Nuclear factor-κB and the hepatic inflammation-fibrosis-cancer axis


  • Ahmed M. Elsharkawy,

    1. Liver Group, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom
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  • Derek A. Mann

    Corresponding author
    1. Liver Group, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom
    • Liver Group, Institute of Cellular Medicine, 4th Floor, Cookson Building, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom
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    • fax: (44) 191 2225455.

  • Potential conflict of interest: Nothing to report.


Nuclear factor-κB (NF-κB) is a transcriptional regulator of genes involved in immunity, inflammatory response, cell fate, and function. Recent attention has focused on the pathophysiological role of NF-κB in the diseased liver. In vivo studies using rodent models of liver disease and cell-targeted perturbation of NF-κB activity have revealed complex and multicellular functions in hepatic inflammation, fibrosis, and the development of hepatocellular carcinoma—a process we have termed the “inflammation-fibrosis-cancer axis”. This review summarizes the current state of knowledge and provides insight into the vast complexity of the hepatic NF-κB signaling system, which should provide a rich source of new therapeutic targets. (HEPATOLOGY 2007;46:590–597.)

The Inflammation-Fibrosis-Cancer Axis

The incidence of hepatocellular carcinoma (HCC) is rising in North America, Europe, and Eastern countries such as China and Japan.1 This increase is largely due to the emergence of hepatitis C virus (HCV), the continued problem of hepatitis B virus (HBV) infection control, and the liver pathologies associated with obesity and chronic alcohol abuse. The increasing levels of obesity in these countries is a particularly significant epidemiological factor that will ensure further worldwide rises in HCC incidence over the next decade as cases of HCC with a natural history of obesity-associated nonalcoholic fatty liver disease continue to increase.2 There is therefore an urgent need to understand how HCC develops in the diseased liver. In this respect, it is often overlooked that 90% of HCC cases have a natural history of unresolved inflammation and severe fibrosis (or cirrhosis) irrespective of the underlying cause of liver disease, though some liver diseases are more closely associated with HCC than others. Approaches to HCC prevention should therefore focus on the molecular regulators of a disease process we have termed the inflammation-fibrosis-cancer (IFC) axis (Fig. 1).

Figure 1.

Illustration of the central role of NF-κB in the IFC axis. p65/p50 heterodimers have been used for illustrative purposes only. Injury-induced apoptosis and necrosis of hepatocytes activates Kupffer cells with subsequent activation of NF-κB. This induces transcription of proinflammatory cytokines, the most important being TNF-α and IL-6. These in turn act on their cognate receptors on both hepatocytes and HSCs. In the former, NF-κB activation results in enhanced cell survival and proliferation, although it also seems to play a role in tumor suppression despite this. In HSCs, TNF-α and IL-6 induce differentiation and activation of the cell again through activation of NF-κB. This leads directly and indirectly to the secretion of profibrogenic and pro-proliferative factors, which result in fibrogenesis and in HCC in the long term.


ECM, extracellular matrix; HM, hepatic myofibroblast; HSC, hepatic stellate cell; IFC, inflammation–fibrosis–cancer; IκB, inhibitor of κB; NEMO, nuclear factor-κB essential modifier; NF-κB, nuclear factor-κB; TLR, Toll-like receptor; TNF-α; tumor necrosis factor-α.

A series of recent high-quality in vivo studies strongly implicate the transcription factor nuclear factor-κB (NF-κB) as a potential master orchestrator of the IFC axis. With seemingly multiple key roles to play at almost every step of the disease process, there is a case to be argued for inhibitors of NF-κB to be applied in the treatment of chronic liver disease. However, before clinical application of 1 or more of the new molecular inhibitors of NF-κB in pharmaceutical development occurs, we must first achieve a careful dissection of the functions of NF-κB in injured/infected hepatocytes, in the immune cells that drive inflammation, and in the fibrogenic cells that promote tissue remodeling and deposition of a collagen-rich extracellular matrix (ECM), which provides an ideal microenvironment for HCC. Consideration must also be given to the complexity of the NF-κB signaling system, the ability of NF-κB to regulate the interplay between immune, fibrogenic, and oncogenic mediators and the potential for this complexity to bear fruit in the form of highly specific therapeutics.

NF-κB—A Highly Complex but Essential Regulator of Inflammation.

NF-κB is a family of dimeric transcription factors that regulate inflammation, innate and adaptive immunity, wound healing responses, and cell fate and function. NF-κB achieves these physiological roles by binding to κB sequences found in the regulatory regions of more than 200 target genes. Mammalian NF-κB dimers are generated via homodimeric and heterodimeric interactions of 5 Rel subunits expressed from distinct but related genes: NF-κB1 (encoding p50 and its precursor p105), NF-κB2 (encoding p52 and its precursor p100), relA (encoding p65), relB (encoding RelB), and c-rel (encoding c-Rel). Crucially each subunit is known to have distinct biological activities that influence the function of any one NF-κB dimer. For example, although p65:p50 is generally an activator of gene transcription, the absence of a transactivation domain in p50 means that p50:p50 dimers lack the ability to activate transcription and unless partnered by coactivators function as transcriptional repressors.

In most resting cells, NF-κB dimers are sequestered in the cytoplasm by inhibitor of κB (IκB) proteins, of which there are at least 6 (IκBα, IκBβ, IκBϵ, Bcl3, and the c-terminus domains of p105 and p100 NF-κB subunits). Activation of NF-κB can occur via at least 2 signal transduction routes known as canonical and noncanonical pathways.

The canonical pathway is activated in response to a wide variety of stimuli, including proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and IL-1 as well as bacterial and viral antigens that act on Toll-like receptors (TLRs). This activation eventually leads to the nuclear accumulation of p65:p50. Prior to stimulation, p65:p50 is held in an inactive complex by IκBα, which masks the nuclear localization and DNA binding domains of the NF-κB dimer. Upon receptor stimulation, IκBα is rapidly phosphorylated at serines 32 and 36. This triggers ubiquitination and 26S proteasome–mediated degradation of the inhibitor enabling nuclear transport of free p65:p50. The pivotal signal transduction event in the canonical pathway is therefore phosphorylation of IκBα, and this is catalyzed by the IKK complex. This complex comprises 2 catalytic subunits, IKKα and IKKβ (also known as IKK1 and IKK2), and a regulatory subunit known as the NF-κB essential modifier (NEMO or IKKγ). IKKβ appears to play the predominant role in the canonical pathway and as such is a strong target for the development of anti-inflammatory drugs.

In contrast, IKKα is the predominant catalytic component of the noncanonical pathway, which is triggered by a more restricted subset of stimuli (for example, CD40 and lymphotoxin β) and requires phosphorylation-induced processing of p100 to generate p52 and subsequent nuclear accumulation of p52:RelB heterodimers. Importantly, p65:p50 and p52:RelB have differential binding affinities for distinct NF-κB DNA binding motifs, which means that they have the ability to differentially regulate subsets of NF-κB target genes. This complexity and diversity within the NF-κB signaling system ensures precise orchestration of target gene expression and enables both cell-specific and stimuli-specific variation in rates of gene transcription.

Further complexity is still provided by differences in the affinity of distinct NF-κB dimers and their associated coregulators for variant κB DNA binding sequences, with reports of variations in a single nucleotide altering binding specificities.3 The NF-κB subunits are also subject to additional regulatory checkpoints, including posttranslational modifications such as phosphorylation, acetylation, and ubiquitination that modulate their transcriptional activity, cellular localization, and stability (for more detailed reviews, see Perkins4 and references therein).

The known physiological roles for NF-κB are expanding as a result of studies investigating a wide and diverse range of developmental, behavioral, and disease phenotypes in mice that have the different Rel subunits, inhibitors of NF-κB, or upstream IKK components knocked out. One theme that has clearly emerged is the cardinal regulatory role played by NF-κB in inflammation and the ability of NF-κB and the IKK complex to control both the initiation and resolution of inflammation. IKKβ-mediated activation of the canonical NF-κB pathway leading to accumulation of nuclear p65:p50 is a requirement for initiation of inflammation and stimulates expression of proinflammatory cytokines and chemokines.5 Many anti-inflammatory drugs function as inhibitors of p65:p50 activation or function.

However, recent studies suggest that components of the IKK/NF-κB system also regulate resolution of inflammation (Fig. 2). For example, IKKα stimulates the turnover of p65 and c-Rel, limiting their residency at proinflammatory gene promoters, and appears to be critical for resolving macrophage activation.6 A further regulatory checkpoint involves the exchange of p50:p50 with p65:p50 dimers at NF-κB DNA binding sites, with p50:p50 promoting the repression of proinflammatory genes and the activation of anti-inflammatory IL-10.7 Notably, the absence of p50:p50 in nfkb1 knockout mice is associated with increased susceptibility to hepatic inflammation and fibrosis in a model of chronic liver disease.8

Figure 2.

Role of the NF-κB system in the resolution of inflammation. During the acute inflammatory state, activation of NF-κB mainly involves p50/p65 heterodimers, which activate transcription through the recruitment of coactivators such as CBP/p300. During the resolution phase of inflammation p65 is degraded and removed from the nucleus in part by phosphorylation by IKKα followed by proteosome-mediated proteolysis. In addition, p50 homodimers mediate the transcription (with the aid of coactivators) of anti-inflammatory cytokines such as IL-10. p50 homodimers also displace p50/p65 heterodimers on the promoters of proinflammatory genes, and by recruiting in corepressors such as HDAC-1 lead to repression of transcription. These concerted events ensure that inflammation does not persist.

Coordinated NF-κB Activation in Parenchymal and Nonparenchymal Cells of the Injured Liver.

Inflammation is believed to be the underlying pathology in 15%-20% of human cancers, including HCC. The majority of liver injuries (alcohol, metabolic, immune-driven, and virus-induced) activate acute inflammatory and wound-healing responses. When the injury is iterative or chronic, liver tissue is exposed to repeated and overlapping waves of inflammation associated with persistently elevated expression of proinflammatory cytokines, chemokines, and matrix metalloproteinases. Unresolved or chronic hepatitis then leads to fibrosis and a precancerous state in which the development of HCC is more likely—though some investigators believe that this is merely a surrogate marker for higher levels of proliferation.

Studies in experimental models have shown that liver injury is associated with activation of NF-κB with the response being significantly influenced by sex (increased in females), age (reduced in older animals), and fat content (increased in mice fed a high-fat diet) of the liver. Studies on hepatic NF-κB in diseased human liver are rare. However, at least 1 report has revealed remarkably elevated hepatic NF-κB activity in alcoholic liver disease, which correlates with the degree of inflammation and fibrosis.9

Injury-induced activation of hepatic NF-κB is observed in a variety of nonparenchymal and parenchymal liver cells, indicating that NF-κB plays a central role in coordinating the inflammatory and wound healing response by stimulating gene transcription in multiple key cellular players. Kupffer cells (the liver's resident macrophages) display powerful NF-κB activation in response to liver injury by alcohol, carbon tetrachloride, endotoxin, and cholestasis, resulting in production and secretion of proinflammatory cytokines (including the hepatomitogens TNF-α and IL-6) that are strongly implicated as promoters of fibrosis and HCC.10In vivo administration of alcohol or lipopolysaccharide induces NF-κB in sinusoidal endothelial cells, concurrent with the induction of inducible NO synthase and cycloxygenase-2 expression.11 NF-κB activation is associated with the transdifferentiation of hepatic stellate cells (HSCs) into scar-forming hepatic myofibroblasts, an event that is considered pivotal in the fibrogenic response.12 NF-κB is activated in HSCs in response to carbon tetrachloride injury and stimulates expression of proinflammatory molecules (IL-6, monocyte chemoattractant protein-1, and intercellular adhesion molecule-1) and antiapoptotic factors (growth arrest and DNA damage-inducible gene 45β) required for HSC function and survival during the fibrogenic response.13

The induction of hepatocyte NF-κB during liver injury has been reported by many laboratories in response to alcohol, endotoxin, TNF-α, and cholestasis (bile duct ligation).11, 14, 15 Activation of hepatocyte NF-κB is generally perceived as a protective response that limits apoptotic loss of the parenchyma and promotes regeneration of hepatocyte mass by stimulating hepatocyte proliferation. However, persistently elevated levels of NF-κB in hepatocytes contribute to a chronic inflammatory state and have also recently been linked to the development of hepatic insulin resistance. Mice maintained on a high-fat diet expressed elevated hepatic NF-κB activity and NF-κB target gene expression (IL-6, IL-1β, and TNF-α) contributing to a low-grade inflammatory condition in the liver. This condition was experimentally mimicked by targeting expression of a constitutively active IKKβ in hepatocytes of transgenic mice; the animals also developed insulin resistance.16 Arkan et al.17 demonstrated that hepatocyte-targeted depletion of IKKβ blunted age-induced reduction in glucose tolerance and protected mice from high-fat diet–induced insulin resistance, though this effect was restricted to the liver (no attenuation of insulin signaling in adipose or muscle tissues was observed). It can therefore be concluded that persistent activation of hepatocyte NF-κB, although able to protect against iterative injury, may also contribute to the development of metabolic disease—something that in itself is associated with an increased risk of hepatic fibrosis and HCC.

Mechanisms of Persistent NF-κB Activation in Liver Disease

Although it is clear that any agent that induces hepatocyte death sets up an inflammatory state in the liver that is associated with NF-κB activation (Fig. 1), the precise molecular players responsible for this have only just begun to be elucidated. One family of receptors that has attracted much attention is the TLRs. There at least 10 mammalian TLRs (1-10) in addition to the IL-1 receptor.18 These receptors function by binding to various bacterial, viral, and other pathogenic components that are collectively known as pathogen-associated molecular patterns. Different pathogen-associated molecular patterns have different affinities for different TLRs. For example, lipopolysaccharide binds and activates TLR4. Upon binding, all TLRs (except for TLR3) recruit the adaptor protein Myd88 and this is crucial for the activation of NF-κB in response to pathogen-associated molecular patterns. Myd88 recruits, amongst other proteins, the activator TRAF6 which via TAK1 activates the IKK complex to induce nuclear translocation of NF-κB as described above.19 In the normal liver expression of TLRs is widespread although most of them are expressed at only low levels.18 Following liver injury, however, there are significant increases in the expression of TLRs on most liver cells and in particular Kupffer cells. This phenomenon along with the known increase in gut permeability in chronic liver disease with subsequent increases in the exposure of the liver to pathogen-associated molecular patterns may help explain the persistent activation of the TLR-Myd88-TRAF6-NF-κB system seen in many hepatic inflammatory conditions.

Oxidative stress is a key mechanism in the hepatocyte damage induced by various metabolic agents that injure the liver.20 Reactive oxygen species are believed to play a role in the activation of NF-κB, though their precise effects may be tissue- and site-specific. Low levels of reactive oxygen species may activate NF-κB partly by activating the IKK complex in the cytoplasm.21 However, high reactive oxygen species levels may inhibit NF-κB activity partly by interfering with DNA binding.22 It is unclear which of these activities predominates in the pathogenesis of liver disease, but the precise role of reactive oxygen species in NF-κB activation in liver disease is an important area that requires further study.23

NF-κB as a Tumor Promoter in Inflammatory Liver Disease.

Experimental evidence from IKK/NF-κB knockout mice indicates that the proinflammatory milieu of the injured liver generates microenvironments in which the development of HCC is favored. Maeda et al.24 showed that induced knockout of Ikkβ in liver and hematopoietic cells substantially reduced tumor development stimulated by diethylnitrosamine. This phenotype was associated with markedly reduced hepatic expression of the macrophage/Kupffer cell–secreted hepatomitogens TNF-α and IL-6, which are normally elevated in diethylnitrosamine-treated mice. This landmark study established that 1 arm of the IFC axis under the control of NF-κB is the production of hepatomitogenic factors by inflammatory cells that would serve to stimulate tumor cell growth. Pikarsky et al.25 also reported the induction of tumor-promoting NF-κB activities in hepatocytes adjacent to regions of inflammation in the livers of mdr2 knockout mice, which spontaneously developed inflammation-driven HCC. On the basis of these studies, IKK/NF-κB inhibitors may be clinically beneficial for the prevention/treatment of HCC. However, this is a simplistic assumption, because NF-κB has a multitude of functions in the physiology of the diseased liver and may operate as both a tumor promoter and a tumor suppressor.

NF-κB as a Tumor Suppressor in Hepatocytes.

Cancers develop as a consequence of an imbalance in tissue homeostasis such that the ratio of cell proliferation to death is increased.26 NF-κB has been reported in numerous studies to operate as a survival factor for hepatocytes. One of the first observations made was that genetic deletion of p65 resulted in embryonic death due to TNF-α–induced hepatocyte apoptosis.27 This phenotype was reproduced in mice with constitutive global deletion of ikkβ or NEMO, confirming that the IKK/NF-κB system plays a critical role in hepatocyte survival, at least during embryogenesis.28 At the same time, data from in vitro studies with human HCC cell lines consistently demonstrate that NF-κB is cytoprotective via its ability to prevent apoptosis. Furthermore, the oncogenic HBV protein HBx and the HCV core and NS5B proteins were reported to induce NF-κB.29–31 The natural conclusion from these observations was that NF-κB operates as a tumor promoter in virus-infected or transformed hepatocytes.

A limited number of descriptive studies in human HCC tissues support this conclusion, with reports of elevated nuclear NF-κB in tumor tissue and in association with virus-infected hepatocytes. It would therefore follow that inhibition of IKK/NF-κB activity should promote apoptosis of infected and transformed hepatocytes and, as suggested above, be of clinical benefit in HCC. However, this scenario must now be reconsidered in the light of more recent work from laboratories employing Cre-lox technology to deliver regulated or cell-targeted genetic manipulation of the IKK/NF-κB system.

The first surprise was that adult transgenic mice expressing an inducible hepatocyte-specific NF-κB inhibitor (ΔN-IκBα) had normal liver function—and although susceptible to hepatocyte apoptosis induced by very high doses of TNF-α, did not display elevated rates of apoptosis in response to partial hepatectomy, which is associated with more physiological levels of TNF-α.14, 32 Similarly, 2 independent laboratories confirmed that mice with hepatocyte-targeted deletion of ikkβ (ikkβhep) develop with normal liver structure and function and do not display elevated susceptibility to hepatocyte apoptosis induced by soluble TNF-α or lipopolysaccharide.33, 34 It is concluded that the canonical IKKβ driven NF-κB pathway is not essential for hepatocyte survival in the normal liver or upon challenge from physiological doses of endotoxin or TNF-α.

However, the canonical activation pathway may regulate hepatocyte survival under other injury conditions where a higher threshold level of NF-κB is required to counteract powerful proapoptotic signals. For example, both ΔN-IκBα and ikkβhep livers were more susceptible to concanavalin-A–induced hepatocyte apoptosis.32, 33 This may reflect the ability of concanavalin-A to trigger activation of both TNFR1 (the receptor for soluble TNF-α) and TNFR2 (the receptor for membrane bound TNF-α), which results in robust and prolonged activation of proapoptotic Jun N-terminal kinase.33 Because concanavalin-A provides a model for T cell–mediated hepatocyte death, it is possible that IKKβ–driven activation of NF-κB functions as an important hepatocyte survival signal in alcoholic and viral liver disease. However, Luedde et al.34 did not observe enhanced susceptibility to concanavalin-A–induced apoptosis in their hepatocyte-targeted ikkβ knockout mice. Two studies published very recently in Gastroenterology provide some answers to this conundrum. Using mice with hepatocyte specific deletions of nemo and p65 respectively, Beraza et al. and Geisler et al. have shown that total inhibition of hepatocyte NF-κB transcriptional activation in response to injury (as opposed to the partial inhibition seen in some ikkβ knockouts) is indeed associated with enhanced hepatocyte apoptosis in response to TNFα.35, 36

Perhaps the most unexpected discovery was that loss of NF-κB activity in hepatocytes might actually promote tumor development. This possibility was demonstrated very recently by the Pasparakis group who again employed Cre-lox technology to target knockout of the NEMO gene to parenchymal cells which in the liver was restricted to hepatocytes and cholangiocytes (NEMOLPC-KO).37 The NEMOLPC-KO mouse is viable and has a functional liver; however, in contrast to ΔN-IκBα or ikkβhep mice, it develops massive and fatal hepatocyte apoptosis in response to TNF/lipopolysaccharide challenge. Inspection of the livers of adult NEMOLPC-KO mice (9 to 12 months) revealed numerous large hepatocellular carcinomas that had developed spontaneously. Intriguingly, HCC in the NEMOLPC-KO mouse was preceded by liver pathologies resembling nonalcoholic steatohepatitis and fibrosis. Why hepatocyte knockout of NEMO should generate this phenotype when it is not observed in ikkβhep mice (and also not in IKKβLPC-KO mice in the Pasparakis study) is unclear, but it may relate to the observation that NEMO-deficient hepatocytes are highly sensitive to TNF-α–induced hepatocyte apoptosis and the fact that the animals were not maintained in pathogen-free conditions. Indeed, crossing of NEMOLPC with a mouse that lacks parenchymal cell FADD (a crucial proapoptotic signaling component of the TNF-α death receptor pathway) prevented the nonalcoholic steatohepatitis phenotype, although the effect of this on the development of tumors is unknown (Manolis Pasparakis, personal communication).

A further key finding in the NEMOLPC mouse was an increased rate of hepatocyte apoptosis that led to highly elevated rates of hepatocyte proliferation. This rebound effect that appears to drive tumor growth was previously reported by Maeda and colleagues,24, 25 who observed compensatory HCC proliferation in ikkβhep mice subjected to diethylnitrosamine injury. Loss of the canonical IKKβ/NF-κB pathway in hepatocytes therefore appears to render the liver more susceptible to HCC in the context of an underlying chronic inflammatory disease, most likely because of an imbalance in normal hepatocyte homeostasis. The spontaneous development of HCC in NEMOLPC may reflect a more profound loss of NF-κB in hepatocytes than that seen in ikkβhep mice (note that IKKα may in part compensate for a lack of IKKβ34). In summary, therapeutic strategies that lead to inhibition of NEMO, IKKβ, or NF-κB in hepatocytes may do more harm than good, and as such, a more fruitful approach may be to target the tumor-promoting activities of NF-κB in the nonparenchymal cellular players of the IFC axis.

NF-κB and Fibrosis/Cirrhosis.

The fact that the vast majority of HCC cases have a natural history of established fibrosis or cirrhosis still must be explained in mechanistic terms. An explanation favored by many is that the fibrotic, collagen-rich ECM provides survival and proliferative stimuli for the developing tumor cells. These stimuli may include ECM–integrin interactions that stimulate gene transcription via focal adhesion kinase, ECM-bound Wnt signaling leading to β-catenin–driven transcription, and elevated hepatomitogen concentrations in the local microenvironment arising from ECM providing a sink for growth factors.

However, what has been overlooked is the potential for ECM-producing fibrogenic hepatic myofibroblasts (HMs) to play a more direct role in HCC development. HMs are rare in the normal liver but become a major cellular population of the diseased liver, where their most well-characterized function is to promote the net deposition of ECM, mainly in the form of fibrillar collagen. HMs are generated locally in response to hepatocyte death and Kupffer cell activation, which stimulate the transdifferentiation of HSCs and periportal fibroblasts toward a smooth muscle myofibroblast-like phenotype. HMs may also be recruited to injured tissue from bone marrow–derived stem cells.38 Irrespective of their cellular origin, and despite a high degree of plasticity that complicates their phenotypic characterization, HMs display many properties compatible with a tumor-promoting cell. HMs are highly proinflammatory and secrete a plethora of cytokines and growth factors, including hepatomitogens directly implicated in HCC growth, such as IL-6 and hepatocyte growth factor.39–41 HMs also express and secrete a wide number of matrix metalloproteinases, including MMP9, which stimulates the growth and spread of transformed hepatocytes.42 A recent paper in Science highlighted the importance of activated HMs in hepatocyte proliferation.43

The persistence and inflammatory/mitogenic phenotype of HMs inclusive of their expression of IL-6 and MMP9 depends on a high constitutive NF-κB activity that is regulated at least in part by transcriptional repression of IκB-α (mediated by CBF-1 and MeCP2) but also by IKK-dependent mechanisms under investigation in our laboratory.44, 45 Suppression of IKK activity in HMs with either a cell-permeable peptide that blocks the interaction of IKKα/β with NEMO or by treatment with sulfasalazine (which inhibits IKKα/β) promotes Jun N-terminal kinase–dependent apoptosis.46In vivo studies have demonstrated the ability of NF-κB inhibitors such as gliotoxin, sulfasalazine, and thalidomide to promote HM apoptosis and stimulate resolution of fibrosis and regeneration of normal liver tissue.13, 46, 47 Angiotensin blockade, an antifibrotic strategy currently in clinical trials, may also act by inhibiting NF-κB activity.48 It would now be of interest to determine the effects of selective and/or inducible knockout of IKK/NF-κB in HMs in the context of models of inflammation-driven HCC to establish if targeting the NF-κB activity of HMs will be of therapeutic benefit. However, several technical hurdles remain to be overcome before this type of study can be undertaken. Firstly, HM-specific promoters are yet to be defined and employed to direct Cre recombinase exclusively to this cellular compartment, although the potential for promoters from the collagen I, desmin, smooth muscle α-actin, or glial fibrillary acidic protein to be used for this purpose is worth exploring. Secondly, the models currently used for HCC in rodents do not accurately represent the human disease, because tumors generally develop in the absence of obvious fibrosis in these models. One answer to this latter problem may be to employ the NEMOLPC mouse, which develops established steatohepatitis and fibrosis before the appearance of HCC.37 Finally, the increasingly recognized roles that various forms of B and T cells play in liver fibrosis and the contribution of NF-κB activities to these is an area that requires investigation in the near future.

Translating Discoveries to Target NF-κB in the IFC Axis.

NF-κB is a driving force of inflammation and is a transcriptional regulator of Kupffer cell–derived hepatocyte mitogens. NF-κB also regulates the production of Kupffer-cell derived activating factors for HSC transdifferentiation to HM. The survival of HMs and their production of tumor-potentiating factors is under the control of NF-κB, as is the normal homeostasis of hepatocytes—which when perturbed increases the risk of progression to HCC. In conclusion, there is little doubt that NF-κB functions as an orchestrator of the IFC axis (Fig. 1). But how we target NF-κB long-term to provide a therapeutic solution to chronic liver disease remains a significant problem, and we are still not ready. Pan-inhibition of NF-κB over months and years would increase the risk of infections and may remove a tumor suppressor role for NF-κB in hepatocytes. Targeting NF-κB inhibitors to the inflammatory and fibrogenic cells of the liver may be a safer option. However, the technology for this type of therapeutic approach is not available; furthermore, there is the important caveat that most of what we know about NF-κB in chronic liver disease is based on experimental animal models, which unfortunately do not provide accurate representations of the pathology of the diseased human liver.

We must therefore focus future attention on confirming that the NF-κB activities and functions described in murine studies translate to how NF-κB operates in liver cells in the context of alcoholic, metabolic, viral, and immune-driven pathologies. The reagents to tackle these questions are now available in the form of specific antibodies that detect molecular markers of NF-κB such as serine phosphorylation events that are well-characterized regulatory checkpoints for nuclear localization, DNA binding, and transcriptional activity. These tools will allow us to determine the cellular location of specific NF-κB activities in diseased tissue and thus enable us to determine whether or not inhibiting NF-κB activity in Kupffer cells or HMs would be useful in human disease. In addition, coupling these reagents with modern pathology technologies such as laser-capture microscopy and DNA microarray may have the potential to provide new diagnostic modalities.

Another concept to consider is whether we really do have sufficient in-depth understanding of the complexities of the hepatic IKK/NF-κB system to be sure that we are ready to produce drugs that will be safe for long-term use. Much of the focus in the NF-κB field is currently placed on the upstream regulatory events—in particular the components of the IKK complex. As discussed previously, unless we can achieve cell targeting of IKK inhibitors, they are likely to cause significant side effects; moreover, it should not be overlooked that there is ever-increasing evidence for NF-κB–independent activities of IKKα, IKKβ, and NEMO, as well as the IκBs.49 In addition, the extensive cross-talk that exists between the NF-κB and other signaling pathways will greatly affect the design of any therapies, though a detailed discussion of this is outside the scope of this review.

An alternative approach would be to refocus attention on the downstream mediators of NF-κB activities, namely the 5 Rel subunits. The subtle differences in the biological and cell-specific functions of the subunits may provide opportunities to target the proinflammatory and tumor-promoting activities of NF-κB while retaining its tumor suppressor functions. For example, will 1 or more of the growing list of phosphorylation modifications of the Rel proteins offer a cell-specific drug target? Future availability of mice carrying cell-specific deletions or functional mutations in the rel genes will help answer this important question.

In conclusion, we have seen genuinely remarkable advances in our understanding of how NF-κB contributes to the pathology of chronic liver disease, and these advances have helped to define the IFC axis. The next challenge is to further refine this knowledge for translation into therapeutics.


We are grateful to various members of the Newcastle University Liver Group for critical reading of the manuscript. We also recognize many outstanding published contributions of investigators in the field that could not be included in this review due to space limitation.