NF-κB Tom Lüdde, Christian Trautwein

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NF- κB

Tom Lüdde, Christian Trautwein

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29.1 Introduction

Nuclear factor (NF)- κB was first described in 1986 as a nuclear factor necessary for immunoglobulin κ light chain transcription [113, 114]. It exists in virtu- ally all known cell types and mitochondria [17, 42], and regulates the transcription of an exceptionally large number of genes including those involved in immune and inflammatory response, cell death, and proliferation [59, 118]. In this chapter, we will give a short introduction into the structure and basic ac- tivation pathways of NF- κB and then focus on its role in the modulation of liver apoptosis, ischemia- reperfusion (I/R) injury, liver regeneration, viral hepatitis and cancer development.

29.2 The NF- κB Transcription Factor Family

The NF- κB signaling pathway was developed early in evolution and is already found in Drosophila and mollusks [86]. In Drosophila, NF- κB-like transcrip- tion factors are activated in order to combat infec- tions [36]. The function of NF- κB for the immune response and also the components of the pathway have been evolutionarily conserved in mammals.

NF- κB is a dimer of members of the Rel family of DNA-binding proteins. Nearly all vertebrate NF- κB proteins have been crystalized and their structures have been determined. The mammalian NF- κB family includes five cellular DNA-binding subunit proteins: p50 (NF- κB1), p52 (NF-κB2), c-Rel (Rel), p65 (RelA) and RelB [43]. The domain architecture of these subunits and the I κB proteins is schemati- cally shown in Fig. 29.1. The NF- κB DNA-binding subunits share an N-terminal Rel homology domain (RHD). It forms a unique butterfly-shaped struc- ture composed of β strands arranged in a pattern similar to immunoglobulin domains. This region is responsible for DNA binding, dimerization, nuclear

translocation and interaction with the inhibitory I κB proteins [42].

p65 (RelA), RelB and c-Rel contain C-terminal transactivation domains that trigger target gene transcription. Of these proteins, p65, which contains two potent transactivation domains (TADs) within its C-terminus, mediates the strongest gene activa- tion [109]. The other two members, p52 and p50, become active, shorter DNA-binding proteins from larger precursors (p105 to p50, p100 to p52) by either constitutive (p105) or regulated (p100) processing steps [2]. They are generally not activators of tran- scription, unless they form heterodimers with p65, RelB or c-Rel. NF- κB commonly refers to a p50/p65 heterodimer, which is the first form of NF- κB re- ported. It is one of the most avidly forming dimers and is the major Rel complex in most cells. Although most NF- κB proteins are transcriptionally active, some combinations such as p50/p50 homodimers and also p52/p52 homodimers are transcriptionally repressive [109]. In the nucleus, NF- κB recognizes the κB sites bearing a consensus sequence 5'GGG.

Pu.N.Py.CC (Pu is purine, Py is pyrimidine, and N is any base) [1].

The activity of NF- κB is controlled by IκBs (IκBα, I κBβ, IκBε, IκBγ, IκBNS and Bcl-3), a family of cyto- plasmic inhibitory proteins that share a number of protein/protein interaction domains called ankyrin repeats. The precursor forms p105 and p100 are also included in this family, since they contain I κB-like repeats and therefore inhibit NF- κB activation [4]

(Fig. 29.1). NF- κB is effectively sequestered in the

cytoplasm by I κB in an inactive state via complex

formation and the ability of I κB to mask the nuclear

localization site (NLS) of NF- κB. As IκBα is an NF-

κB target gene, it also terminates NF-κB activation

at transcriptional level: increased synthesis of I κBα

shuts down NF- κB-induced gene expression by

I κBα-mediated nuclear export of the DNA-binding

subunits, thereby acting within a negative feedback

loop [3].

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29.3 Control of NF- κB Activation: General Aspects In order to activate NF- κB, different pathways have evolved, which all lead to the generation of DNA- binding dimers. At present, the so-called canonical and non-canonical pathways and the DNA dam- age-induced NF- κB pathway have been identified.

Moreover, further mechanisms such as p65 post- translational modifications regulate the activity of this transcription factor.

The canonical pathway is the best described and probably most important mediator of NF- κB activa- tion in response to cytokines. An essential step dur- ing this pathway is the disruption of cytoplasmic NF- κB:IκB complexes, initiated by the phosphor- ylation of the most important I κB family member, I κBα, at serine 32 and 36 through a high molecular I κB kinase (IKK) complex. This phosphorylation is the prerequisite for the subsequent polyubiquiti- nation of I κB-α by a specific, constitutively active ubiquitin ligase belonging to the SCF family [56, 137]. The ubiquitin-marked I κB proteins are then rapidly degraded by the 26S proteasome, leading to the unmasking of the nuclear localization site (NLS) of NF- κB and thus allowing nuclear entry, DNA binding and transcriptional activity of NF- κB. Since numerous studies have implicated a central role of this pathway in liver physiology and pathology, it will be discussed in detail later in this chapter.

Another form of NF- κB activation, the so-called non-canonical pathway, has been described particu- larly in B cells. The activation of this I κB-independ- ent pathway involves the IKK subunit IKK1 and re- sults in the release of p52/RelB and p50/RelB dimers [88, 115, 133]. It is induced for example by lympho- toxin β (LTβ) and leads to NIK- and IKK1-depend-

ent processing of the p100 precursor protein, which results in the release of p52 [100, 115]. LT β employs canonical and non-canonical pathways. Whereas in the canonical pathways, signaling is abrogated quickly by induction of I κBα and subsequent re- moval of p65 from its cognate DNA, the non-canoni- cal pathway comprises slower p100 processing, thus inducing a delayed NF- κB activation [19, 22, 88]. At present, the significance of this pathway in hepato- cytes is not clear, but a recent study has shown that this pathway is also employed by lipopolysaccharide (LPS), which can act as a damaging agent to the liver [87].

DNA-damage-induced NF- κB activation, in con- trast to the previously described pathways, occurs in an IKK-independent manner. It has been observed after doxorubicin stimulation or UV radiation and involves mitogen-activated protein kinase (MAPK)- dependent alternative I κBα phosphorylation [61, 96, 110, 127].

A growing number of studies have suggested that besides the formation and nuclear transloca- tion of NF- κB dimers, post-translational modifica- tions of NF- κB subunits might also influence NF-κB activation. In particular, the phosphorylation of p65 and also its acetylation appear to modify its tran- scriptional activity significantly. Numerous phos- phorylation sites have been described on the p65 protein, which are targeted either by a single or by several kinases. Serine 276 is the best-characterized phosphorylation site of p65. Fibroblasts containing a mutant p65 with serine 276 replaced by alanine instead of the wild-type form showed an impaired tumor necrosis factor α (TNF)-induced expres- sion of the NF- κB target gene interleukin (IL)-6, and these cells lost their NF- κB-dependent protec- tion against TNF-induced apoptosis [94]. Whereas both the catalytic subunit of PKA, PKAc and MSK-1

Fig. 29.1. Structure of the NF-κB and IκB proteins. RHD Rel-homology domain, TAD transactivation domain.

(From M.L. Schmitz, I. Mattioli, H. Buss, M. Kracht. NF-κB: a multi-faceted transcription factor regulated at several levels. Submitted for publication. With kind permission from the authors.)

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have been identified as serine 276 kinases [94, 129], phosphorylation at serine 536 appears to involve the IKK complex itself [82, 107], demonstrating an addi- tional function of this complex in NF- κB activation besides I κB phosphorylation. Another way of post- translational modification of NF- κB is acetylation of p65. Acetylation of p65 was first detected in vivo by [3H]acetate radiolabeling of overexpressed p65, and further studies revealed that acetylation of endog- enous p65 is induced by stimuli such as TNF [14, 63].

This process involves the histone acetyl transferases CBP and p300 and might enhance the transactiva- tion potential of p65 [41, 98, 116]. However, further studies are needed to evaluate the significance of post-translational modifications of p65 in vivo.

29.4 NF- κB in the Regulation of Liver Apoptosis

29.4.1

Death Receptor Cytokines in Liver Failure

Hepatic failure takes place when the amount of func- tioning hepatocytes decreases until the organ is not capable of fulfilling both its metabolic and synthetic functions. Generally, there are different concepts describing how a cell is driven into death. Necrotic cell death is the result of acute metabolic disruption with ATP depletion, ion dysregulation, mitochon- drial and cellular swelling and activation of degra- dative enzymes. This culminates in rupture of the plasma membrane and loss of intracellular proteins, metabolites and ions [80]. On the other hand, ap- optosis, synonymously used for programmed cell death, is an important mode of cell death when spe- cific cells need to be removed during development or normal tissue turnover [62]. It leads to the orderly resorption of target cells without severe impairment of cellular metabolism and its onset is triggered by specific and active signaling through activation of a cascade of cysteine-aspartate proteases called cas- pases [108].

Excessive apoptosis has been implicated in a number of acute and chronic liver diseases, e.g., vi- ral and autoimmune hepatitis, cholestatic disease, alcoholic or drug/toxin-induced liver injury and transplantation-associated liver damage, including graft rejection [93]. Studies in patients and animal models have strongly implicated that death recep- tor ligands such as TNF or Fas ligand (FasL) are in- volved in the induction of apoptosis and in trigger- ing destruction of the liver [78].

TNF was originally identified by its capacity to induce hemorrhagic necrosis in mice tumors [10], but severe side-effects led to a failure of its use as a systemic anticancer chemotherapeutic agent [34, 64]. A very prominent effect was the direct cyto- toxic role of TNF for human hepatocytes, resulting in increased levels of serum transaminases and bi- lirubin. Since then, many clinical studies have un- derlined the crucial role of TNF in fulminant hepat- ic failure and other liver diseases. TNF participates in many forms of hepatic pathology, including I/R injury, alcoholic and viral hepatitis, and injury from hepatotoxins [18, 35, 44, 71] (see Chapter 10).

Exogenous TNF- α induces fulminant hepatic failure (FHF) and hepatocyte apoptosis in combi- nation with other toxins [71]. TNF serum levels are clearly elevated in patients with FHF [89]. In another study, it was shown that serum TNF levels were sig- nificantly higher in FHF patients who died than in patients who survived [8]. We could show that serum TNF, TNF-receptor 1 (TNF-R1) and TNF-R2 levels were increased markedly in patients with fulminant hepatic failure and that these changes directly cor- related with disease activity. In explanted livers of patients with FHF, infiltrating mononuclear cells expressed high amounts of TNF and hepatocytes overexpressed TNF-R1. Moreover, we found that ap- optotic hepatocytes were significantly increased in FHF, and there was strong correlation with TNF- α expression [119]. Thus, it is very likely that the TNF system is involved in the pathogenesis of FHF in humans, and its significance has also been shown clearly in several animal models of hepatic failure, e.g., the endotoxin/d-galactosamine (GalN) and the concanavalin A (ConA) model [39, 99].

29.4.2

TNF-Dependent NF-

κB Activation

Tumor necrosis factor facilitates programmed cell

death by activation of caspases. It signals through

two distinct cell surface receptors, TNF-R1 and TNF-

R2, of which TNF-R1 initiates the majority of TNF's

biological activities. Binding of TNF to its receptor

leads to the release of the inhibitory protein silencer

of death domains (SODD) from TNF-R1's intracel-

lular domain. This leads to the recognition of the in-

tracellular TNF-R1 domain by the adaptor protein

TNF receptor-associated death domain (TRADD),

which in turn recruits Fas-associated death domain

(FADD). FADD recruits caspase-8 to the TNF-R1

complex, where it becomes activated and initiates

the protease cascade leading to activation of execu-

tioner caspases and apoptosis (Fig. 29.2) [13] (see

Chapter 10).

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Next to activation of caspases, ligand binding of TNF to its receptor also leads to the activation of the NF- κB pathway (Fig. 29.2). As outlined in Sect. 29.3, TNF-induced activation of NF- κB relies on phos- phorylation of two conserved serines (S32 and S36 in human I κBα) in the N-terminal regulatory domain of I κBs. After phosphorylation, the IκBs undergo a second post-translational modification: polyu- biquitination by a cascade of enzymatic reactions, mediated by the β-TrCP-SCF complex (or the E3IκB ubiquitin ligase complex). This process is followed by the degradation of I κB proteins by the protea- some, thus releasing NF- κB from its inhibitory IκB- binding partner, so it can translocate to the nucleus and activate transcription of NF- κB-dependent tar- get genes [56, 135]. Since the enzymes that catalyze the ubiquitination of I κB are constitutively active, the only regulated step in NF- κB activation appears to be in most cases the phosphorylation of I κB mol- ecules.

A high-molecular-weight complex that mediates the phosphorylation of I κB has been purified and characterized. This complex consists of three tight- ly associated IKK polypeptides: IKK1 (also called IKK α) and IKK2 (IKKβ) are the catalytic subunits of the kinase complex and have very similar pri- mary structures with 52% overall similarity [28, 57, 84, 102]. Moreover, it contains a regulatory subunit called NEMO (NF- κB Essential Modulator), IKKγ or IKKAP-1 [83, 103, 136]. In vitro, IKK1 and IKK2 can form homo- and heterodimers [138]. Both IKK1 and IKK2 are able to phosphorylate I κB in vitro, but IKK2 has a higher kinase activity in vitro compared with IKK1 [23, 83, 84, 131, 139].

Activation of the IKK complex upon TNF stimu- lation involves IKK recruitment to the TNF-R1 [26, 27, 140]. Next to TNF-R1, this process involves TNF- receptor-associated-factor 2 (TRAF2) and the death- domain kinase receptor-interacting protein (RIP).

In response to TNF treatment, TRAF2 recruits the IKK complex to TNF-R1 via the interaction of the RING-finger motifs of TRAF2 with the leucin-zip- per motif of both IKK1 and IKK2 [26, 27]. RIP can directly interact with NEMO and mediate IKK acti- vation, although the enzymatic activity of RIP is not required for this process [140]. The mechanism by which recruitment of the IKK complex to the TNF receptor leads to IKK activation is not clear, but might involve NEMO-induced autophosphoryla- tion of the IKK complex. Moreover, ubiquitination of multiple factors that regulate the IKK complex, like TRAF6/TAK1 or c-IAP1, an inhibitor of apop- tosis that is also part of the TNF receptor complex, modulate the activity of the NF- κB pathway [135].

29.4.3

Anti-apoptotic Function of NF-

κB in the Liver Numerous studies have shown that NF- κB provides survival signals in the context of death receptor-in- duced apoptosis in the liver. This process is assumed to involve the transcriptional induction of various apoptotic suppressors [76]. Evidence that NF- κB governs critical anti-apoptotic proteins comes from well-described animal models. Injection of TNF into mice and addition of TNF to hepatic cells resulted in activation of NF- κB binding [37]. In contrast to Fas-mediated apoptosis, hepatocytes are resistant to apoptosis induced by TNF or LPS, a potent induc- tor for endogenous TNF in the liver, unless they are treated with inhibitors of transcription of (anti-ap- optotic) proteins like cycloheximide or actinomycin D [69–71]. Specific blockade of NF- κB activation by adenoviral-directed overexpression of the NF- κB super-repressor I κB (that retains the dimeric NF-κB in the cytoplasm since it contains mutations at the phosphorylation sites) significantly enhanced TNF- mediated apoptosis of hepatocytes [90]. A similar result was obtained by treatment of hepatocytes with the proteasome inhibitor lactacystin, which prevents degradation of I κBs, and an antibody against p65 protein [7].

Further evidence for the central role of the NF- κB pathway in preventing apoptosis in hepatocytes came from genetic experiments. Knockout mice lacking the p65 subunit of NF- κB die between days E15 and E16 post-coitum as a result of fetal hepato- cyte apoptosis [6]. This is caused by increased sensi- tivity towards TNF, since TNF/p65 double-deficient

Fig. 29.2. TNF-dependent activation of NF-κB.

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mice are rescued from embryonic lethality [29].

c-Rel may partially compensate for p65 in block- ing liver apoptosis in that c-rel/p65 double knock- out mice show liver degeneration approximately 1.5 days earlier than p65 single knockout mice [45].

Genetic experiments have also highlighted the differential functions of the IKK subunits in TNF- mediated liver apoptosis. Mice lacking Ikk1 die shortly after birth and display a phenotype marked by thickening of skin and limb as well as skeletal de- fects. Stimulation of embryonic fibroblasts or liver tissue with IL-1, TNF or endotoxin results in normal IKK activation and I κBα degradation [51, 124]. In contrast, Ikk2

^–/–

mice die in utero approximately at embryonic day 12.5 as a result of massive apoptosis in the liver, and fibroblasts from these mice show no activation of NF- κB in response to TNF and IL-1 [72, 73, 125]. A similar phenotype was noted in mice lacking the regulatory subunit NEMO, which also die from massive apoptosis in the liver and show a defect in NF- κB activation upon TNF stimulation in primary murine embryonic fibroblast (MEF) cells [105]. Therefore, at least during embryogenesis, IKK2 and NEMO appear to be the critical subunits for NF- κB activation and protection of liver cells from proinflammatory cytokines like TNF.

The role of the IKK subunits in the adult animal is controversial, but might vary from the situation during embryogenesis. Experiments with adenovi- ruses expressing dominant negative IKK forms in primary hepatocytes showed that overexpression of a dominant negative IKK2 mutant can totally block TNF-dependent NF- κB activation and lead to hepa- tocyte apoptosis [111]. Conditional knockout mice based on the cre/loxP system have emerged as new powerful tools to study gene functions in the adult animal in vivo. In a recent study we could show that hepatocyte-specific ablation of IKK2 did not lead to impaired activation of NF- κB or increased apoptosis after TNF stimulation, probably because IKK1 ho- modimers can take over this function in the absence of IKK2 in the adult mouse. In contrast, conditional knockout of NEMO resulted in complete block of NF- κB activation and massive hepatocyte apoptosis (T. Lüdde et al., submitted for publication), underlin- ing that NEMO is the only irreplaceable IKK subunit for prevention of TNF-mediated liver apoptosis.

Besides its anti-apoptotic function in TNF-me- diated liver apoptosis, recent reports suggest that NF- κB also plays a role in other apoptotic pathways.

Concavalin A (ConA) activates T lymphocytes in vitro and causes T-cell-dependent hepatic injury in mice, characterized by apoptotic cell death [40].

ConA exerts cytotoxic effects through cell-bound TNF, which activates TNF-R1 and TNF-R2 [79].

ConA stimulation in mice leads to activation of NF-

κB, and this pathway is blocked by anti-TNF [128].

Inhibition of NF- κB by degradation-resistant IκBα (I κBα super-repressor) increases susceptibility to ConA-mediated liver apoptosis [68]. Fas, also called APO-1 or CD95, is a cell surface receptor belonging to the TNF receptor that is expressed in hepatocytes and plays a role in liver failure [91, 106]. The func- tion of NF- κB in Fas-mediated apoptosis in hepa- tocytes is controversial. In hepatocyte-derived cell lines, inactivation of NF- κB made these cells more susceptible to apoptosis induced by Fas stimulation [81]. In contrast, in a model of adenoviral hepatitis in mice, NF- κB mediates the pro-apoptotic function of Fas [66], underlining that the pro- or anti-apop- totic role of NF- κB is determined by the nature of the death stimulus.

29.4.4

Mediators of the Anti-Apoptotic NF-

κB Function After binding to its responsive DNA elements, NF- κB induces a variety of target genes that mediate its anti-apoptotic function (Fig. 29.3). Among these genes are the cellular inhibitors of apoptosis (c- IAPs), e.g., c-IAP1 and c-IAP2, which directly bind and inhibit effector caspases, such as caspase-3 and caspase-7, and prevent activation of pro-caspase-6 and pro-caspase-9 [25]. Another cIAP regulated by NF- κB is X chromosome-linked IAP (XIAP), which inhibits caspase-3 and caspase-7 and might prevent activation of pro-caspase-9 [24, 32]. c-FLIP (FLICE inhibitory protein) is an NF- κB-regulated protein that inhibits apoptosis by interfering with pro-cas- pase-8 activation [59]. Moreover, anti-apoptotic Bcl-2 family members (A1 and Bcl-xL) and TRAF1 and TRAF2 are induced upon TNF stimulation in an NF- κB-dependent manner [5, 59].

Recent studies have revealed that the NF- κB and

the c-Jun N-terminal kinase (JNK) pathways are

functionally connected. The JNK cascade is impor-

tant in regulating cell death decisions and is strong-

ly activated in TNF-mediated apoptosis via TRAF2

and RIP [75, 78]. The effects of JNK on TNF-induced

apoptosis have been enigmatic, and numerous stud-

ies have suggested pro- or anti-apoptotic functions

[21, 67, 74, 126]. Functional interconnection be-

tween NF- κB and JNK was revealed by two studies,

showing that TNF-induced activation of JNK is pro-

longed in cells that are deficient in NF- κB activation

(p65 and Ikk2 knockout MEFs and cells stably ex-

pressing degradation-resistant I κBα) and prolonged

JNK activation in these cells promoted apoptosis

[21, 126]. Only ectopic expression of XIAP, but not

other anti-apoptotic molecules, could inhibit pro-

longed JNK activation in TNF-treated p65 knockout

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MEFs [126]. This interaction was further confirmed in primary rat hepatocytes, where inhibition of NF- κB by an IκB super-repressor led to prolonged JNK activation. Inhibition of JNK by a chemical inhibi- tor could inhibit TNF-induced, but not Fas-induced apoptosis in these cells [112], underlining that one important mechanism by which NF- κB mediates its anti-apoptotic function in hepatocytes is suppres- sion of JNK activity.

The way this inhibition is exerted is unclear.

GADD45 β (growth arrest and DNA damage-in- duced protein β) is one possible mediator of this in- teraction, but its involvement seems to be restricted to specific cell types [92, 97]. Other possible candi- dates for mediation of NF- κB effects on JNK activity are reactive oxygen species (ROS). Two studies have provided evidence for the fact that in hepatocytes, prolonged JNK activation induced by TNF is de- pendent on the production of ROS and is negatively regulated by NF- κB, which blocks ROS accumula- tion [12, 79]. The NF- κB-dependent genes that in- hibit ROS accumulation still have to be defined.

29.5 The Role of NF- κB

in Hepatic Ischemia-Reperfusion Injury

Despite these linear, death receptor-dependent pathways that can be well examined in experimen- tal models such as TNF or FasL stimulation in mice,

experimental hepatic I/R injury in rodents repre- sents a complex model that reflects liver damage after organ transplantation, tissue resections or he- morrhagic shock and whose molecular mechanisms are poorly understood. In most studies, the injury detected after temporal clamping of hepatic blood flow and the subsequent reperfusion leads to an ex- cessive inflammatory response followed by necrotic cell death [53]. Instead of linear apoptotic signal- ing, it has been proposed that I/R injury consists of two different phases, one displaying acute cellular injury and a secondary, subacute phase resulting from inflammatory responses [52, 54, 55]. Genetic experiments identified TNF but not Fas as a criti- cal component in the mediation of I/R injury to the liver [104]. The role of NF- κB in this model is not clear, but recent studies have shown that NF- κB is activated after I/R in the liver and might withhold a specific function in this context.

NF- κB DNA binding occurs quickly upon he- patic I/R [141], but it has been unclear how NF- κB is activated in this model. It has been suggested that this process occurs alternatively to that during death receptor-dependent signaling. Whereas TNF stimulation leads to IKK-dependent phosphoryla- tion of both I κBα and IκBβ on serine residues, re- cent studies have suggested that during I/R injury, NF- κB gets activated by c-Src-dependent tyrosine phosphorylation of I κBα but not IκBβ on Tyr42, and this process takes place in the absence of I κBα ubiquitin-dependent degradation [30, 31], thus rep- resenting an alternative NF- κB activation pathway

Fig. 29.3. Induction of NF-κB target genes and mediation of their anti-apoptotic function by interaction with the TNF pathway.

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to the ones listed in Sect. 29.3. Data from our own laboratory suggest that this process is still depend- ent on IKK2, since conditional knockout mice for IKK2 show a defect in NF- κB activation after I/R.

It has also been unclear whether NF- κB-dependent signaling withholds a protective or damaging role in I/R injury. Since NF- κB withholds both proinflam- matory and anti-apoptotic properties in the liver [32], inhibition of its function after I/R might turn the fate of hepatocytes in both directions. In fact, in- hibition of NF- κB activation protects from liver in- jury due to I/R [31], thus underlining the complexity and organ-specific differences in NF- κB function.

29.6 NF- κB in Liver Regeneration

In mammals, the liver stands out against other or- gans by its capacity to regenerate lost parenchymal mass in response to surgical resection, viral injury or toxic exposure, for example [85]. Therefore, it has been a major challenge to evaluate the extra- and intracellular events that orchestrate this highly complex process that drives quiescent, fully differ- entiated hepatocytes through distinct stages includ- ing priming of hepatocytes, cell cycle progression, proliferation and cessation of regeneration [33]. A well-established model for studying liver regenera- tion is that of partial (two thirds) hepatectomy (PH) in rodents.

NF- κB was first identified in the liver as a fac- tor that is rapidly activated within 30 min after PH [20]. The importance of NF- κB and TNF signaling was further confirmed by the fact that liver re- generation is defective in TNFR-1 knockout mice, which do not show hepatic NF- κB activation after PH [134]. The question remained whether NF- κB could directly promote hepatocyte proliferation in this model. It has been shown that NF- κB can di- rectly stimulate the transcription of genes that en- code G1 cyclins, and a κB site is present within the cyclin D1 promoter [46, 50]. However, recent results questioned a direct involvement in hepatocytes and highlighted its role in non-parenchymal cells (NPC) like Kupffer cells. Chaisson et al. used transgenic mice that expressed the non-degradable I κBα su- per-repressor specifically in hepatocytes and these mice showed normal hepatocyte proliferation after PH [11]. Moreover, mice lacking the IKK β subunit in hepatocytes showed normal liver regeneration [79]. TNF-induced NF- κB activation in NPC leads to the production of cytokines such as IL-6. IL-6 is not absolutely necessary for liver regeneration, but has a protective, anti-apoptotic function in liver re-

generation [132], underlining the fact that instead of a direct proliferative effect, TNF-dependent NF- κB activation in NPC is part of an IL-6 production pathway that protects hepatocytes from apoptosis during liver regeneration.

29.7 NF- κB in Viral Hepatitis and Cancer

Many viruses, such as Epstein-Barr virus (EBV) [48], influenza virus [95] or human immunodefi- ciency virus type 1 (HIV-1) [38] have regulatory pro- teins that activate NF- κB. For some viruses, activa- tion of NF- κB prevents host cell apoptosis, whereas for other viruses this process is associated with the induction of apoptosis [16, 48]. The hepatitis B virus (HBV) remains the leading cause of hepatic failure worldwide, followed by hepatitis C virus (HCV) in- fection [101]. Both HBV and HCV modulate the NF- κB pathway [15, 77, 117, 123]. Activation of NF-κB by HBV has been shown to be mediated by an inter- action of the viral HBx protein with I κBα and the precursor/inhibitor subunit p105 [15, 120]. In this context, NF- κB suppresses HBx-mediated apopto- sis [121]. Regarding HCV-mediated NF- κB modula- tion, studies have shown conflicting results. While Shrivastava et al. showed that stable cell transfect- ants expressing the HCV core protein suppressed TNF-induced NF- κB activation [117], Tai et al. pro- vided evidence that HCV infection may cause anti- apoptosis by activation of NF- κB, which may lead to viral persistence and possibly hepatocarcinogenesis [123].

According to Hanahan and Weinberg, tumori- genesis requires six essential alterations to normal cell physiology: self-sufficiency in growth signals;

insensitivity to growth inhibition; evasion of apop-

tosis; immortalization; sustained angiogenesis; and

tissue invasion and metastasis [47]. As outlined in

this chapter, NF- κB is able to induce several of these

cellular alterations. NF- κB can also attenuate the

apoptotic response to genotoxic anticancer drugs

and ionizing radiation. Tumor cells in which NF- κB

is constitutively active are highly resistant to anti-

cancer drugs or ionizing radiation, and inhibition of

NF- κB in these cells greatly increases their sensitivi-

ty to such treatments [130]. In addition to conferring

resistance to cancer therapies, the anti-apoptotic

activity of NF- κB can also have an important role

in the emergence of neoplasms, by preventing the

death of cells that have undergone chromosomal re-

arrangements or other types of DNA damage. More-

over, at least one NF- κB-regulated chemokine, IL-8,

has been shown to promote angiogenesis [65] and

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NF- κB activation has been reported to contribute to extracellular matrix destruction by cancer cells [9].

A recent study demonstrated constitutive activation of NF- κB in hepatocellular carcinoma (HCC) [122].

Therefore, further experiments, e.g., studies in knockout animals for the relevant NF- κB molecules in the liver, are needed to evaluate the pathogenic relevance of NF- κB in HCC development.

29.8 NF- κB as a Therapeutic Target

As outlined above, a growing number of studies have implicated NF- κB-dependent pathways in the development of liver failure, chronic liver disease, viral hepatitis, hepatic inflammation and liver car- cinogenesis. Numerous chemical compounds and viral vectors that inhibit NF- κB have been devel- oped or are under development [58]. Nevertheless, because of the ubiquitous presence of NF- κB in vir- tually all cells and its involvement in many differ- ent cellular pathways and functions, application of these drugs to inhibit NF- κB non-specifically in a certain context might comprise the danger of un- wanted side-effects. With the help of new powerful tools such as constitutive or conditional knockout mice, differential functions for components of the NF- κB pathway could be revealed. These experi- ments have especially pointed towards the subunits of the IKK complex as therapeutic targets for devel- opment of anti-inflammatory and anticancer agents [60]. As most liver diseases such as liver failure or HCC still have very limited causative treatment op- tions at present, more basic and clinical studies are needed that might lead to the establishment of a new generation of molecular drugs almost 20 years after the discovery of NF- κB.

Selected Reading

Karin M, Lin A. NF-kappaB at the crossroads of life and death.

Nat Immunol 2002;3:221–227. (A hallmark article about the regulation of NF- B.)

Pahl HL. Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 1999;18:6853–6866. (In this article numerous NF- B target genes are compiled.)

Jaeschke H. Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning. Am J Physiol Gastrointest Liver Physiol 2003;284:G15–G26. (This article gives a com- pact summary about the complex molecular mechanisms during hepatic I/R injury.)

Karin M, Yamamoto Y, Wang QM. The IKK NF-kappa B system:

a treasure trove for drug development. Nat Rev Drug Disc 2004;3:17–26. (In this article, recent advances in the development of pharmacological strategies influencing the NF- B pathway are summarized.)

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