF-B. Mol Cell Biol 20:1626 Madrid LV, Mayo MW, Reuther JY, Baldwin AS Jr (2001) Akt stimulates the transactivation potential of the RelA/p65 subunit of NF- B through utilization of the I B kinase and activation of the mitogen-activated protein kinase p38. J Biol Chem 276:18934 Pahl HL (1999) Activators and target genes of Rel/NF-B transcription factors. Oncogene 18:6853 DiDonato JA, Hayakawa M, Rothwarf DM, Zandi E, Karin M (1997) A cytokine-responsive IB kinase that activates the transcription factor NF-B. Nature 388:548 Mercurio F, Zhu H, Murray BW, et al. (1997) IKK-1 and IKK-2: cytokine-activated IB kinases essential for NF-B activation. Science 278:860 Regnier CH, Song HY, Gao X, Goeddel DV, Cao Z, Rothe M (1997) Identification and characterization of an IB kinase. Cell 90:373 Woronicz JD, Gao X, Cao Z, Rothe M, Goeddel DV (1997) IB kinase-: NF-B activation and complex formation with IB kinase- and NIK. Science 278:866 Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M (1997) The IB kinase complex (IKK) contains two kinase subunits, IKK and IKK, necessary for IB phosphorylation and NF-B; activation. Cell 91:243 Yamaoka S, Courtois G, Bessia C, et al. (1998) Complementation cloning of NEMO, a component of the IB kinase complex essential for NF-B activation. Cell 93:1231 Yujiri T, Ware M, Widmann C, et al. (2000) MEK kinase 1 gene disruption alters cell migration and c-Jun NH2-terminal kinase regulation but does not cause a measurable defect in NF- B activation. Proc Natl Acad Sci U S A 97:7272 Yin L, Wu L, Wesche H, et al. (2001) Defective lymphotoxin- receptor-induced NF-B transcriptional activity in NIK-deficient mice. Science 291:2162 Matsushima A, Kaisho T, Rennert PD, et al. (2001) Essential role of nuclear factor (NF)-B–inducing kinase and inhibitor of B (IB) kinase  in NF-B activation through lymphotoxin  receptor, but not through tumor necrosis factor receptor I. J Exp Med 193:631 Xiao G, Harhaj EW, Sun SC (2001) NF-B–inducing kinase regulates the processing of NF-B2 p100. Mol Cell 7:401 Yang J, Lin Y, Guo Z, et al. (2001) The essential role of MEKK3 in TNF-induced NF-B activation. Nat Immunol 2:620

293

38. Li Q, Van Antwerp D, Mercurio F, Lee KF, Verma IM (1999) Severe liver degeneration in mice lacking the IB kinase 2 gene. Science 284:321 39. Li ZW, Chu W, Hu Y, et al. (1999) The IKK subunit of IB kinase (IKK) is essential for nuclear factor B activation and prevention of apoptosis. J Exp Med 189:1839 40. Tanaka M, Fuentes ME, Yamaguchi K, et al. (1999) Embryonic lethality, liver degeneration, and impaired NF- B activation in IKK–deficient mice. Immunity 10:421 41. Li Q, Estepa G, Memet S, Israel A, Verma IM (2000) Complete lack of NF-B activity in IKK1 and IKK2 double-deficient mice: additional defect in neurulation. Genes Dev 14:1729 42. Hu Y, Baud V, Delhase M, et al. (1999) Abnormal morphogenesis but intact IKK activation in mice lacking the IKK subunit of IB kinase. Science 284:316 43. Li Q, Lu Q, Hwang JY, et al. (1999) IKK1-deficient mice exhibit abnormal development of skin and skeleton. Genes Dev 13:1322 44. Takeda K, Takeuchi O, Tsujimura T, et al. (1999) Limb and skin abnormalities in mice lacking IKK. Science 284:313 45. Rudolph D, Yeh WC, Wakeham A, et al. (2000) Severe liver degeneration and lack of NF-B activation in NEMO/IKK-deficient mice. Genes Dev 14:854 46. Smahi A, Courtois G, Vabres P, et al., The International Incontinentia Pigmenti (IP) Consortium (2000) Genomic rearrangement in NEMO impairs NF-B activation and is a cause of incontinentia pigmenti. Nature 405:466 47. Makris C, Godfrey VL, Krahn-Senftleben G, et al. (2000) Female mice heterozygous for IKK /NEMO deficiencies develop a dermatopathy similar to the human X-linked disorder incontinentia pigmenti. Mol Cell 5:969 48. Schmidt-Supprian M, Bloch W, Courtois G, et al. (2000) NEMO/ IKK -deficient mice model incontinentia pigmenti. Mol Cell 5:981 49. Peters RT, Maniatis T (2001) A new family of IKK-related kinases may function as I  B kinase kinases. Biochim Biophys Acta 1471:M57 50. Shimada T, Kawai T, Takeda K, et al. (1999) IKK-i, a novel lipopolysaccharide-inducible kinase that is related to IB kinases. Int Immunol 11:1357 51. Pomerantz JL, Baltimore D (1999) NF-B activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase. EMBO J 18:6694 52. Tojima Y, Fujimoto A, Delhase M, et al. (2000) NAK is an IB kinase-activating kinase. Nature 404:778 53. Nomura F, Kawai T, Nakanishi K, Akira S (2000) NF-B activation through IKK-I–dependent I-TRAF/TANK phosphorylation. Genes Cells 5:191 54. Bonnard M, Mirtsos C, Suzuki S, et al. (2000) Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-B–dependent gene transcription. EMBO J 19:4976 55. Koul D, Yao Y, Abbruzzese JL, Yung WK, Reddy SA (2001) Tumor suppressor MMAC/PTEN inhibits cytokine-induced NFB activation without interfering with the IB degradation pathway. J Biol Chem 276:11402 56. Sizemore N, Lerner N, Dombrowski N, Sakurai H, Stark GR (2002) Distinct roles of the IB kinase  and  subunits in liberating nuclear factor B (NF-B) from IB and in phosphorylating the p65 subunit of NF-B. J Biol Chem 277:3863 57. Medzhitov R, Preston-Hurlburt P, Janeway CA Jr (1997) A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388:394 58. Rock FL, Hardiman G, Timans JC, Kastelein RA, Bazan JF (1998) A family of human receptors structurally related to Drosophila Toll. Proc Natl Acad Sci U S A 95:588 59. Takeuchi O, Kawai T, Sanjo H, et al. (1999) TLR6: A novel member of an expanding toll-like receptor family. Gene 231:59 60. Chuang TH, Ulevitch RJ (2000) Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8 and hTLR9. Eur Cytokine Netw 11:372

294

X. Li and G.R. Stark/Experimental Hematology 30 (2002) 285–296

61. Hemmi H, Takeuchi O, Kawai T, et al. (2000) A Toll-like receptor recognizes bacterial DNA. Nature 408:740 62. Tartaglia LA, Goeddel DV (1992) Two TNF receptors. Immunol Today 13:151 63. Hsu H, Xiong J, Goeddel DV (1995) The TNF receptor 1–associated protein TRADD signals cell death and NF- B activation. Cell 81:495 64. Hsu H, Shu HB, Pan MG, Goeddel DV (1996) TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84:299 65. Hsu H, Huang J, Shu HB, Baichwal V, Goeddel DV (1996) TNFdependent recruitment of the protein kinase RIP to the TNF receptor1 signaling complex. Immunity 4:387 66. McCarthy JV, Ni J, Dixit VM (1998) RIP2 is a novel NF-B–activating and cell death–inducing kinase. J Biol Chem 273:16968 67. Sun X, Lee J, Navas T, Baldwin DT, Stewart TA, Dixit VM (1999) RIP3, a novel apoptosis-inducing kinase. J Biol Chem 274:16871 68. Greenfeder SA, Nunes P, Kwee L, Labow M, Chizzonite RA, Ju G (1995) Molecular cloning and characterization of a second subunit of the interleukin 1 receptor complex. J Biol Chem 270:13757 69. Huang J, Gao X, Li S, Cao Z (1997) Recruitment of IRAK to the interleukin 1 receptor complex requires interleukin 1 receptor accessory protein. Proc Natl Acad Sci U S A 94:12829 70. Korherr C, Hofmeister R, Wesche H, Falk W (1997) A critical role for interleukin-1 receptor accessory protein in interleukin-1 signaling. Eur J Immunol 27:262 71. Wesche H, Korherr C, Kracht M, Falk W, Resch K, Martin MU (1997) The interleukin-1 receptor accessory protein (IL-1RAcP) is essential for IL-1–induced activation of interleukin-1 receptor–associated kinase (IRAK) and stress-activated protein kinases (SAP kinases). J Biol Chem 272:7727 72. Lord KA, Hoffman-Liebermann B, Liebermann DA (1990) Nucleotide sequence and expression of a cDNA encoding MyD88, a novel myeloid differentiation primary response gene induced by IL-6. Oncogene 5:1095 73. Wesche H, Henzel WJ, Shillinglaw W, Li S, Cao Z (1997) MyD88: an adapter that recruits IRAK to the IL-1 receptor complex. Immunity 7:837 74. Cao Z, Henzel WJ, Gao X (1996) IRAK: a kinase associated with the interleukin-1 receptor. Science 271:1128 75. Burns K, Clatworthy J, Martin L, et al. (2000) Tollip, a new component of the IL-1RI pathway, links IRAK to the IL-1 receptor. Nat Cell Biol 2:346 76. Cao Z, Xiong J, Takeuchi M, Kurama T, Goeddel DV (1996) TRAF6 is a signal transducer for interleukin-1. Nature 383:443 77. Muzio M, Ni J, Feng P, Dixit VM (1997) IRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling. Science 278:1612 78. Wesche H, Gao X, Li X, Kirschning CJ, Stark GR, Cao Z (1999) IRAK-M is a novel member of the Pelle/interleukin-1 receptor–associated kinase (IRAK) family. J Biol Chem 274:19403 79. Arch RH, Gedrich RW, Thompson CB (1998) Tumor necrosis factor receptor–associated factors (TRAFs)—a family of adapter proteins that regulates life and death. Genes Dev 12:2821 80. Kishimoto K, Matsumoto K, Ninomiya-Tsuji J (2000) TAK1 mitogen-activated protein kinase kinase kinase is activated by autophosphorylation within its activation loop. J Biol Chem 275:7359 81. Ninomiya-Tsuji J, Kishimoto K, Hiyama A, Inoue J, Cao Z, Matsumoto K (1999) The kinase TAK1 can activate the NIK-I B as well as the MAP kinase cascade in the IL-1 signalling pathway. Nature 398:252 82. Irie T, Muta T, Takeshige K (2000) TAK1 mediates an activation signal from toll-like receptor(s) to nuclear factor-B in lipopolysaccharide-stimulated macrophages. FEBS Lett 467:160 83. Takaesu G, Kishida S, Hiyama A, et al. (2000) TAB2, a novel adaptor protein, mediates activation of TAK1 MAPKKK by linking

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

99.

100.

101. 102.

103. 104.

TAK1 to TRAF6 in the IL-1 signal transduction pathway. Mol Cell 5:649 Takaesu G, Ninomiya-Tsuji J, Kishida S, Li X, Stark GR, Matsumoto K (2001) Interleukin-1 (IL-1) receptor–associated kinase leads to activation of TAK1 by inducing TAB2 translocation in the IL-1 signaling pathway. Mol Cell Biol 21:2475 Qian Y, Commane M, Ninomiya-Tsuji J, Matsumoto K, Li X (2001) IRAK-mediated translocation of TRAF6 and TAB2 in the interleukin-1–induced activation of NF B. J Biol Chem 276:41661 Deng L, Wang C, Spencer E, et al. (2000) Activation of the IB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103:351 Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ (2001) TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412:346 Irie T, Muta T, Takeshige K (2000) TAK1 mediates an activation signal from toll-like receptor(s) to nuclear factor-B in lipopolysaccharide-stimulated macrophages. FEBS Lett 467:160 Lee J, Mira-Arbibe L, Ulevitch RJ (2000) TAK1 regulates multiple protein kinase cascades activated by bacterial lipopolysaccharide. J Leukoc Biol 68:909 Sanz L, Diaz-Meco MT, Nakano H, Moscat J (2000) The atypical PKCinteracting protein p62 channels NF-B activation by the IL-1–TRAF6 pathway. EMBO J 19:1576 Bowie A, O’Neill LA (2000) The interleukin-1 receptor/Toll-like receptor superfamily: signal generators for pro-inflammatory interleukins and microbial products. J Leukoc Biol 67:508 Poltorak A, He X, Smirnova I, et al. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085 Takeuchi O, Hoshino K, Kawai T, et al. (1999) Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11:443 Takeuchi O, Kaufmann A, Grote K, et al. (2000) Cutting edge: preferentially the R-stereoisomer of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 activates immune cells through a tolllike receptor 2– and MyD88-dependent signaling pathway. J Immunol 164:554 Underhill DM, Ozinsky A, Hajjar AM, et al. (1999) The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature 401:811 Underhill DM, Ozinsky A, Smith KD, Aderem A (1999) Toll-like receptor-2 mediates mycobacteria-induced proinflammatory signaling in macrophages. Proc Natl Acad Sci U S A 96:14459 Sebastiani G, Leveque G, Lariviere L, et al. (2000) Cloning and characterization of the murine toll-like receptor 5 (Tlr5) gene: sequence and mRNA expression studies in Salmonella-susceptible MOLF/Ei mice. Genomics 64:230 Alexopoulou L, Holt AC, Medzhitov R, Flavell RA (2001) Recognition of double-stranded RNA and activation of NF-B by Toll-like receptor 3. Nature 413:732 Thomassen E, Renshaw BR, Sims JE (1999) Identification and characterization of SIGIRR, a molecule representing a novel subtype of the IL-1R superfamily. Cytokine 11:389 Adachi O, Kawai T, Takeda K, et al. (1998) Targeted disruption of the MyD88 gene results in loss of IL-1– and IL-18–mediated function. Immunity 9:143 Kawai T, Adachi O, Ogawa T, Takeda K, Akira S (1999) Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11:115 Fitzgerald KA, Palsson-McDermott EM, Bowie AG, et al. (2001) Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction. Nature 413:78 Horng T, Barton GM, Medzhitov R (2001) TIRAP: an adapter molecule in the Toll signaling pathway. Nat Immunol 2:835 Locksley RM, Killeen N, Lenardo MJ (2001) The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104:487

X. Li and G.R. Stark/Experimental Hematology 30 (2002) 285–296 105. Schwandner R, Yamaguchi K, Cao Z (2000) Requirement of tumor necrosis factor receptor–associated factor (TRAF)6 in interleukin 17 signal transduction. J Exp Med 191:1233 106. Inohara N, Ogura Y, Chen FF, Muto A, Nunez G (2001) Human Nod1 confers responsiveness to bacterial lipopolysaccharides. J Biol Chem 276:2551 107. Girardin SE, Tournebize R, Mavris M, et al. (2001) CARD4/Nod1 mediates NF-B and JNK activation by invasive Shigella flexneri. EMBO Rep 2:736 108. Beutler B (2001) Autoimmunity and apoptosis: the Crohn’s connection. Immunity 15:5 109. Ogura Y, Bonen DK, Inohara N, et al. (2001) A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 411:603 110. Hugot JP, Chamaillard M, Zouali H, et al. (2001) Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 411:599 111. Berlett BS, Stadtman ER (1997) Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 272:20313 112. Sen CK, Packer L (1996) Antioxidant and redox regulation of gene transcription. FASEB J 10:709 113. Li N, Karin M (1999) Is NF-B the sensor of oxidative stress? FASEB J 13:1137 114. Schreck R, Rieber P, Baeuerle PA (1991) Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF- B transcription factor and HIV-1. EMBO J 10:2247 115. Manna SK, Zhang HJ, Yan T, Oberley LW, Aggarwal BB (1998) Overexpression of manganese superoxide dismutase suppresses tumor necrosis factor–induced apoptosis and activation of nuclear transcription factor-B and activated protein-1. J Biol Chem 273:13245 116. Meyer M, Schreck R, Baeuerle PA (1993) H2O2 and antioxidants have opposite effects on activation of NF- B and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J 12:2005 117. Anderson MT, Staal FJ, Gitler C, Herzenberg LA, Herzenberg LA (1994) Separation of oxidant-initiated and redox-regulated steps in the NF- B signal transduction pathway. Proc Natl Acad Sci U S A 91:11527 118. Kretz-Remy C, Bates EE, Arrigo AP (1998) Amino acid analogs activate NF-B through redox-dependent IB- degradation by the proteasome without apparent IB- phosphorylation. Consequence on HIV-1 long terminal repeat activation. J Biol Chem 273:3180 119. Kretz-Remy C, Mehlen P, Mirault ME, Arrigo AP (1996) Inhibition of I  B- phosphorylation and degradation and subsequent NF- B activation by glutathione peroxidase overexpression. J Cell Biol 133:1083 120. Li N, Karin M (1998) Ionizing radiation and short wavelength UV activate NF-B through two distinct mechanisms. Proc Natl Acad Sci U S A 95:13012 121. Bender K, Gottlicher M, Whiteside S, Rahmsdorf HJ, Herrlich P (1998) Sequential DNA damage-independent and -dependent activation of NF-B by UV. EMBO J 17:5170 122. Imbert V, Rupec RA, Livolsi A, et al. (1996) Tyrosine phosphorylation of I  B- activates NF- B without proteolytic degradation of I  B-. Cell 86:787 123. Singh S, Darnay BG, Aggarwal BB (1996) Site-specific tyrosine phosphorylation of IB negatively regulates its inducible phosphorylation and degradation. J Biol Chem 271:31049 124. Sonenshein GE (1997) Rel/NF- B transcription factors and the control of apoptosis. Semin Cancer Biol 8:113 125. Wu M, Arsura M, Bellas RE, et al. (1996) Inhibition of c-myc expression induces apoptosis of WEHI 231 murine B cells. Mol Cell Biol 16:5015 126. Wu M, Lee H, Bellas RE, et al. (1996) Inhibition of NF-B/Rel induces apoptosis of murine B cells. EMBO J 15:4682 127. Lee H, Arsura M, Wu M, Duyao M, Buckler AJ, Sonenshein GE

128.

129.

130.

131.

132.

133. 134.

135. 136. 137. 138.

139.

140.

141.

142.

143.

144. 145.

146.

147.

148.

295

(1995) Role of Rel-related factors in control of c-myc gene transcription in receptor-mediated apoptosis of the murine B cell WEHI 231 line. J Exp Med 181:1169 Arsura M, Wu M, Sonenshein GE (1996) TGF  1 inhibits NF- B/ Rel activity inducing apoptosis of B cells: transcriptional activation of I  B . Immunity 5:31 Tan JE, Wong SC, Gan SK, Xu S, Lam KP (2001) The adaptor protein BLNK is required for B cell antigen receptor–induced activation of nuclear factor- B and cell cycle entry and survival of B lymphocytes. J Biol Chem 276:20055 Petro JB, Rahman SM, Ballard DW, Khan WN (2000) Bruton’s tyrosine kinase is required for activation of IB kinase and nuclear factor B in response to B cell receptor engagement. J Exp Med 191:1745 Petro JB, Khan WN (2001) Phospholipase C- 2 couples Bruton’s tyrosine kinase to the NF-B signaling pathway in B lymphocytes. J Biol Chem 276:1715 Jumaa H, Wollscheid B, Mitterer M, Wienands J, Reth M, Nielsen PJ (1999) Abnormal development and function of B lymphocytes in mice deficient for the signaling adaptor protein SLP-65. Immunity 11:547 Pappu R, Cheng AM, Li B, et al. (1999) Requirement for B cell linker protein (BLNK) in B cell development. Science 286:1949 Hayashi K, Nittono R, Okamoto N, et al. (2000) The B cell–restricted adaptor BASH is required for normal development and antigen receptor–mediated activation of B cells. Proc Natl Acad Sci U S A 97:2755 Khan WN, Alt FW, Gerstein RM, et al. (1995) Defective B cell development and function in Btk-deficient mice. Immunity 3:283 Kerner JD, Appleby MW, Mohr RN, et al. (1995) Impaired expansion of mouse B cell progenitors lacking Btk. Immunity 3:301 Wang D, Feng J, Wen R, et al. (2000) Phospholipase C2 is essential in the functions of B cell and several Fc receptors. Immunity 13:25 Ruland J, Duncan GS, Elia A, et al. (2001) bcl-10 is a positive regulator of antigen receptor–induced activation of NF-B and neural tube closure. Cell 104:33 Lucas PC, Yonezumi M, Inohara N, et al. (2001) bcl-10 and MALT1, independent targets of chromosomal translocation in malt lymphoma, cooperate in a novel NF- B signaling pathway. J Biol Chem 276:19012 Yoneda T, Imaizumi K, Maeda M, et al. (2000) Regulatory mechanisms of TRAF2-mediated signal transduction by bcl-10, a MALT lymphoma-associated protein. J Biol Chem 275:11114 Aifantis I, Gounari F, Scorrano L, Borowski C, von Boehmer H (2001) Constitutive pre-TCR signaling promotes differentiation through Ca2 mobilization and activation of NF-B and NFAT. Nat Immunol 2:403 Voll RE, Jimi E, Phillips RJ, et al. (2000) NF- B activation by the pre-T cell receptor serves as a selective survival signal in T lymphocyte development. Immunity 13:677 Boldin MP, Goncharov TM, Goltsev YV, Wallach D (1996) Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/ APO-1– and TNF receptor–induced cell death. Cell 85:803 Hsu H, Xiong J, Goeddel DV (1995) The TNF receptor 1–associated protein TRADD signals cell death and NF- B activation. Cell 81:495 Hsu H, Shu HB, Pan MG, Goeddel DV (1996) TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84:299 Chinnaiyan AM, O’Rourke K, Tewari M, Dixit VM (1995) FADD, a novel death domain–containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81:505 Chinnaiyan AM, Tepper CG, Seldin MF, et al. (1996) FADD/ MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor–induced apoptosis. J Biol Chem 271:4961 Tartaglia LA, Ayres TM, Wong GH, Goeddel DV (1993) A novel domain within the 55 kd TNF receptor signals cell death. Cell 74:845

296

X. Li and G.R. Stark/Experimental Hematology 30 (2002) 285–296

149. Itoh N, Nagata S (1993) A novel protein domain required for apoptosis. Mutational analysis of human Fas antigen. J Biol Chem 268:10932 150. Lee SY, Lee SY, Kandala G, Liou ML, Liou HC, Choi Y (1996) CD30/TNF receptor–associated factor interaction: NF- B activation and binding specificity. Proc Natl Acad Sci U S A 93:9699 151. Force WR, Glass AA, Benedict CA, Cheung TC, Lama J, Ware CF (2000) Discrete signaling regions in the lymphotoxin- receptor for tumor necrosis factor receptor–associated factor binding, subcellular localization, and activation of cell death and NF-B pathways. J Biol Chem 275:11121 152. Aliprantis AO, Yang RB, Mark MR, et al. (1999) Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science 285:736 153. Wang CY, Mayo MW, Baldwin AS Jr (1996) TNF- and cancer therapy–induced apoptosis: potentiation by inhibition of NF-B. Science 274:784 154. Micheau O, Lens S, Gaide O, Alevizopoulos K, Tschopp J (2001) NF-B signals induce the expression of c-FLIP. Mol Cell Biol 21:5299

155. Doi TS, Marino MW, Takahashi T, et al. (1999) Absence of tumor necrosis factor rescues RelA-deficient mice from embryonic lethality. Proc Natl Acad Sci U S A 96:2994 156. Gilmore TD (1999) Multiple mutations contribute to the oncogenicity of the retroviral oncoprotein v-Rel. Oncogene 18:6925 157. Rayet B, Gelinas C (1999) Aberrant rel/nfkb genes and activity in human cancer. Oncogene 18:6938 158. Pahl HL (1999) Activators and target genes of Rel/NF-B transcription factors. Oncogene 18:6853 159. Newton TR, Patel NM, Bhat-Nakshatri P, Stauss CR, Goulet RJ Jr, Nakshatri H (1999) Negative regulation of transactivation function but not DNA binding of NF-B and AP-1 by IB1 in breast cancer cells. J Biol Chem 274:18827 160. Gerondakis S, Grossmann M, Nakamura Y, Pohl T, Grumont R (1999) Genetic approaches in mice to understand Rel/NF-B and IB function: transgenics and knockouts. Oncogene 18:6888 161. Franzoso G, Carlson L, Xing L, et al. (1997) Requirement for NF-B in osteoclast and B-cell development. Genes Dev 11:3482