Hepatic Tumor Necrosis Factor Signaling and Nuclear Factor- B ...

0163-769X/07/$20.00/0 Printed in U.S.A.

Endocrine Reviews 28(4):365–386 Copyright © 2007 by The Endocrine Society doi: 10.1210/er.2006-0031

Hepatic Tumor Necrosis Factor Signaling and Nuclear Factor-␬B: Effects on Liver Homeostasis and Beyond Andy Wullaert, Geert van Loo, Karen Heyninck, and Rudi Beyaert Unit of Molecular Signal Transduction in Inflammation, Department for Molecular Biomedical Research-VIB, and Department of Molecular Biology, Ghent University, Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium The proinflammatory cytokine TNF has a pivotal role in liver pathophysiology because it holds the capacity to induce both hepatocyte cell death and hepatocyte proliferation. This dual effect of TNF on hepatocytes reflects its ability to induce both nuclear factor ␬B (NF-␬B)-dependent gene expression and cell death. Multiple studies have demonstrated the crucial role of the transcription factor NF-␬B in the decision between life and death of a hepatocyte. Massive hepatocyte apoptosis preceding embryonic lethality in NF-␬B-deficient mice constituted the first indication of an essential antiapoptotic function of NF-␬B in the liver. Although many studies confirmed this crucial cytoprotective role of NF-␬B in adult liver, a number of genetic studies recently obtained conflicting results on the exact role of NF-␬B in different mouse models of TNF hepatotoxicity, demonstrating that caution should be taken

when interpreting studies using different NF-␬B-deficient mice in distinct models of liver injury. Recent reports showing a role for hepatic NF-␬B activation in the proliferation of malignant cells during hepatocarcinogenesis, and in the progression of fatty liver diseases to insulin resistance and type 2 diabetes mellitus demonstrate that NF-␬B can also have more detrimental effects in the liver. Moreover, its role in the development of the metabolic syndrome emphasizes that hepatic NF-␬B activation might also have adverse effects on the endocrine system. Therefore, understanding the regulation of hepatic TNF signaling and NF-␬B activation is of critical therapeutic importance. In this review, we summarize how studies on the role of NF-␬B in different mouse models of liver pathologies have contributed to this understanding. (Endocrine Reviews 28: 365–386, 2007)

I. Introduction II. Role of TNF in Liver Diseases III. TNF-Induced Signaling Pathways in Hepatocytes A. TNF signaling to the activation of NF-␬B and MAPKs B. TNF-induced signaling pathways leading to hepatocyte apoptosis IV. Crucial Role for NF-␬B in Regulating the Hepatocyte’s Response to TNF V. Role of NF-␬B Activation in Mouse Models of TNFMediated Hepatic Diseases A. Role of NF-␬B activation in mouse models of TNFmediated liver toxicity B. Role of NF-␬B activation in liver regeneration C. Role of NF-␬B activation in mouse models of hepatocarcinogenesis

D. Role of hepatic NF-␬B activation in the metabolic syndrome E. Role of NF-␬B-dependent inflammation in hereditary hemochromatosis VI. Mechanisms of the Antiapoptotic Effect of NF-␬B A. NF-␬B-dependent inhibition of caspase activation B. NF-␬B-dependent inhibition of JNK activation C. Mechanisms of crosstalk between TNF-induced activation of NF-␬B and JNK VII. Concluding Remarks I. Introduction

L

IVER CELL INJURY and cell death are prominent features in all liver disease processes. In healthy liver, constitutive production of cytokines is absent or very low. During liver inflammation, however, hepatocytes are exposed to increased levels of cytokines such as TNF, IL-1␤, and interferon ␥, as well as oxidative stress and bile acids. Excessive exposure to these agents will eventually cause hepatocyte cell death, which can contribute to acute liver injuries, such as fulminant hepatitis and ischemia/reperfusion (I/R) damage, or even chronic sustained injuries, such as alcoholic liver disease, cholestatic liver disease, and viral hepatitis (1, 2). Conversely, insufficient hepatocyte apoptosis, associated with failure to remove mutated cells or with unrestrained proliferation within the context of a chronic inflammatory milieu, can promote the development of liver cancer. Paradoxically, hepatic carcinogenesis can also arise from sustained hepatocyte cell death due to the high rate of compensatory regeneration invoked in the tissue, thus elevating the risk of mitotic errors (3). These liver pathologies resulting either from excessive hepatocyte cell death or from

First Published Online April 12, 2007 Abbreviations: ABIN, A20 binding inhibitor of NF-␬B; ASK1, apoptosis signal-regulating kinase 1; A-SMase, acidic sphingomyelinase; c-IAP, cellular inhibitor of apoptosis; Con A, concanavalin A; DEN, diethylnitrosamine; DISC, death-inducing signaling complex; FADD, Fas-associated death domain; GADD45␤, growth arrest DNA damageinducible gene 45␤; GalN, D-(⫹)-galactosamine; I␬B, inhibitor of ␬B; I␬B␣s, I␬B␣ superrepressor; IKK, I␬B kinase; I/R, ischemia/reperfusion; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MKK, MAPK kinase; MKP, MAPK phosphatase; MPT, mitochondrial permeability transition; NAFLD, nonalcoholic fatty liver disease; NF-␬B, nuclear factor-␬B; NKT cells, natural killer T cells; N-SMase, neutral sphingomyelinase; RIP, receptor interacting protein; ROS, reactive oxygen species; TAB, TAK1-binding protein; TAK1, TGF-␤ activated kinase 1; tBid, truncated Bid; TNF-R, TNF-receptor; TRADD, TNF-R-associated death domain; TRAF, TNF-R-associated factor; Trx, thioredoxin; XIAP, Xlinked inhibitor of apoptosis. Endocrine Reviews is published by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

365

366

Endocrine Reviews, June 2007, 28(4):365–386

a lack thereof emphasize the crucial role of the balance between hepatocyte survival and death in preserving liver homeostasis. The proinflammatory cytokine TNF is a key regulator of this vital balance because TNF can induce hepatocyte proliferation as well as hepatocyte cell death (Fig. 1) (2). Therefore, accurate knowledge of the TNF-induced signal transduction pathways implicated in the prevention as well as the execution of hepatocyte cell death is of major importance for understanding the development of liver diseases. Moreover, an improved understanding of the TNFinduced molecular pathways that determine the hepatocyte’s destiny, either survival or cell death, will undoubtedly contribute to the development of novel therapeutic strategies for preventing hepatocyte cell death in liver injury, or selectively killing malignant cells in liver tumors. In this review, we will focus on the intracellular signal transduction pathways that mediate the opposing effects of TNF in hepatocytes. We will discuss how the balance between these TNFinduced signaling pathways influences liver homeostasis. In addition, we will summarize how disturbing this balance promotes the development of liver pathologies, as well as how this affects the endocrine system and, as such, contributes to the metabolic syndrome and hemochromatosis.

II. Role of TNF in Liver Diseases

TNF induction is known to be one of the earliest events in hepatic inflammation, triggering a cascade of other cytokines that cooperate in recruiting inflammatory cells, killing hepatocytes, and initiating a wound-healing response. As such, TNF is involved in the pathogenesis of various inflammatory liver diseases (2). For instance, it has been well established that serum and hepatic TNF levels are increased in patients with acute or chronic hepatitis B virus and hepatitis C virus infection (4 –9). Also, patients suffering from fulminant liver failure show significantly increased serum TNF levels (10). Moreover, elevated TNF levels in alcoholic hepatitis patients play a crucial role in mediating hepatocyte damage and correlate inversely with patient survival (11, 12). This detrimental role for TNF in alcoholic liver injury has also been shown in animal models. For instance, neutralizing antibodies to TNF efficiently suppress alcohol-induced liver damage, and consistently, mice deficient in TNF signaling show significantly reduced liver injury upon intragastric ethanol delivery (13, 14). In addition to these inflammatory liver diseases, also I/R of the liver leads to the production of TNF, which results in subsequent hepatocyte apoptosis (15). Although the above observations clearly demonstrate that TNF can contribute to hepatocyte cell death during liver diseases, the healthy liver has well-developed defense mechanisms that permit hepatocytes to adapt to TNF-initiated stress. Indeed, acute treatment with exogenous TNF will normally be well tolerated by the liver and will not lead to hepatocyte injury. In fact, systemic administration of TNF will induce healthy hepatocytes to proliferate rather than die (16). This proliferative response also occurs when the liver is confronted with partial hepatectomy, which acutely elicits hepatic production of TNF. Multiple studies have shown that this increased production of TNF is necessary for the rem-

Wullaert et al. • TNF Signaling in the Liver

FIG. 1. Role of TNF in liver homeostasis. TNF can induce both hepatocyte proliferation and hepatocyte cell death. Because excessive hepatocyte cell death contributes to liver injury and excessive hepatocyte proliferation leads to liver cancer, TNF has a crucial role in preserving liver homeostasis.

nant liver to regenerate, pointing to an essential role for TNF in the proliferation of hepatocytes after partial hepatectomy (17–19). These studies show that depending on the context, exposure of hepatocytes to TNF induces signals that mediate cell death, or alternatively, survival pathways that allow hepatocytes to tolerate tissue damage or even recover from it. Although at first sight paradoxical, the activation of both apoptotic and survival pathways in response to the same stimulus ensures that neither aberrant cellular survival nor excessive cell death arises and, in doing so, preserves proper liver homeostasis. Moreover, next to its role in deciding between life and death of a hepatocyte, TNF also affects the metabolic functions of the liver. Indeed, TNF can cause insulin resistance and affects glucose metabolism in hepatocytes (20). Also hemochromatosis depends, at least partially, on the inflammatory activity of TNF (21). These observations add to the impressive activity range of TNF in the liver and emphasize the need of studying the intracellular signaling pathways that regulate cellular responses to TNF, because only this will help us to understand how a single cytokine, such as TNF, can exert such pleiotropic effects in the liver. III. TNF-Induced Signaling Pathways in Hepatocytes

TNF exerts its biological effects by binding two plasma membrane receptors, TNF-receptor 1 (TNF-R1) and TNF-R2. The majority of the biological effects of TNF are mediated by TNF-R1 triggering, which can initiate signaling pathways leading to the activation of the transcription factor nuclear factor-␬B (NF-␬B), the initiation of MAPK cascades, as well as cell death. This diversity of signaling pathways initiated by TNF-R1 is accomplished by the recruitment of different adapter proteins to its cytoplasmic part. According to a recent model (Fig. 2), TNF signaling starts when TNF-R-associated death domain (TRADD), TNF-R associated factor (TRAF)2, and receptor interacting protein (RIP)1 are re-


Endocrine Reviews, June 2007, 28(4):365–386 367

FIG. 2. TNF-R1-induced signaling pathways via complex I and complex II. TNF binding to TNF-R1 leads to the recruitment of TRADD, TRAF2, and RIP1, forming complex I (1). TRAF2 catalyzes Lys-63-linked polyubiquitination of itself and RIP1, allowing RIP1 to recruit the TAK1 complex (2). This subsequently activates specific MKKs (3) as well as the IKK complex (4). Whereas MKKs activate the p38 and JNK MAPKs, the IKK␤ catalytic subunit of the IKK complex phosphorylates the NF-␬B-bound I␬Ba (5), leading to its Lys-48-linked polyubiquitination and subsequent proteasomal degradation (6). This allows NF-␬B to translocate to the nucleus (7), where it induces the transcription of genes with an NF-␬B consensus binding site in their promoter. Binding of FADD and procaspase-8 to the TRADD/TRAF2/RIP1 complex results in the formation of the cytosolic complex II, which will signal toward apoptosis (8). See text for further details. DD, Death domain; Ub, ubiquitin; P, phosphate.

368


cruited to TNF-R1 to form complex I, which signals to the activation of NF-␬B, as well as the MAPKs p38 and c-Jun N-terminal kinase (JNK). Once released from the receptor, the cytosolic complex II further recruits Fas-associated death domain (FADD) and pro-caspase-8, which will result in cell death (22). Alternatively, this cell death-inducing complex II has been suggested to result from internalization of the TNF-R1 receptosome (23). Whereas TNF-R1 is the main receptor for soluble TNF, TNF-R2 generally mediates the effects of the membranebound TNF precursor and seems mostly restricted to T cells, where it can directly induce specific gene expression and cell proliferation or apoptosis (24 –26). In nonlymphoid cells, TNF-R2-induced responses are mainly attributed to the indirect stimulation of TNF-R1 via ligand passing from TNF-R2 to TNF-R1 (27) or via the TNF-R2-induced secretion of endogenous membrane-bound TNF and autotropic or paratropic activation of TNF-R1 (28). Although the role of TNF-R2 in the liver has remained more elusive than the prominent role of TNF-R1 in this organ, TNF-R2 signaling was shown to be essential for the development of concanavalin A (ConA)-induced liver injury in mice. In addition, mice that are incapable of processing membrane-bound TNF to its soluble form were shown to retain full sensitivity to this model of inflammatory hepatitis (29). Taken together, these observations suggest that triggering of TNF-R2 by membrane-bound TNF is sufficient for initiating lethal ConAinduced hepatitis in mice. Therefore, the role of TNF-R2 in liver pathologies should not be neglected. Furthermore and in support of a pathogenic role of TNF-R2 in the liver, mice with a human Tnfr2 transgene develop inflammation in various organs including the liver (30). Despite these indications that TNF-R2 signaling might also contribute to the development of liver diseases, information on the general signaling mechanisms of TNF-R2 and its specific role in the liver has remained rather sparse. Therefore, this review will mainly focus on the effects of TNF-R1 signaling in liver pathophysiology, but we will draw attention to the potential role of TNF-R2 wherever possible. Before embarking on the complex regulation of interplay between the different TNF-induced intracellular signaling pathways, which will eventually determine the hepatocyte’s biological response to the TNF stimulus, we will first focus on some important mechanisms and molecules in each of the individual pathways. It should be stressed that our current knowledge of TNF signaling is still largely fragmentary and based on studies with different cell types and experimental set-ups. It can therefore be expected that the effect of specific adaptor proteins and kinases might sometimes be cell typespecific or depend on the TNF concentration that was used. A. TNF signaling to the activation of NF-␬B and MAPKs

NF-␬B is a transcription factor that is normally held in the cytoplasm by members of the inhibitor of ␬B (I␬B) protein family. The prototypical NF-␬B complex consists of a p65/ p50 heterodimer and is bound to I␬B␣. TNF-induced signaling to NF-␬B involves the activation of the I␬B kinase (IKK) complex, which consists of two catalytic subunits, IKK␣ (or IKK1) and IKK␤ (or IKK2), and a regulatory subunit, IKK␥


(or NEMO). Once activated, the IKK␤ subunit phosphorylates I␬B␣, leading to its ubiquitination and subsequent proteasomal degradation. This allows the p65/p50 complex to translocate to the nucleus, where it can activate transcription of NF-␬B responsive genes by binding to ␬B sites in their promoter regions. Although an “alternative” NF-␬B pathway that proceeds independent of IKK␤ and IKK␥ can be induced by some other members of the TNF family, TNF itself is believed to be a specific activator of the “classical” IKK␤/IKK␥-mediated NF-␬B pathway. We will therefore only focus on this pathway for our discussion of the more upstream signaling components (31). TNF-induced signaling toward IKK activation is initiated by the sequential recruitment of the adapter proteins TRADD, TRAF2, and RIP1 to the cytoplasmic part of TNFR1. Once this receptor complex I is formed, TRAF2 and RIP1 cooperate to recruit the TGF-␤ activated kinase (TAK)1 complex. TRAF2 contains a ubiquitin ligase activity by which it catalyzes the attachment of ubiquitin Lys-63-linked polyubiquitin chains to itself as well as RIP1. In contrast to Lys48-linked polyubiquitination, which labels proteins for proteasome-mediated degradation, Lys-63-linked polyubiquitination has emerged as an important signaling mechanism controlling various physiological processes such as the NF-␬B activation pathway (32, 33). The RIP1-associated Lys63-linked polyubiquitin chains are recognized by TAK1binding protein (TAB)2 and TAB3, both of which mediate the recruitment of TAK1 (34, 35). This event is crucial to TNFinduced IKK activation, because activated TAK1 will phosphorylate critical residues in IKK␤, leading to its activation (36 –38). Because TAK1 is actually a member of the MAP3K family, recruitment of TAK1 to the TNF-R1 complex mediates not only TNF-induced activation of NF-␬B, but also TNF-induced activation of the p38 and JNK MAPKs (36 –38). Although other members of the MAP3K family have also been suggested to play a role in TNF-induced activation of p38 and JNK, gene targeting studies could not confirm their role in TNF-induced MAPK activation. Possibly, different MAP3Ks make partial and additive contributions to TNF-induced JNK and p38 activation in a cell-type-specific manner. The activated MAP3Ks in turn activate the MAPK kinase (MKK)3/6 and MKK4/7 members of the MAP2K family that are responsible for the activation of p38 and JNK, respectively. B. TNF-induced signaling pathways leading to hepatocyte apoptosis

Triggering TNF-R1 can lead to hepatocyte apoptosis by recruiting the adapter proteins TRADD and FADD to its cytoplasmic part. FADD contains a dead effector domain by which it subsequently recruits procaspase-8, thus constituting the death-inducing signaling complex (DISC) where clustering of pro-caspase-8 results in its autoactivation. The active caspase-8 is then able to proteolytically activate several effector caspases, which are responsible for many of the destructive cellular events that result in apoptosis. Whether recruitment of FADD and activation of caspase-8 really occur at the level of the TNF-R1 at the cell membrane or as part of a separate intracellular signaling complex is still a matter of


debate (22, 23). In the case of hepatocytes, only a small amount of active caspase-8 is formed at the DISC, which has only a minor contribution to TNF-induced hepatocyte cell death. However, next to the direct activation of downstream executioner caspases by caspase-8 (the so-called type I cell death pathway), hepatocytes and some other cell types (all referred to as type II cells) require a second pathway that involves a mitochondrial amplification loop to undergo apoptotic cell death (39 – 42). This mitochondrial pathway involves the release of numerous apoptogenic factors from the mitochondrial intermembrane space into the cytosol, including cytochrome c. Together with Apaf-1, deoxy-ATP, and procaspase-9, cytochrome c forms the apoptosome, a highmolecular weight protein complex that activates caspase-9. Activated caspase-9 then proteolytically activates effector caspases, which in turn amplify the apoptotic signal by activating procaspase-8 and -9. The full-blown caspase activity that emerges from this amplification loop ultimately kills the cell. In hepatocytes, the mitochondrial pathway appears to be central in TNF-induced cell death because all signaling events directly or indirectly target the mitochondria (Fig. 3). Normally, a delicate balance between the antiapoptotic members of the Bcl-2 protein family, such as Bcl-2 itself and Bcl-xL, and its proapoptotic members, such as Bid, Bak, and Bax, preserves mitochondrial homeostasis. However, TNFinduced DISC formation leads to caspase-8 mediated cleavage of Bid, resulting in its active truncated form, tBid. This event establishes a molecular link between the DISC and the initiation of the mitochondrial pathway, because tBid will translocate to the mitochondria to induce the release of cytochrome c into the cytosol, thus shifting the Bcl-2 balance toward apoptosis. Several mechanisms have been proposed by which tBid disrupts the integrity of the mitochondrial membrane. For instance, tBid has been suggested to promote insertion and oligomerization of Bak and/or Bax into the outer mitochondrial membrane, thus leading to the formation of pores and resulting in the mitochondrial release of cytochrome c (43– 45). In addition, tBid has been shown to mediate TNF-induced opening of the permeability transition pore in the outer mitochondrial membrane, a phenomenon that is called the mitochondrial permeability transition (MPT). MPT appears to be essential to TNF-induced hepatocyte apoptosis because specific inhibitors of MPT prevent the hepatotoxic effects of TNF in vitro as well as in vivo (40, 45, 46). Next to these direct mechanisms of inducing mitochondrial permeability, tBid also indirectly contributes to TNF-induced mitochondrial dysfunction via the release of cathepsin B from the lysosomes. Cathepsin B was previously shown to critically mediate TNF-induced mitochondrial release of cytochrome c in hepatocytes. Consistently, cathepsin B-deficient hepatocytes are more resistant to TNF-induced apoptosis than wild-type hepatocytes (47), and mice with a targeted deletion of the cathepsin B gene display less TNFinduced liver injury than their wild-type counterparts (48). The observation that TNF stimulation does not induce lysosomal permeabilization in Bid-deficient hepatocytes suggested that the reduced release of mitochondrial cytochrome c observed in these cells is due to the absence of cytosolic cathepsin B. Indeed, the release of cathepsin B into the cytosol


leads to the activation of caspase-2, which subsequently facilitates efficient mitochondrial cytochrome c release. The mechanism by which active caspase-2 achieves this mitochondrial permeabilization is unclear (49). These studies demonstrate a central role for tBid in the induction of multiple pathways, all of which directly or indirectly converge on the mitochondrial release of cytochrome c and thus contribute to TNF-induced hepatocyte apoptosis. Besides the release of cytochrome c, another important consequence of tBid-induced mitochondrial dysfunction is the generation of reactive oxygen species (ROS). TNF-induced mitochondrial dysfunction leads to a rapid decrease in the mitochondrial membrane potential due to disruption of the electron transport chain. This causes the escape of electrons from the respiratory chain, which contributes to the formation of ROS, including superoxide anions (O2⫺), hydroxyl radicals (OH), and peroxide (H2O2). Because deletion of Bid impairs the majority of this ROS generation, the mitochondria seem to be the major source for TNF-induced ROS during hepatocyte cell death (50). The critical role for ROS in TNF-induced hepatocyte apoptosis was shown in studies using antioxidants, which alleviate hepatocyte cell death induced by TNF both in vitro and in vivo (50 –52). The mechanisms by which ROS regulate TNF-induced hepatocyte apoptosis are complex and not fully understood, but mitochondrial ROS might contribute to apoptosis by promoting peroxidation of outer mitochondrial membrane lipids, leading to cristae reorganization and thus augmenting cytochrome c release (50). Although the studies above demonstrate a central role for Bid in the generation of ROS as well as the release of mitochondrial cytochrome c into the cytosol, Bid-deficient hepatocytes are not completely resistant to TNF-induced apoptosis. The absence of Bid significantly delays TNFinduced hepatocyte apoptosis but does not prevent it (45). Moreover, Bid-deficient mice are only partially protected against TNF-mediated liver injury (44). These observations suggest that Bid-independent pathways also contribute to TNF-induced hepatocyte apoptosis. In this respect, TNF-induced activation of acidic and neutral sphingomyelinase (A-SMase and N-SMase), both of which catalyze sphingomyelin degradation and formation of ceramide, has been implicated in hepatocyte cell death. A critical role for A-SMase in this process was demonstrated in A-SMase-deficient hepatocytes, which produce substantially less ceramide upon TNF treatment than wildtype hepatocytes, resulting in reduced cell death. Consistently, A-SMase-deficient mice are resistant to TNFinduced liver injury (53). Also N-SMase has been suggested to play a role in TNF-induced hepatotoxicity because mice with a targeted deletion of FAN, the adapter protein that mediates TNF-induced activation of N-SMase, are partially protected against TNF-mediated liver injury (54). These studies indicate that the intracellular level of ceramide is an important factor that determines the susceptibility of hepatocytes to TNF-induced apoptosis. Enhanced levels of ceramide seem to contribute to TNFinduced hepatocyte apoptosis by down-regulating expression of methionine adenosyltransferases, leading to depletion of intracellular S-adenosyl-L-methionine. This

370



FIG. 3. Signaling pathways in TNF-induced hepatocyte cell death: all roads lead to the mitochondria. After binding of TNF to TNF-R1, complex II is formed and caspase-8 is activated. Because hepatocytes are type II cells, the amount of active caspase-8 that is generated is not sufficient to directly activate caspase-3 (dashed line). Therefore, caspase-8 initiates the mitochondrial pathway by cleaving Bid to tBid, which induces mitochondrial permeabilization via three distinct mechanisms: 1) tBid mediates opening of the permeability transition pore (PTP), leading to MPT; 2) tBid mediates the insertion of Bak and Bax in the mitochondrial outer membrane, leading to the formation of the Bak/Bax pore; and 3) tBid mediates lysosomal permeabilization, which leads to mitochondrial permeabilization via a cathepsin B- and caspase-2-dependent mechanism. MPT can also be induced independently of tBid, via N-SMase/A-SMase-mediated generation of ceramide (4). These events result in the mitochondrial release of cytochrome c and ROS. Whereas cytochrome c contributes to apoptosis via activation of caspase-9 and subsequent activation of caspase-3, ROS mainly contribute to necrosis. See text for further details. DD, Death domain; pro, prodomain.


results in depletion of mitochondrial reduced glutathione (55), which sensitizes mitochondria to the harmful effects of ganglioside GD3, a glycosphingolipid that is synthesized from ceramide. GD3 traffics to the mitochondria and directly induces ROS production, followed by MPT, cytochrome c release, and caspase activation, thus contributing to hepatocyte cell death (56 –58). Besides this ceramide-mediated pathway for ROS generation, other Bidindependent extramitochondrial sources of ROS might contribute to TNF-induced hepatocyte cell death, including ROS resulting from the 5-lipoxygenase-mediated metabolization of arachidonic acid that is produced upon triggering of cytosolic phospholipase A2 by TNF (59, 60). It should be mentioned, however, that this TNF-induced release of arachidonic acid, which also seems to involve cathepsin B (61), is thought to be a late process during TNF-induced cell death, occurring downstream of mitochondrial changes and effector caspase activation (62). Considering the dense network of intracellular signaling pathways that TNF can induce in hepatocytes, it is hard to imagine the complexity that emerges if one also takes into account the signaling mechanisms that result from other factors that are indirectly induced by TNF. TNF undoubtedly induces the release of many cytokines and other factors that additively or synergistically induce cell injury, or instead try to limit the destructive activities of TNF. The most obvious example of the former is TNFinduced expression and release of TNF itself, which allows paracrine effects of TNF to gradually convert a localized inflammatory reaction into widespread tissue damage. However, TNF also induces many negative feedback mechanisms for preventing this exacerbation of the inflammatory response. In this context, TNF is an important inducer of acute phase proteins in the liver. These proteins together provoke a systemic antiinflammatory reaction known as the acute phase response, which has been shown to limit tissue damage in various models of liver injury. For instance, mice incapable of responding to IL-6, the most potent inducer of the acute phase response, are hypersensitive to lipopolysaccharide (LPS)-induced liver damage (63). Furthermore, induction of the acute phase response by IL-6 protects mice against I/R-induced liver injury and promotes the regeneration of hepatocytes (64). From the above, it is clear that increased expression of TNF in the liver induces benign systemic responses that protect the organism from excessive damage. However, although individual acute phase proteins such as ␣1-antitrypsin and ␣1-acid glycoprotein have been shown to exert hepatoprotective activities in TNF-mediated liver injury (65, 66), they fail to protect hepatocytes from the cytotoxic effects of TNF in vitro (67). This indicates that on their turn, these acute phase proteins employ indirect mechanisms for protecting hepatocytes against cell death. Although all these indirect and systemic effects of TNF undoubtedly contribute to the regulation of liver homeostasis, we have limited ourselves to review only the aforementioned TNF-induced intracellular, and thus cell autonomous, signaling pathways in hepatocytes that affect the overall response of the liver to TNF.


IV. Crucial Role for NF-␬B in Regulating the Hepatocyte’s Response to TNF

Despite the fact that binding of TNF to TNF-R1 can simultaneously activate each of the above-mentioned signaling pathways, switching on an impressive cell death-inducing machinery, healthy hepatocytes are completely resistant to the cytotoxic effects of TNF. However, in the setting of global transcriptional arrest, such as by treatment with actinomycin D in vitro or D-(⫹)-galactosamine (GalN) in vivo, hepatocytes rapidly undergo cell death upon TNF stimulation. This indicates that hepatocytes are refractory to TNFinduced cell death due to the ability to up-regulate transcription of essential protective genes. Multiple studies have shown that NF-␬B activation is at least partially responsible for the induction of these cell death inhibitory factors. Mice deficient in the p65 NF-␬B subunit provided the first indication that NF-␬B is important to protect hepatocytes against TNF-induced cell death. These mice, as well as mice without the essential IKK␤ or IKK␥ subunits of the IKK complex, die during midgestation due to massive hepatocyte apoptosis (68 –71). Embryonic hepatocyte apoptosis was later shown to depend on TNF signaling because additional deletion of the TNF or the TNF-R1 gene rescued NF-␬B-deficient mice from embryonic lethality (72–74). The essential protective function of NF-␬B against TNF-induced hepatocyte apoptosis was confirmed by in vitro studies in which cultured hepatocytes are transfected with a mutant I␬B␣ that lacks the normal phosphorylation sites (Ser 32 and Ser36) for IKK␤ so that it can no longer be marked for proteasomal degradation and thus acts as a superrepressor (I␬B␣s) of NF-␬B activation. In this case, I␬B␣s expression converts the hepatocyte’s response to TNF from proliferation to apoptosis (75, 76). In addition to this hepatoprotective role of NF-␬B in cultured hepatocytes and the developing embryo, several reports have shown that adult mice also depend on NF-␬B activation to avoid TNF-induced hepatocyte apoptosis (see Sections V.A and V.B) (77–79). Consequently, given the abundance of TNF during liver diseases together with the crucial role of NF-␬B in protecting hepatocytes against the cytotoxic effects of TNF, it is not surprising that the activation status of NF-␬B critically influences the pathogenesis of several TNF-mediated hepatic diseases. As such, the role of NF-␬B has been studied extensively in various mouse models of TNF-mediated liver injury.

V. Role of NF-␬B Activation in Mouse Models of TNFMediated Hepatic Diseases A. Role of NF-␬B activation in mouse models of TNFmediated liver toxicity

Several mouse models have been developed to study TNFmediated liver injury. Although TNF as such does not cause severe liver toxicity when injected in healthy mice, coadministration of GalN, a hepatotoxin that blocks transcription specifically in hepatocytes, drastically sensitizes mice to TNF hepatotoxicity and lethality (80, 81). Similarly, GalN sensitizes mice to LPS, which induces several cells to produce TNF, which then acts in a paracrine manner to induce liver

372



injury. As such, administration of TNF/GalN or LPS/GalN causes an inflammatory hepatitis, characterized by infiltration of neutrophils and macrophages, and massive hepatocyte apoptosis and necrosis, which together result in lethality. TNF-R1-deficient mice are completely resistant against the lethal effects of TNF/GalN as well as LPS/GalN, demonstrating the essential role of TNF-R1 in these models (82, 83). It should be mentioned that the use of GalN may grossly exaggerate the effects of TNF, emphasizing the need for more advanced models of TNF-mediated liver injury to confirm the pathophysiological relevance of some of the findings made with the above-mentioned models. Nevertheless, the TNF/GalN-induced model of liver injury provides an elegant setting in which the direct effects of TNF signaling in hepatocytes can be studied in an in vivo context, without the interference of indirect effects of other cytokines or factors. Another model that is frequently used to study TNF-mediated liver injury is the administration of the T cell mitogenic plant lectin Con A (84). Con A induces a T cell-mediated hepatitis that, in contrast to the GalN-based models of liver injury, depends on both TNF-R1 and TNF-R2 (29). A beneficial role for NF-␬B activation when facing TNFmediated liver injury was already suggested when pretreatment of mice with NF-␬B activating stimuli, such as IL-1 or TNF itself, was found to confer protection against subsequent TNF/GalN-induced liver injury (85). Mice compromised in NF-␬B activation do not display this desensitization to the hepatotoxic effects of TNF (77), proving that NF-␬B activation induces potent defense mechanisms against TNFinduced liver injury. In the meantime, several mice deficient in NF-␬B activation, either by targeted deletion of essential NF-␬B signaling molecules or by transgenic expression of NF-␬B inhibitory proteins, have confirmed the hepatoprotective effects of NF-␬B. The responses of these various NF␬B-deficient mice in different models of TNF-mediated liver injury are summarized in Table 1 and will be discussed in detail below. Mice with hepatocyte-specific expression of an I␬B␣s suffer from severe hepatocyte apoptosis after injection of a TNF

dose that is well-tolerated by wild-type mice (78). Similarly, inducible hepatocyte-specific expression of an I␬B␣s leads to far more deleterious liver damage after Con A injection (86). In addition, these I␬B␣s transgenic mice are more sensitive to infection with Listeria, which mainly affects the liver. Due to Listeria-induced TNF production, mice expressing an I␬B␣s display more necrotic and apoptotic hepatocytes after Listeria infection and fail to eliminate the bacteria, eventually resulting in lethality (86). In line with the detrimental effects of I␬B␣s expression in these models of TNF-mediated liver injury, deletion of IKK␥ in hepatocytes also renders mice much more sensitive than wild-type mice to TNF (79). Hepatocytespecific deletion of IKK␤, however, did not inhibit TNFinduced NF-␬B activation, probably because of compensatory signaling by IKK␣ in the absence of IKK␤ (79, 87). In line with the intact NF-␬B signaling in the liver of these IKK␤deficient mice, no sensitization to liver injury induced by TNF itself, or by TNF-inducing treatments such as LPS/GalN and Con A, could be observed (79). In contrast, another study, using a different conditional hepatocyte-specific knockout of IKK␤, found that loss of IKK␤ almost completely blocks TNF-induced NF-␬B activation (88). The reason for the discrepancy between these studies regarding the role of IKK␤ in TNF-induced NF-␬B activation in hepatocytes is thus far unclear, but it might reflect different experimental conditions such as the use of different hepatocyte-specific Cre transgenic lines to delete IKK␤ in both studies. Importantly, despite impaired hepatic NF-␬B activation, Maeda et al. (88) could observe little or even no sensitization of their hepatocyte-specific IKK␤-deficient mice to LPS/GalN or LPS, respectively. Because no difference in LPS-induced circulating or hepatic TNF levels could be observed in IKK␤deficient vs. wild-type mice, these findings challenge the paradigm of NF-␬B being an essential protective factor against TNF in the liver. Indeed, this observation is in sharp contrast with studies involving NF-␬B-deficient mice, as well as studies using transgenic or adenoviral expression of an I␬B␣s, all of which have clearly demonstrated an inverse correlation between NF-␬B activation and susceptibility to

TABLE 1. Response of NF-␬B-deficient mice in models of TNF-mediated liver injury Model of liver injury

TNF LPS

Mouse genotype

Phenotype vs. wild-type mice

IKK␤⫺/⫺, hepatocyte specific, Alfp-Cre IKK␥⫺/⫺, inducible deletion, Mx-Cre ⌬N-I␬B␣ tg, inducible hepatocyte specific IKK␤⫺/⫺, hepatocyte specific, Alb-Cre

⫽ ⫹⫹⫹ ⫹⫹⫹ ⫽

IKK␤⫺/⫺, hepatocyte specific, Alfp-Cre ⫺/⫺

IKK␥ LPS/GalN Con A

Listeria

, hepatocyte specific, Alfp-Cre

IKK␤⫺/⫺, IKK␤⫺/⫺, IKK␤⫺/⫺, IKK␤⫺/⫺,

hepatocyte hepatocyte hepatocyte hepatocyte

specific, specific, specific, specific,

Alb-Cre Alfp-Cre Alfp-Cre Alb-Cre

⌬N-I␬B␣ tg, inducible hepatocyte specific ⌬N-I␬B␣ tg, inducible hepatocyte specific

⫽ ⫹⫹⫹ ⫹ ⫽ ⫽ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹

Remarks

No inhibition of NF-␬B activation after TNF More apoptosis and necrosis More apoptosis No difference in TNF levels, modest increase in apoptotic hepatocytes No inhibition of NF-␬B activation after TNF, no hepatocyte apoptosis No NF-␬B activation after LPS, massive hepatocyte apoptosis Higher lethality, more apoptotic hepatocytes No inhibition of NF-␬B activation after TNF No inhibition of NF-␬B activation after TNF No difference in TNF levels, more apoptosis and necrosis, higher JNK activation, higher ROS levels Higher lethality, more necrosis Failure to eliminate bacteria, higher lethality, more hepatocyte necrosis and apoptosis

Ref.

79 79 78 88 79, 87 87 88 79 79 88, 178 86 86

Alb, Albumin; Alfp, albumin regulatory elements and ␣-fetoprotein enhancers; tg, transgenic; ⫽, no difference; ⫹, slightly more sensitive; ⫹⫹⫹, much more sensitive.


TNF-induced liver injury (48, 77–79). Therefore, the lack of sensitization of IKK␤-deficient mice to LPS-induced liver injury as observed by Maeda et al. (88) is very puzzling. As a possible explanation, the authors suggest that NF-␬B activation in hepatocytes only protects against the cytotoxic effects of membrane-bound TNF, which preferentially binds TNF-R2, and not against soluble TNF that is produced after LPS injection. In support of this hypothesis, their IKK␤deficient mice are highly susceptible to liver injury induced by Con A, which induces expression of membrane-bound TNF and critically depends on the presence of TNF-R2 (29). However, although both LPS and Con A-induced liver injury are clearly mediated by induction of TNF synthesis, these agents can also induce a range of other signaling pathways in different cell types of the liver. Differences between these additional LPS- and Con A-induced pathways in the liver might also account for the different response of the IKK␤deficient mice in LPS- vs. Con A-induced liver damage. For instance, an alternative LPS-induced signaling pathway might confer the NF-␬B-deficient hepatocytes protection against the cytotoxic effect of TNF. On the other hand, the lack of sensitization of the IKK␤-deficient mice to LPS could simply reflect a TNF dosage effect. Indeed, it has been shown that the concentration threshold of TNF needed to induce hepatic NF-␬B activation in wild-type mice is lower than the amount of TNF needed to induce hepatocyte apoptosis in NF-␬B-deficient mice (78). Therefore, it is possible that LPS did not induce sufficient amounts of TNF to kill the IKK␤deficient hepatocytes. Alternatively, the threshold of NF-␬B activation needed to protect hepatocytes against LPS-induced liver injury might be lower than the amount of NF-␬B activation needed for protection against Con A. Because the IKK␤-deficient mice used by Maeda et al. (88) still show some residual NF-␬B activation, this might have been sufficient to prevent LPS-induced but not Con A-induced hepatocyte death. Unfortunately, these authors did not test whether challenging their IKK␤-deficient mice with TNF alone leads to severe liver injury, an experiment that could have revealed whether the lack of sensitization to LPS in IKK␤-deficient mice results from the inability of NF-␬B to protect hepatocytes against circulating TNF. Remarkably, in some models of TNF-mediated hepatotoxicity, inhibition of NF-␬B activation has been reported to be protective rather than sensitizing (summarized in Table 2). Although at first sight this contrasts with the well-established role of NF-␬B in the liver as an antiapoptotic factor, a more detailed look reveals that in these models protection by


NF-␬B inhibition is associated with decreased TNF levels, bypassing the danger of sensitizing hepatocytes to TNF cytotoxicity. Because TNF production in the liver is mainly derived from Kupffer cells, inhibition of hepatic NF-␬B activation can only be beneficial when it occurs in these cells. For instance, NF-␬B decoy oligonucleotides delivered by liposomes predominantly inhibit NF-␬B activation in Kupffer cells. Consequently, these NF-␬B decoys efficiently block LPS-induced production of TNF, thereby preventing cell death of hepatocytes sensitized by prior infection with Propionibacterium acnes (89). Because systemic administration of adenoviruses leads to infection of hepatocytes as well as Kupffer cells (90), adenoviral gene transfer of an I␬B␣s is another opportunity to block hepatic TNF production. Indeed, adenoviral expression of an I␬B␣s has been shown to protect mice against ethanol-induced liver injury. Chronic administration of ethanol leads to increased levels of gutderived LPS in the portal circulation, thereby activating Kupffer cells to produce TNF. Adenoviral expression of an I␬B␣s prevents this TNF synthesis by Kupffer cells and as such impairs subsequent liver injury (91). Also after I/R, Kupffer cell stimulation leading to TNF production is essential for the development of liver injury (15). As a result, adenoviral expression of an I␬B␣s abrogated TNF production and liver injury after I/R (92), suggesting that in this model Kupffer cell NF-␬B activation needs to be blocked to prevent liver damage. However, also hepatocyte-specific NF-␬B-deficient mice display less hepatic TNF production after I/R of the liver, resulting in decreased liver damage (79). This indicates that after I/R, the initial trigger that activates Kupffer cells to secrete TNF might be produced by hepatocytes in an NF-␬B-dependent manner. Remarkably, the hepatocyte-specific IKK␤-deficient mice that were used in this study show inhibition of NF-␬B activation after I/R, but not after TNF (79), indicating that the mechanisms by which TNF and I/R lead to NF-␬B activation in the liver are different. In support of this notion, mice in which an I␬B␤ transgene replaces the endogenous I␬B␣ gene display reduced hepatic NF-␬B activation after I/R, leading to decreased TNF levels and diminished liver injury (93). In contrast, TNF-induced NF-␬B activation in these mice is not altered (94). Thus, while I␬B␣ and I␬B␤ are redundant for regulating TNF-induced NF-␬B activation, I␬B␣ seems to have a unique function in regulating I/R-induced NF-␬B activation. In this respect, tyrosine phosphorylation of I␬B␣ has been proposed to mediate I/Rinduced activation of NF-␬B (93, 95). Because IKK␤-deficient mice display impaired NF-␬B activation after I/R, the IKK

TABLE 2. Protective effect of inhibition of NF-␬B activation in models of TNF-mediated liver injury Model of liver injury

I/R

Method of NF-␬B inhibition

IKK␤⫺/⫺, hepatocyte specific, Alfp-Cre I␬B␤ knock-in, replacing the I␬B␣ gene I␬B␣s adenovirus

Chronic ethanol

I␬B␣s adenovirus

LPS/P. acnes

NF-␬B decoy oligonucleotides

Remarks

Ref.

Inhibition of NF-␬B activation after I/R, decreased TNF levels, less necrosis, less inflammation Reduced NF-␬B activation after I/R, decreased TNF levels, reduced inflammation, increased survival Inhibition of NF-␬B activation after I/R, decreased iNOS levels, no hepatocyte apoptosis Decreased TNF levels, liver to body weight ratio, ALT levels, inflammation and necrosis Decreased TNF levels, hepatocyte apoptosis and necrosis, and lethality

79 93 92 91 89

Alfp, Albumin regulatory elements and ␣-fetoprotein enhancers; iNOS, inducible nitric oxide synthase; ALT, alanine aminotransferase.

374


complex also seems to play a role in this pathway. However, the mechanism by which IKK␤ influences tyrosine phosphorylation of I␬B␣ is not clear yet. Taken together, the above observations show that despite the well-established antiapoptotic role of NF-␬B in the liver, inhibition of NF-␬B activity can be beneficial in different conditions of TNFmediated liver injury. B. Role of NF-␬B activation in liver regeneration

Liver regeneration after partial hepatectomy is a fundamental parameter of the liver’s response to injury (96). After partial hepatectomy, NF-␬B is rapidly activated and appears to play a central role in initiating proliferation of the remnant hepatocytes as well as protecting them from apoptosis. This was shown by the observation that adenoviral expression of an I␬B␣s as well as pharmacological inhibition of NF-␬B activation impairs liver regeneration and leads to hepatocyte apoptosis (97–99). Originally, it was thought that TNF-induced NF-␬B activation in hepatocytes is important for initiating their proliferation because TNF-R1-deficient mice also fail to start a regenerative response after partial hepatectomy (18). Although it has been reported that TNF-induced DNA replication in hepatic cells does not occur in the absence of NF-␬B activation (100), it is now generally accepted that the beneficial effects of TNF-induced NF-␬B activation in liver regeneration are mainly attributed to the induction of IL-6 (101). Studies with NF-␬B reporter mice have shown that NF-␬B activation after partial hepatectomy occurs primarily in Kupffer cells (102). Moreover, the notion that Kuppfer cell NF-␬B activation is sufficient for inducing liver regeneration is supported by the observation that mice expressing a hepatocyte-specific I␬B␣s show a normal regenerative response after partial hepatectomy (78). Nevertheless, because adenoviral expression of an I␬B␣s not only prevents hepatocyte proliferation but also results in hepatocyte apoptosis, hepatocyte NF-␬B activation is important after partial hepatectomy as well. Indeed, this indicates that the increased levels of TNF that occur during liver regeneration can kill hepatocytes in the absence of NF-␬B activation. These studies supporting an antiapoptotic effect of NF-␬B in proliferating hepatocytes contrast with the observation that mice specifically impaired in hepatocyte NF-␬B activation do not display increased hepatocyte apoptosis after partial hepatectomy (78). Two potential explanations for this contradictory observation are the following. First, in hepatocyte-specific I␬B␣s transgenic mice, nonparenchymal cells of the liver might provide an indirect mechanism for protecting hepatocytes against TNF. This cytoprotective effect might be abolished by adenoviral inhibition of NF-␬B activation because adenoviruses infect both hepatocytes and nonparenchymal cells of the liver (90, 103). Second, it should be noted that the I␬B␣s transgenic mice used in the study by Chaisson et al. (78) express this NF-␬B inhibitor only in 45% of their hepatocytes. This efficiency of transgene expression is sufficient to sensitize hepatocytes to injection of recombinant TNF, but not to the level of TNF elicited after partial hepatectomy, raising the possibility that the combination of only partial inhibition of hepatic NF-␬B activation and only limited amounts of TNF


after partial hepatectomy is inadequate to invoke hepatocyte cell death. C. Role of NF-␬B activation in mouse models of hepatocarcinogenesis

The studies described above clearly demonstrate that NF-␬B activation in hepatocytes is essential for protection against apoptosis during TNF-mediated liver injury, whereas activation of NF-␬B in nonparenchymal cells of the liver is essential to stimulate hepatocyte proliferation during liver regeneration. Although both of these activities of NF-␬B are beneficial in these respective situations, they can also have detrimental effects. Indeed, considering the fact that both proliferation and evasion of apoptosis are important hallmarks of cancer (104), it is not surprising that NF-␬B activation in the liver was recently associated with an increased risk for hepatocarcinogenesis. Moreover, other hallmarks of cancer such as sustained angiogenesis, tissue invasion, and metastasis were also reported to be at least partially dependent on NF-␬B signaling (105, 106). A major link between NF-␬B-dependent inflammation and hepatocarcinogenesis came from studies with genetically altered mice. In multidrug resistance gene 2-deficient mice, bile duct inflammation is followed by the appearance of hepatocarcinoma due to prolonged NF-␬B activation in hepatocytes (107). In this model of inflammation-associated cancer, TNF produced by the nonparenchymal cells of the liver activates NF-␬B in hepatocytes. Hepatocyte-specific expression of an I␬B␣s as well as suppressing TNF activity resulted in apoptosis of transformed hepatocytes and failure to progress to carcinomas. This impaired tumorigenesis was only apparent when NF-␬B activity was inhibited between 7 and 14 months after birth, whereas its inactivation during the first 7 months caused no beneficial effects. These observations indicate that TNF-induced activation of NF-␬B in hepatocytes plays a detrimental role during late phases of tumor development by increasing survival of dysplastic hepatocytes. In contrast, in the diethylnitrosamine (DEN)-induced model of hepatocarcinogenesis, the number and the size of hepatocarcinomas is significantly increased in hepatocytespecific IKK␤-deficient mice, pointing to a beneficial effect for hepatocyte NF-␬B activation (108). In these mice, DEN causes increased hepatocyte necrosis due to impaired hepatocyte NF-␬B activation, leading to more compensatory regeneration. The latter is enhanced by NF-␬B activation in Kupffer cells because additional ablation of IKK␤ in these cells resulted in less and smaller DEN-induced tumors. Thus, in this chemically induced model of hepatocarcinogenesis, hepatocyte NF-␬B activation avoids the initiation of tumorigenesis by preventing hepatocyte necrosis. However, once tumorigenesis has started, NF-␬B activation in the surrounding inflammatory cells acts as the driving force of further tumor development by promoting proliferation of the transformed hepatocytes. Finally, a recent study using hepatocyte-specific IKK␥deficient mice reveals a role for NF-␬B as a tumor suppressor in the liver because ablation of IKK␥ causes the spontaneous development of chronic hepatitis resembling human nonal-


coholic steatohepatitis that results in hepatocellular carcinoma. This carcinogenic process was shown to be initiated by hypersensitivity of the IKK␥-deficient hepatocytes to oxidative stress-dependent, FADD-mediated apoptosis, triggering compensatory hepatocyte proliferation, inflammation, and activation of liver progenitor cells (87). Thus, similar to hepatocyte-specific IKK␤-deficient mice in the DEN-induced model of liver cancer, hepatocyte-specific IKK␥-deficient mice also indicate an essential role for the antiapoptotic effect of NF-␬B in the liver in avoiding compensatory proliferation leading to carcinogenesis. Moreover, in both of these models excessive JNK activation resulting from NF-␬B deficiency was suggested to play a causative role in hepatocyte apoptosis and consequent tumor development. DEN-induced carcinogenesis in mice with hepatocyte-specific deletion of both IKK␤ and JNK1 is completely abolished, showing that JNK activity is a principal mechanism by which IKK␤ deficiency in hepatocytes results in increased chemical hepatocarcinogenesis (109). In addition, IKK␥-deficient hepatocytes display a constitutive activation of JNK, suggesting that in these mice persistent activation of JNK also has a role in initiating hepatocyte apoptosis (87). Taken together, the studies described above demonstrate a dual role for hepatocyte NF-␬B activation in hepatocarcinogenesis (Fig. 4). In early stages of tumorigenesis, its cytoprotective effect is beneficial by preventing hepatocyte cell death and thus avoiding compensatory proliferation, whereas in late stages of tumorigenesis it supports malignancy by promoting survival of the transformed hepatocytes. In nonparenchymal cells of the liver, NF-␬B activation during hepatocarcinogenesis is detrimental because it provides the tumor cells with essential growth factors, allowing them to keep on proliferating. D. Role of hepatic NF-␬B activation in the metabolic syndrome

The liver plays an important role in whole body lipid metabolism. When the rate of synthesis or import of fatty acids by hepatocytes exceeds the rate of export or catabolism, lipids will start accumulating in the liver. As ethanol metabolism stimulates the synthesis of fatty acids and counteracts their oxidation, the consumption of alcohol can disrupt the lipid metabolic balance and can cause alcoholic fatty liver disease (110). However, fatty liver diseases can also develop in the absence of alcohol abuse. These nonalcoholic fatty liver diseases (NAFLDs) encompass a whole spectrum of liver pathologies ranging from simple fatty liver (steatosis) to liver inflammation (nonalcoholic steatohepatitis), fibrosis, and finally cirrhosis. Importantly, population studies have shown that all of these NAFLDs are linked with obesity, insulin resistance, and type 2 diabetes mellitus, features that are collectively referred to as the metabolic syndrome. This strong association of NAFLD with metabolic abnormalities has lead to the assumption that NAFLD is in fact the hepatic manifestation of the metabolic syndrome (111). NAFLDs are associated with a chronic subacute inflammatory state, and growing evidence links this inflammation to the development of the metabolic syndrome. The findings that TNF is overexpressed in adipose tissue of obese rodents


and that TNF can cause insulin resistance constituted the first indications for this notion (112, 113). Further studies showed that antiinflammatory drugs can reverse insulin resistance because high doses of salicylates improved glycemia, insulin sensitivity, and hyperlipidemia in mice as well as in patients (114 –116). Because salicylates are potent inhibitors of IKK␤ activity, these observations suggested an important role for NF-␬B activation in acquiring insulin resistance. Subsequent genetic evidence that IKK-dependent NF-␬B activation is implicated in the development of insulin resistance came from mice heterozygous for an IKK␤ null allele, which are partially protected from insulin resistance on an ob/ob background as well as after a high-fat diet (114). In contrast to this observation, another study could not detect an effect of IKK␤ heterozygosity in obesity-induced insulin resistance (117). Although a definite explanation for the conflicting results obtained in these studies is still lacking, it is possible that opposing contributions of NF-␬B activity in different organs obscure the effect of overall NF-␬B inhibition on insulin sensitivity. Despite the controversy over the role of systemic NF-␬B activation in insulin resistance, two research groups have recently linked hepatic NF-␬B activation to the development of the metabolic syndrome. These studies applied transgenic approaches in genetic as well as high-fat diet-induced models of obesity to demonstrate a role for hepatic NF-␬B activation in the progression of fatty liver to steatohepatitis, insulin resistance, and type 2 diabetes mellitus (118, 119). Cai et al. (118) generated mice that express constitutively active IKK␤ in hepatocytes, which leads to hepatic production of proinflammatory cytokines such as TNF, IL-1␤, and IL-6. The subacute hepatic NF-␬B activation in these transgenic mice caused profound hepatic and more moderate systemic insulin resistance. Inhibition of NF-␬B activation by hepatocyte-specific expression of I␬B␣s in these transgenic mice reversed this phenotype, reinforcing the notion that insulin resistance in these mice is a result of hepatic NF-␬B activation (118). Complementary to this study, Arkan et al. (119) used mice lacking IKK␤ specifically in hepatocytes. These mice retain insulin responsiveness and glucose tolerance in the liver, but develop peripheral insulin resistance in muscle and fat in response to high-fat diet, obesity, or aging (119). Taken together, these two studies show that NF-␬B activation in hepatocytes has a causative role in developing hepatic insulin resistance. However, the observations that excessive NF-␬B activation in the liver leads to systemic insulin resistance and that lack of NF-␬B activation in hepatocytes could not prevent the development of insulin resistance in muscle and fat tissue suggest that different cell types could be involved in regulating systemic insulin sensitivity. In this context, it was shown that myeloid-specific deletion of IKK␤ leads to a global improvement of insulin sensitivity in mice on a highfat diet (119). Because myeloid-specific deletion of IKK␤ prevents NF-␬B activation in macrophages, including Kupffer cells, this observation not only indicates a role for myeloid cells in regulating systemic insulin sensitivity but also suggests a role for Kupffer cells in the development of hepatic insulin resistance. Originally, adipose tissue was appreciated as the source for inflammatory cytokines that cause insulin

376



FIG. 4. Role of NF-␬B activation in hepatocarcinogenesis. NF-␬B activation in Kupffer cells leads to the production of TNF, which can cause either NF-␬B activation or cell death in hepatocytes, as well as IL-6 and other growth factors (GF), which can initiate hepatocyte proliferation. During early tumorigenesis, hepatocyte NF-␬B activation is beneficial because it protects against cell death and consequently prevents compensatory proliferation. If hepatocyte NF-␬B activation is impaired, TNF induces cell death, which leads to compensatory proliferation of other hepatocytes. This increases the risk of DNA mutations, which can give rise to tumor cells. During late tumorigenesis, NF-␬B activation protects the tumor cells against cell death. Moreover, the tumor cells are provided with essential growth factors due to NF-␬B activation in nonparenchymal cells of the liver, such as Kupffer cells. DD, Death domain.


resistance (20, 112). However, hepatocyte lipid accumulation represents a second potentially important site for inflammatory cytokine production (118). These proinflammatory cytokines, either produced by abdominal fat tissue and delivered via portal circulation, or produced by hepatocytes during steatosis, may then activate Kupffer cells. These activated Kupffer cells could be responsible for the subacute inflammation in the liver that causes progression to the metabolic syndrome (20). Overall, these findings suggest that IKK␤-dependent NF-␬B activation in hepatocytes most likely acts in a paracrine manner to down-modulate insulin sensitivity in liver because production of inflammatory cytokines by hepatocytes that results from NF-␬B activation and leads to Kupffer cell activation causes hepatic insulin resistance. In contrast, systemic insulin resistance is caused by myeloid cells because IKK␤-dependent NF-␬B activation in myeloid cells affects insulin action in all tissues (liver, fat, muscle). It is important to mention that in line with the previously discussed models of TNF-mediated liver injury, during NAFLD NF-␬B activation is also essential to protect hepatocytes against TNF-induced cell death. In humans, obesity has been shown to increase the risk and severity of liver damage in alcoholics (120). Recently, inhibition of NF-␬B activation was shown to be responsible for this sensitizing effect of obesity in a mouse model for alcohol-induced liver injury. Ob/ob mice display reduced hepatic NF-␬B activation after chronic administration of ethanol, which unleashes the apoptotic effects of the simultaneously increased levels of hepatic TNF, resulting in more hepatocyte apoptosis in these obese mice in comparison with lean mice (121). Furthermore, a high-fat diet also sensitizes mice to TNF-mediated liver injury by diminishing NF-␬B activation. Indeed, feeding mice with a high-fat diet was shown to increase expression of I␬B␣ in the liver, thus decreasing activation of NF-␬B and predisposing mice to increased hepatocyte apoptosis after partial hepatectomy (122). Although these studies are in support of a major role for NF-␬B inhibition in obesity-mediated sensitization to liver injury, one has to realize that leptin deficiency has a broad effect on innate as well as adaptive immunity, for instance by causing depletion of hepatic natural killer (NK) T cells (123). Moreover, depletion of these NKT cells not only occurs in genetically obese mice, but also after feeding wild-type mice a high-fat diet (124). Because hepatic NKT cell depletion promotes the production of proinflammatory cytokines in the liver, partially because of deregulated cytokine production by Kupffer cells (125), this phenomenon might be an important factor in the sensitization to TNF-mediated liver injury. Indeed, ob/ob mice as well as mice on a high-fat diet are extremely vulnerable to LPS-induced liver injury (126). In contrast to this observation, ob/ob mice are protected against Con A-induced T cell-mediated liver injury, which is associated with decreased hepatic production of TNF (127). Together, these data on the role of obesity in different models of TNF-mediated liver injury are quite puzzling. Given the systemic leptin deficiency of ob/ob mice, different effects of leptin on different cell types could account for some of these confusing results. Whereas leptin insufficiency could modulate the immune system such that, depending on the cell types in-


volved, cytokine production in the liver is either more proinflammatory or more hepatoprotective, its effect on hepatocytes could decrease NF-␬B activity and sensitize these cells to second hits of liver injury. Taken together, the above observations underscore the delicate balance of NF-␬B activation in hepatocytes during NAFLD, because excessive NF-␬B activation and subsequent inflammation may form the trigger for development and progression of some key features of the metabolic syndrome. On the other hand, NAFLD can predispose to certain second hits of TNF-mediated liver injury, which could partially be explained by deregulated cytokine production by nonparenchymal cells together with sudden drops in hepatocyte NF-␬B activity. E. Role of NF-␬B-dependent inflammation in hereditary hemochromatosis

Hereditary hemochromatosis is a genetic disorder characterized by progressive iron overload in parenchymal tissue and is associated with a high risk of liver cirrhosis, development of hepatocellular carcinoma, cardiomyopathy, and diabetes (128, 129). In 90% of all patients, hereditary hemochromatosis is due to homozygosity for the point mutation C282Y in the HFE gene, which encodes for a major histocompatibility complex class I-like protein (130). Mice deficient in HFE or homozygous for the missense C282Y mutation develop iron overload that recapitulates hereditary hemochromatosis in humans, confirming that hereditary hemochromatosis arises from loss of HFE function (128, 131). In normal adults, storage iron is deposited in hepatocytes and tissue macrophages and mobilized in response to acute need. Erythrocytes are the major consumers of iron, and their demand normally exceeds the capacity of storage cells to mobilize iron for erythropoiesis, which enhances intestinal absorption of dietary iron and macrophage recycling of iron from hemoglobin. However, nonhematopoietic tissues easily assimilate iron, resulting in vigorous intestinal absorption and consequent deposition of excess iron in parenchymal cells (128). Tight regulation of iron levels is thus mandatory and is determined by both intestinal absorption and macrophage release of iron. One key regulator of both intestinal iron absorption and macrophage iron release is hepcidin (132–135), a peptide that is produced almost exclusively by hepatocytes in response to inflammatory stimuli and iron load (134, 136). Mice deficient in Usf2, a transcription factor essential for hepcidin production, lack hepcidin expression and demonstrate massive iron overload resembling the hemochromatosis phenotype in humans (137). On the other hand, mice with a hepcidin transgene develop severe iron deficiency anemia (138). These genetic studies in mice confirm the importance of regulating hepcidin synthesis for maintaining an appropriate iron balance. Induction of hepcidin expression by inflammatory stimuli seems to be mediated mainly by the inflammatory cytokine IL-6 produced by Kupffer cells, which triggers hepcidin transcription in nearby hepatocytes (139, 140). Kupffer cells are required for the activation of hepcidin synthesis during inflammation but are dispensable for the regulatory activity exerted by iron on hepatic hepcidin (141). Besides

378


this crucial role for IL-6, TNF was also shown to regulate cellular iron distribution and as such modulates the severity of liver iron overload and damage (21, 142–144). The involvement of inflammatory cytokines and Kupffer cells in regulating the hepatic iron balance suggests a delicate role for NF-␬B activation in this process. Indeed, production of inflammatory cytokines resulting from NF-␬B activation in Kupffer cells causes in a paracrine way an increase of hepatocyte-derived hepcidin, which in turn augments the hepatic iron concentrations. Interestingly, chronic iron overload has been shown to trigger oxidative stress leading to activation of NF-␬B (145), suggesting that increased iron production in the liver can act as an amplifier of NF-␬B activation and hence of hepatic inflammation. Particularly important in this context, administration of iron to Kupffer cells was shown to activate NF-␬B and TNF promoter activity through enhancement of IKK activity (146). As such, uncontrolled NF-␬B activation and iron release form a feed-forward loop that can lead to excessive production of hepcidin and can result in serum anemia of inflammation. On the other hand, situations where NF-␬B activation is compromised might lead to insufficient hepcidin production and may therefore contribute to the pathology of hereditary hemochromatosis. VI. Mechanisms of the Antiapoptotic Effect of NF-␬B

The above-described observations on the role of NF-␬B in various models of hepatic diseases identify NF-␬B as the master switch between life and death in the liver. NF-␬B activation exerts an antiapoptotic effect by which it protects hepatocytes against apoptosis during liver injury and liver regeneration. In addition, this cytoprotective effect of NF-␬B also has a role in hepatocarcinogenesis, where it can either be beneficial by preventing tumorigenesis during early stages or detrimental by maintaining malignant transformation of hepatocytes during later stages of tumor development. Given this central role for NF-␬B in the decision between hepatocyte survival and cell death in these liver diseases, elucidating its molecular mechanism of action is of considerable therapeutic interest. Understanding the underlying molecular mechanisms of the antiapoptotic effects of NF-␬B can facilitate us to mimic these cytoprotective actions to protect hepatocytes from cell death during liver injury. Conversely, such knowledge can also give us clues on how to specifically block these antiapoptotic mechanisms to sensitize malignant hepatocytes to the toxic effects of TNF or chemotherapeutics. Therefore, in the next part of this review, we will focus on the molecular mechanisms by which NF-␬B protects cells against apoptosis. We will also try to put each of these proposed mechanisms into a “hepatic perspective” by discussing their role in hepatocytes and TNF-mediated liver diseases. A. NF-␬B-dependent inhibition of caspase activation

The cytoprotective effect of NF-␬B is associated with the induction of several antiapoptotic genes, such as the caspase-8 inhibitor c-FLIPL, the Bcl-2 family members Bcl-xL and A1/Bfl-1, X-linked inhibitor of apoptosis (XIAP), and the


simultaneous up-regulation of TRAF1, TRAF2, cellular inhibitor of apoptosis (c-IAP)1, and c-IAP2 (147–152). These proteins interfere with TNF-induced apoptosis at various levels in the signaling pathways leading to caspase activation. For instance, c-FLIPL competes with caspase-8 for binding to the TNF-R1 complex II, thus impairing the formation of the DISC (22). Bcl-xL and A1/Bfl-1 act at the mitochondria, where they prevent tBid-induced MPT and mitochondrial depolarization, thus reducing the production of ROS and cytochrome c-mediated caspase-9 activation (153). XIAP as well as the c-IAPs act more downstream, because they directly bind distinct caspases and thereby inhibit their proteolytic activity (154, 155). Some of these antiapoptotic proteins have already been shown to exert beneficial effects during TNF-mediated liver injury. For instance, transgenic mice overexpressing Bcl-xL are partially protected against TNF/GalN-induced liver injury (156). For a long time, these NF-␬B induced cytoprotective factors were thought to be fully responsible for the cell deathinhibiting effect of NF-␬B activation. However, a detailed look into the expression pattern of these antiapoptotic proteins and their cytoprotective effect in NF-␬B-deficient cells made it clear that the NF-␬B-dependent up-regulation of antiapoptotic genes could not solely account for the complete protection of hepatocytes against TNF-induced cell death. For instance, A1/Bfl-1 as well as TRAFs and c-IAPs can only partially inhibit TNF-induced cell death when NF-␬B activation is blocked (152, 157, 158). In addition, expression of TRAF1 and A1/Bfl-1 is restricted to certain tissues, and several types of cells appear to express TRAF1 in the absence of TRAF2 (149, 159), which is needed for the recruitment of TRAF1 to the TNF-R, indicating that these proteins cannot protect every cell type against TNF-induced cell death. The cell type-specific effects of these antiapoptotic proteins, together with their inability to protect NF-␬B-deficient cells fully against apoptosis, indicate that either cooperation of several of these proteins is needed to prevent TNF-induced apoptosis efficiently, or alternatively, that other NF-␬B-dependent events contribute to the resistance of hepatocytes to TNF-induced apoptosis. B. NF-␬B-dependent inhibition of JNK activation

Multiple studies have shown that both the level and the duration of TNF-induced JNK activation determine the biological outcome of TNF stimulation. Whereas transient and modest activation of JNK is associated with cellular survival, prolonged and robust activation of JNK plays an important role in TNF-induced cell death. Moreover, the level of TNFinduced JNK activation seems to be controlled by NF-␬B activation. In cells lacking p65 or IKK␤, or cells stably expressing an I␬B␣s, JNK activation in response to TNF is sustained. This prolonged JNK activation contributes to subsequent cell death because inhibition of JNK by pharmacological agents or dominant-negative kinase mutants effectively rescues these NF-␬B-deficient cells from TNF-induced cytotoxicity (160 –163). These observations indicate that at least some of the protective effects of NF-␬B activation in TNF-induced cytotoxicity are mediated by rapidly terminating TNF-induced JNK activation.


The proapoptotic role of JNK activation has also been demonstrated in mouse hepatocytes. NF-␬B-deficient embryos, which normally die due to hepatocyte apoptosis, were shown to survive longer when the gene encoding JNK1 is also deleted (164). In another study using specific knockout mice for either JNK1 or JNK2, Wang et al. (165) could demonstrate that JNK2 knockout mice show a significantly increased survival and a lack of hepatocyte apoptosis in the TNF/GalN and LPS/GalN mouse model of liver toxicity. Interestingly, in the same study JNK1 knockout mice showed similar levels of liver injury as control mice, demonstrating clearly that in these models JNK2 is essential for TNF-mediated apoptosis, whereas JNK1 is not involved in this pathway at all. It is worth mentioning that loss of JNK2 was not associated with any decrease in JNK activity, supporting the conclusion that hepatocellular injury in response to TNF is promoted by JNK2 through a mechanism unrelated to the classical c-Jun kinase activity of this protein (see also below). Similar to the previously reported effect of NF-␬B inhibition on the kinetics of JNK activation, the study of Wang et al. (165) illustrated that GalN also converted LPS-induced JNK signaling from a transient to prolonged activation. An association between sustained activation of JNK and enhanced TNF-induced apoptosis has also been shown in I␬B␣s-expressing hepatocytes, which show prolonged TNF-induced activation of JNK, and inhibition of this response blocks TNF-induced cell death (166 –168). The proapoptotic effect of JNK activation has also been suggested to contribute to other TNF-mediated liver injuries. For instance, prolonged activation of JNK seems to contribute to reperfusion injury associated with liver transplantation because specific JNK inhibitors prevent both hepatocyte apoptosis and necrosis in models of I/R-induced liver injury (169 –171). Also in the Con A-induced model of liver injury, TNF-induced activation of JNK correlates with liver cell damage (172, 173). Interestingly, hepatocyte-specific deletion of IKK␤ enhances the activation of JNK by Con A, and suppression of this response by additional ablation of JNK1 or JNK2 reduces Con A-induced liver injury (88), indicating that the antiapoptotic effects of NF-␬B activation in this model of TNF-mediated liver injury are mediated by attenuating JNK activation. In this study, binding of membrane-bound TNF to TNF-R2 was suggested to be responsible for the increased Con A-induced JNK activation. In contrast with this suggestion, human TNF was shown to be capable of inducing prolonged JNK activation and subsequent apoptosis in mouse primary hepatocytes (167). Because human TNF only binds TNF-R1 on murine cells, this study suggests that TNF-R1 triggering is sufficient for inducing sustained activation of JNK. However, one has to realize that autocrine signaling of murine TNF could still occur in these hepatocytes. Therefore, only a similar experiment evaluating the effect of human TNF on JNK activation in TNF-deficient murine hepatocytes would unequivocally discriminate between both TNF receptors. Despite the controversy that still exists on the mechanisms of sustained JNK activation in the absence of NF-␬B activation, its proapoptotic role is generally accepted. In addition, a molecular link between JNK activation and cell death was recently suggested because JNK1 was shown to induce proteasomal degradation of the caspase-8 inhibitor c-FLIPL


(164). In this study, activated JNK1 was shown to phosphorylate and thereby activate the ubiquitin ligase Itch, which subsequently catalyzes the attachment of Lys-48-linked polyubiquitin chains to c-FLIPL. This targets the latter for proteasomal degradation. As c-FLIPL normally prevents the association of procaspase-8 to the TNF-R complex I, its degradation allows the molecular shift to complex II, an event that is a prerequisite to initiate cell death signaling. In contrast, Wang et al. (165) showed that there is no c-FLIP regulation or degradation during LPS/GalN-induced liver damage in JNK2 knockout mice (165). However, they showed that JNK2 mediates direct caspase-8 activation via a yet unknown mechanism that seems to be independent of the c-Jun kinase activity of JNK2. Altogether, these data can be interpreted in a way that both JNK1 and JNK2 have important proapoptotic functions in LPS- and TNF-mediated hepatotoxicity, although via slightly different molecular mechanisms. C. Mechanisms of crosstalk between TNF-induced activation of NF-␬B and JNK

The proapoptotic role of sustained JNK activation in hepatocytes shows that the mechanisms controlling the level of JNK activity serve as key modulators of the biological outcome of hepatocyte responses to TNF. Various mechanisms have been proposed by which NF-␬B activation controls the length of TNF-induced JNK activation (Fig. 5). Three NF-␬B-dependent genes have been put forward as potential candidates that shut down TNF-induced JNK activation: growth arrest DNA damage-inducible gene 45␤ (GADD45␤), the already mentioned caspase inhibitor XIAP, and the zinc finger protein A20 (161, 162, 174). However, a JNK inhibitory role for GADD45␤ and XIAP could not be confirmed by knockout studies and has not been observed in hepatocytes yet. In contrast, A20-deficient cells show prolonged activation of JNK upon TNF treatment. Although such a JNK inhibitory role of A20 in hepatocytes remains to be shown, its antiapoptotic effects have already proven to be beneficial in TNF-mediated liver injury. Indeed, adenoviral gene transfer of A20 protects mice against LPS/GalN-induced liver failure by decreasing hepatocyte apoptosis (175). Moreover, adenoviral gene transfer of A20-binding inhibitor of NF-␬B (ABIN)-1 and ABIN-3, which have been suggested to contribute to the biological effects of A20, were also shown to protect mice from TNF/GalN- and LPS/GalN-induced acute liver failure and lethality, respectively (176, 177). Next to up-regulating the expression of JNK inhibitory proteins, NF-␬B activation also inhibits prolonged JNK activation by preventing accumulation of ROS. Indeed, pretreatment of NF-␬B-deficient cells with antioxidants abolishes extended JNK activation and subsequently protects against TNF-induced cell death (178). ROS-mediated JNK activation was also suggested to be involved in TNF-mediated liver injury because protection of mice against Con A-induced liver failure by an antioxidant diet is associated with prevention of ROS accumulation as well as inhibition of prolonged JNK activation (179). Several studies suggest that TNF-induced ROS shift the balance toward prolonged activation of JNK by promoting the activation of the JNK-acti-

380



FIG. 5. Regulation of TNF-induced JNK activation by NF-␬B activation. A, In the presence of NF-␬B activation, the NF-␬B-dependent proteins GADD45␤, XIAP, and A20 prevent phosphorylation of JNK. Although the molecular mechanisms responsible for JNK inhibition by A20 (1) and XIAP (2) are still not clear, GADD45␤ blocks the catalytic activity of the upstream MKK7 (3). The NF-␬B dependent antioxidants MnSOD (4) and FHC (5) prevent accumulation of ROS, thus inhibiting its actions (dashed lines). Trx keeps ASK1 inactive, and MKPs can dephosphorylate JNK, leading to a transient activation of JNK in response to TNF. B, In the absence of NF-␬B activation, A20, XIAP, and GADD45␤ do not prevent JNK phosphorylation; and ROS accumulate because of the absence of the antioxidants FHC and MnSOD (dashed lines). ROS oxidize Trx (1), causing its release from ASK1 (2), which is thereby activated. In addition, ROS oxidize and thereby inactivate MKPs (3). Together, this leads to sustained activation of JNK in response to TNF. See text for further details. DD, Death domain; P, phosphate; FHC, ferritin heavy chain; MnSOD, manganese superoxide dismutase.

vating apoptosis signal-regulating kinase 1 (ASK1) and simultaneously blocking the inhibitory MAPK phosphatases (MKPs) that are essential for dephosphorylating activated JNK (179 –181). Although the mechanism of ROS-induced prolonged JNK activation in hepatocytes remains to be demonstrated, inhibition of ASK1 is necessary to block hepatocyte apoptosis fully (182). In addition, overexpression of SOCS1, a ubiquitin ligase capable of inducing proteasomal degradation of ASK1, protects primary hepatocytes against TNF-induced apoptosis (183, 184). Also, overexpression of thioredoxin (Trx), the ASK1 inhibitor that is inactivated by ROS (180), has been shown to protect hepatocytes from TNF-

induced cytotoxicity in vitro as well as in vivo (51, 185). These observations demonstrate a pro- and antiapoptotic role for ASK1 and Trx in hepatocytes, respectively, which would be in line with inactivation of Trx followed by activation of ASK1 as a mechanism by which ROS contribute to prolonged JNK activation and subsequent TNF-mediated liver injury.

VII. Concluding Remarks

This overview shows that the hepatocyte’s fates of death or survival are intimately linked by NF-␬B activation. It is


generally accepted that NF-␬B activation in hepatocytes exerts a cytoprotective effect, whereas NF-␬B activation in nonparenchymal cells of the liver promotes hepatocyte proliferation. Given this central role of NF-␬B activation in the life and death of a hepatocyte, the activation status of NF-␬B crucially influences liver homeostasis and the development of numerous liver diseases. Therefore, understanding the underlying molecular mechanisms that activate NF-␬B as well as the mechanisms by which NF-␬B exerts its cytoprotective effects is of great therapeutic value. Because NF-␬B activation has a key role in the initiation of the inflammatory response, NF-␬B was originally considered as an attractive therapeutic target for inflammatory liver diseases. In addition, because of the importance of NF-␬B signaling in the inflammatory induction of insulin resistance, NF-␬B is also an attractive target for treatment of the subacute inflammation that underlies the metabolic syndrome. However, the discovery of its cytoprotective effects has made the use of NF-␬B inhibitors in inflammatory and metabolic disorders more complicated because inhibition of NF-␬B would also sensitize hepatocytes to cell death. For instance, inhibition of NF-␬B activation in the intestine has been shown to alleviate inflammation but at the same time cause apoptosis in intestinal epithelial cells (186). Consequently, for an NF-␬B inhibitory therapeutic strategy to be safe, hepatocyte cell death should simultaneously be blocked. Mimicking the antiapoptotic machinery of NF-␬B might provide a means to avoid the hepatotoxicity that is normally associated with NF-␬B inhibition. However, several studies have shown that caspase inhibitors can enhance ROS-dependent necrosis both in vitro and in vivo (187, 188), excluding caspase inhibitors as safe cell death inhibitory adjuvants in NF-␬B-inhibiting therapies. Fortunately, elucidating the molecular mechanisms by which NF-␬B exerts its cytoprotective effects recently provided us with novel possibilities to accomplish prevention of hepatocyte cell death. NF-␬B activation prevents both apoptosis and necrosis by abrogating prolonged JNK activation and/or excessive ROS generation in response to TNF. Therefore, targeting the signaling pathways that lead to JNK activation and/or ROS generation might be a safer approach to avoid the hepatotoxic effects of NF-␬B inhibition. Moreover, because sustained JNK activation is observed in dietary and genetic models of obesity, and JNK deficiency is protective in different models of obesityinduced insulin resistance (189 –191), specific JNK1 and JNK2 inhibitors may not only be useful in inflammatory liver diseases but might also provide new opportunities for the treatment of obesity and related metabolic disorders. In the search for appropriate factors that can be used as safe antiinflammatory agents in the liver, the zinc finger protein A20 may be a very promising lead. A20 can inhibit NF-␬B activation as well as TNF-induced cell death (174), and this cytoprotective effect of A20 has already proven its efficacy in a mouse model of TNF-mediated liver injury (175). More importantly, despite its NF-␬B inhibiting effect, which is normally associated with increased hepatocyte apoptosis during liver regeneration, A20 does not exert detrimental effects after partial hepatectomy but instead improves liver regeneration (192). This shows that A20 can inhibit NF-␬B activation in hepatocytes without sensitizing to TNF cyto-


toxicity. Because A20 is suggested to exert its cytoprotective effect through inhibition of sustained JNK activation (174, 193), this argues for targeting the JNK pathway along with inhibiting NF-␬B in TNF-mediated liver injury. Interestingly, also the A20-binding protein ABIN-1 was recently shown to possess both an NF-␬B inhibitory and an antiapoptotic effect in hepatocytes, allowing it to completely protect mice against TNF-induced liver injury (176). Although the mechanism by which ABIN-1 exerts its antiapoptotic effect remains to be elucidated, this observation suggests that ABIN-1 might also be able to prevent hepatic NF-␬B activation without sensitizing to the cytotoxic effects of TNF. Although a lot of research still needs to be done, molecules with such activities will be the method of choice to treat inflammatory liver diseases in the future.

Acknowledgments We are grateful to Dr. Sophie Janssens for her help in preparing the figures. Address all correspondence and requests for reprints to: Rudi Beyaert, Department for Molecular Biomedical Research, VIB, Ghent University, Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium. E-mail: [email protected] Research in the authors’ laboratory is supported by grants from the “Interuniversitaire Attractiepolen” (IAP5/12 and IAP6/18), the Fund for Scientific Research (FWO; Grant 3G010505), the ‘Geconcerteerde Onderzoeksacties’ (GOA; Grant 01G06B6) of the Ghent University, and a Marie Curie Reintegration Grant (FP6-ERG-2004-Mobility-11 nr.031063; to G.v.L.). K.H. and G.v.L. are postdoctoral research associates with the FWO-Flanders. Present address for A.W.: Department for Mouse Genetics and Inflammation, Institute for Genetics, University of Cologne, Zu¨lpicher Str. 47, D-50674 Cologne, Germany. Disclosure Statement: The authors have nothing to disclose.

References 1. Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ 2002 Mechanisms of hepatotoxicity. Toxicol Sci 65: 166 –176 2. Bradham CA, Plumpe J, Manns MP, Brenner DA, Trautwein C 1998 Mechanisms of hepatic toxicity. I. TNF-induced liver injury. Am J Physiol 275:G387–G392 3. Karin M, Greten FR 2005 NF-␬B: linking inflammation and immunity to cancer development and progression. Nat Rev Immunol 5:749 –759 4. Kallinowski B, Haseroth K, Marinos G, Hanck C, Stremmel W, Theilmann L, Singer MV, Rossol S 1998 Induction of tumour necrosis factor (TNF) receptor type p55 and p75 in patients with chronic hepatitis C virus (HCV) infection. Clin Exp Immunol 111: 269 –277 5. Nelson DR, Lim HL, Marousis CG, Fang JW, Davis GL, Shen L, Urdea MS, Kolberg JA, Lau JY 1997 Activation of tumor necrosis factor-␣ system in chronic hepatitis C virus infection. Dig Dis Sci 42:2487–2494 6. Sheron N, Lau J, Daniels H, Goka J, Eddleston A, Alexander GJ, Williams R 1991 Increased production of tumour necrosis factor ␣ in chronic hepatitis B virus infection. J Hepatol 12:241–245 7. Yoshioka K, Kakumu S, Arao M, Tsutsumi Y, Inoue M, Wakita T, Ishikawa T, Mizokami M 1990 Immunohistochemical studies of intrahepatic tumour necrosis factor ␣ in chronic liver disease. J Clin Pathol 43:298 –302 8. Spengler U, Zachoval R, Gallati H, Jung MC, Hoffmann R, Riethmuller G, Pape G 1996 Serum levels and in situ expression of

382

9.

10. 11. 12.

13.

14. 15. 16. 17.

18.

19.

20. 21.

22. 23.

24. 25. 26.

27. 28.

Endocrine Reviews, June 2007, 28(4):365–386 TNF-␣ and TNF-␣ binding proteins in inflammatory liver diseases. Cytokine 8:864 – 872 Gonzalez-Amaro R, Garcia-Monzon C, Garcia-Buey L, MorenoOtero R, Alonso JL, Yague E, Pivel JP, Lopez-Cabrera M, Fernandez-Ruiz E, Sanchez-Madrid F 1994 Induction of tumor necrosis factor ␣ production by human hepatocytes in chronic viral hepatitis. J Exp Med 179:841– 848 Muto Y, Nouri-Aria KT, Meager A, Alexander GJ, Eddleston AL, Williams R 1988 Enhanced tumour necrosis factor and interleukin-1 in fulminant hepatic failure. Lancet 2:72–74 Bird GL, Sheron N, Goka AK, Alexander GJ, Williams RS 1990 Increased plasma tumor necrosis factor in severe alcoholic hepatitis. Ann Intern Med 112:917–920 McClain CJ, Song Z, Barve SS, Hill DB, Deaciuc I 2004 Recent advances in alcoholic liver disease. IV. Dysregulated cytokine metabolism in alcoholic liver disease. Am J Physiol Gastrointest Liver Physiol 287:G497–G502 Iimuro Y, Gallucci RM, Luster MI, Kono H, Thurman RG 1997 Antibodies to tumor necrosis factor ␣ attenuate hepatic necrosis and inflammation caused by chronic exposure to ethanol in the rat. Hepatology 26:1530 –1537 Yin M, Wheeler MD, Kono H, Bradford BU, Gallucci RM, Luster MI, Thurman RG 1999 Essential role of tumor necrosis factor ␣ in alcohol-induced liver injury in mice. Gastroenterology 117:942–952 Rudiger HA, Clavien PA 2002 Tumor necrosis factor ␣, but not Fas, mediates hepatocellular apoptosis in the murine ischemic liver. Gastroenterology 122:202–210 Feingold KR, Soued M, Grunfeld C 1988 Tumor necrosis factor stimulates DNA synthesis in the liver of intact rats. Biochem Biophys Res Commun 153:576 –582 Akerman P, Cote P, Yang SQ, McClain C, Nelson S, Bagby GJ, Diehl AM 1992 Antibodies to tumor necrosis factor-␣ inhibit liver regeneration after partial hepatectomy. Am J Physiol 263:G579 – G585 Yamada Y, Kirillova I, Peschon JJ, Fausto N 1997 Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor. Proc Natl Acad Sci USA 94:1441–1446 Yamada Y, Webber EM, Kirillova I, Peschon JJ, Fausto N 1998 Analysis of liver regeneration in mice lacking type 1 or type 2 tumor necrosis factor receptor: requirement for type 1 but not type 2 receptor. Hepatology 28:959 –970 Shoelson SE, Lee J, Goldfine AB 2006 Inflammation and insulin resistance. J Clin Invest 116:1793–1801 Fargion S, Valenti L, Dongiovanni P, Scaccabarozzi A, Fracanzani AL, Taioli E, Mattioli M, Sampietro M, Fiorelli G 2001 Tumor necrosis factor ␣ promoter polymorphisms influence the phenotypic expression of hereditary hemochromatosis. Blood 97:3707– 3712 Micheau O, Tschopp J 2003 Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 114:181–190 Schneider-Brachert W, Tchikov V, Neumeyer J, Jakob M, WinotoMorbach S, Held-Feindt J, Heinrich M, Merkel O, Ehrenschwender M, Adam D, Mentlein R, Kabelitz D, Schutze S 2004 Compartmentalization of TNF receptor 1 signaling: internalized TNF receptosomes as death signaling vesicles. Immunity 21:415– 428 Zheng L, Fisher G, Miller RE, Peschon J, Lynch DH, Lenardo MJ 1995 Induction of apoptosis in mature T cells by tumour necrosis factor. Nature 377:348 –351 Kim EY, Teh HS 2001 TNF type 2 receptor (p75) lowers the threshold of T cell activation. J Immunol 167:6812– 6820 Depuydt B, van Loo G, Vandenabeele P, Declercq W 2005 Induction of apoptosis by TNF receptor 2 in a T-cell hybridoma is FADD dependent and blocked by caspase-8 inhibitors. J Cell Sci 118:497–504 Tartaglia LA, Pennica D, Goeddel DV 1993 Ligand passing: the 75-kDa tumor necrosis factor (TNF) receptor recruits TNF for signaling by the 55-kDa TNF receptor. J Biol Chem 268:18542–18548 Grell M, Zimmermann G, Gottfried E, Chen CM, Grunwald U, Huang DC, Wu Lee YH, Durkop H, Engelmann H, Scheurich P, Wajant H, Strasser A 1999 Induction of cell death by tumour necrosis factor (TNF) receptor 2, CD40 and CD30: a role for TNF-R1


29.

30. 31. 32. 33. 34. 35. 36.

37.

38. 39. 40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

activation by endogenous membrane-anchored TNF. EMBO J 18: 3034 –3043 Kusters S, Tiegs G, Alexopoulou L, Pasparakis M, Douni E, Kunstle G, Bluethmann H, Wendel A, Pfizenmaier K, Kollias G, Grell M 1997 In vivo evidence for a functional role of both tumor necrosis factor (TNF) receptors and transmembrane TNF in experimental hepatitis. Eur J Immunol 27:2870 –2875 Douni E, Kollias G 1998 A critical role of the p75 tumor necrosis factor receptor (p75TNF-R) in organ inflammation independent of TNF, lymphotoxin ␣, or the p55TNF-R. J Exp Med 188:1343–1352 Hayden MS, Ghosh S 2004 Signaling to NF-␬B. Genes Dev 18: 2195–2224 Sun L, Chen ZJ 2004 The novel functions of ubiquitination in signaling. Curr Opin Cell Biol 16:119 –126 Wullaert A, Heyninck K, Janssens S, Beyaert R 2006 Ubiquitin: tool and target for intracellular NF-␬B inhibitors. Trends Immunol 27:533–540 Chen G, Goeddel DV 2002 TNF-R1 signaling: a beautiful pathway. Science 296:1634 –1635 Chen ZJ 2005 Ubiquitin signalling in the NF-␬B pathway. Nat Cell Biol 7:758 –765 Sato S, Sanjo H, Takeda K, Ninomiya-Tsuji J, Yamamoto M, Kawai T, Matsumoto K, Takeuchi O, Akira S 2005 Essential function for the kinase TAK1 in innate and adaptive immune responses. Nat Immunol 6:1087–1095 Shim JH, Xiao C, Paschal AE, Bailey ST, Rao P, Hayden MS, Lee KY, Bussey C, Steckel M, Tanaka N, Yamada G, Akira S, Matsumoto K, Ghosh S 2005 TAK1, but not TAB1 or TAB2, plays an essential role in multiple signaling pathways in vivo. Genes Dev 19:2668 –2681 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 –351 Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME 1998 Two CD95 (APO-1/ Fas) signaling pathways. EMBO J 17:1675–1687 Bradham CA, Qian T, Streetz K, Trautwein C, Brenner DA, Lemasters JJ 1998 The mitochondrial permeability transition is required for tumor necrosis factor ␣-mediated apoptosis and cytochrome c release. Mol Cell Biol 18:6353– 6364 Osawa Y, Banno Y, Nagaki M, Nozawa Y, Moriwaki H, Nakashima S 2001 Caspase activation during hepatocyte apoptosis induced by tumor necrosis factor-␣ in galactosamine-sensitized mice. Liver 21:309 –319 Imao M, Nagaki M, Imose M, Moriwaki H 2006 Differential caspase-9-dependent signaling pathway between tumor necrosis factor receptor- and Fas-mediated hepatocyte apoptosis in mice. Liver Int 26:137–146 Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ, Roth KA, MacGregor GR, Thompson CB, Korsmeyer SJ 2001 Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292:727–730 Zhao Y, Li S, Childs EE, Kuharsky DK, Yin XM 2001 Activation of pro-death Bcl-2 family proteins and mitochondria apoptosis pathway in tumor necrosis factor-␣-induced liver injury. J Biol Chem 276:27432–27440 Zhao Y, Ding WX, Qian T, Watkins S, Lemasters JJ, Yin XM 2003 Bid activates multiple mitochondrial apoptotic mechanisms in primary hepatocytes after death receptor engagement. Gastroenterology 125:854 – 867 Soriano ME, Nicolosi L, Bernardi P 2004 Desensitization of the permeability transition pore by cyclosporin a prevents activation of the mitochondrial apoptotic pathway and liver damage by tumor necrosis factor-␣. J Biol Chem 279:36803–36808 Guicciardi ME, Deussing J, Miyoshi H, Bronk SF, Svingen PA, Peters C, Kaufmann SH, Gores GJ 2000 Cathepsin B contributes to TNF-␣-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Invest 106:1127–1137 Guicciardi ME, Miyoshi H, Bronk SF, Gores GJ 2001 Cathepsin B knockout mice are resistant to tumor necrosis factor-␣-mediated hepatocyte apoptosis and liver injury: implications for therapeutic applications. Am J Pathol 159:2045–2054 Guicciardi ME, Bronk SF, Werneburg NW, Yin XM, Gores GJ


50.

51.

52.

53.

54.

55.

56.

57.

58. 59.

60.

61. 62.

63.

64.

65. 66. 67.

2005 Bid is upstream of lysosome-mediated caspase 2 activation in tumor necrosis factor ␣-induced hepatocyte apoptosis. Gastroenterology 129:269 –284 Ding WX, Ni HM, DiFrancesca D, Stolz DB, Yin XM 2004 Biddependent generation of oxygen radicals promotes death receptor activation-induced apoptosis in murine hepatocytes. Hepatology 40:403– 413 Okuyama H, Nakamura H, Shimahara Y, Araya S, Kawada N, Yamaoka Y, Yodoi J 2003 Overexpression of thioredoxin prevents acute hepatitis caused by thioacetamide or lipopolysaccharide in mice. Hepatology 37:1015–1025 Wu J, Liu L, Yen RD, Catana A, Nantz MH, Zern MA 2004 Liposome-mediated extracellular superoxide dismutase gene delivery protects against acute liver injury in mice. Hepatology 40:195– 204 Garcia-Ruiz C, Colell A, Mari M, Morales A, Calvo M, Enrich C, Fernandez-Checa JC 2003 Defective TNF-␣-mediated hepatocellular apoptosis and liver damage in acidic sphingomyelinase knockout mice. J Clin Invest 111:197–208 Malagarie-Cazenave S, Segui B, Leveque S, Garcia V, Carpentier S, Altie MF, Brouchet A, Gouaze V, Andrieu-Abadie N, Barreira Y, Benoist H, Levade T 2004 Role of FAN in tumor necrosis factor-␣ and lipopolysaccharide-induced interleukin-6 secretion and lethality in D-galactosamine-sensitized mice. J Biol Chem 279:18648 – 18655 Mari M, Colell A, Morales A, Paneda C, Varela-Nieto I, GarciaRuiz C, Fernandez-Checa JC 2004 Acidic sphingomyelinase downregulates the liver-specific methionine adenosyltransferase 1A, contributing to tumor necrosis factor-induced lethal hepatitis. J Clin Invest 113:895–904 Garcia-Ruiz C, Colell A, Mari M, Morales A, Fernandez-Checa JC 1997 Direct effect of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen species. Role of mitochondrial glutathione. J Biol Chem 272:11369 –11377 Garcia-Ruiz C, Colell A, Paris R, Fernandez-Checa JC 2000 Direct interaction of GD3 ganglioside with mitochondria generates reactive oxygen species followed by mitochondrial permeability transition, cytochrome c release, and caspase activation. FASEB J 14: 847– 858 Garcia-Ruiz C, Colell A, Morales A, Calvo M, Enrich C, Fernandez-Checa JC 2002 Trafficking of ganglioside GD3 to mitochondria by tumor necrosis factor-␣. J Biol Chem 277:36443–36448 Woo CH, Eom YW, Yoo MH, You HJ, Han HJ, Song WK, Yoo YJ, Chun JS, Kim JH 2000 Tumor necrosis factor-␣ generates reactive oxygen species via a cytosolic phospholipase A2-linked cascade. J Biol Chem 275:32357–32362 O’Donnell VB, Spycher S, Azzi A 1995 Involvement of oxidants and oxidant-generating enzyme(s) in tumour-necrosis-factor-␣mediated apoptosis: role for lipoxygenase pathway but not mitochondrial respiratory chain. Biochem J 310 (Pt 1):133–141 Foghsgaard L, Lademann U, Wissing D, Poulsen B, Jaattela M 2002 Cathepsin B mediates tumor necrosis factor-induced arachidonic acid release in tumor cells. J Biol Chem 277:39499 –39506 Wissing D, Mouritzen H, Egeblad M, Poirier GG, Jaattela M 1997 Involvement of caspase-dependent activation of cytosolic phospholipase A2 in tumor necrosis factor-induced apoptosis. Proc Natl Acad Sci USA 94:5073–5077 Streetz KL, Wustefeld T, Klein C, Kallen KJ, Tronche F, Betz UA, Schutz G, Manns MP, Muller W, Trautwein C 2003 Lack of gp130 expression in hepatocytes promotes liver injury. Gastroenterology 125:532–543 Camargo Jr CA, Madden JF, Gao W, Selvan RS, Clavien PA 1997 Interleukin-6 protects liver against warm ischemia/reperfusion injury and promotes hepatocyte proliferation in the rodent. Hepatology 26:1513–1520 Libert C, Brouckaert P, Fiers W 1994 Protection by ␣ 1-acid glycoprotein against tumor necrosis factor-induced lethality. J Exp Med 180:1571–1575 Libert C, Van Molle W, Brouckaert P, Fiers W 1996 ␣1-Antitrypsin inhibits the lethal response to TNF in mice. J Immunol 157:5126 – 5129 Van Molle W, Libert C, Fiers W, Brouckaert P 1997 ␣1-Acid glycoprotein and ␣ 1-antitrypsin inhibit TNF-induced but not anti-


68. 69.

70.

71.

72.

73. 74.

75. 76.

77.

78.

79.

80. 81.

82.

83.

84. 85.

Fas-induced apoptosis of hepatocytes in mice. J Immunol 159:3555– 3564 Beg AA, Sha WC, Bronson RT, Ghosh S, Baltimore D 1995 Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-␬B. Nature 376:167–170 Tanaka M, Fuentes ME, Yamaguchi K, Durnin MH, Dalrymple SA, Hardy KL, Goeddel DV 1999 Embryonic lethality, liver degeneration, and impaired NF-␬B activation in IKK-␤-deficient mice. Immunity 10:421– 429 Li ZW, Chu W, Hu Y, Delhase M, Deerinck T, Ellisman M, Johnson R, Karin M 1999 The IKK␤ subunit of I␬B kinase (IKK) is essential for nuclear factor ␬B activation and prevention of apoptosis. J Exp Med 189:1839 –1845 Rudolph D, Yeh WC, Wakeham A, Rudolph B, Nallainathan D, Potter J, Elia AJ, Mak TW 2000 Severe liver degeneration and lack of NF-␬B activation in NEMO/IKK␥-deficient mice. Genes Dev 14:854 – 862 Doi TS, Marino MW, Takahashi T, Yoshida T, Sakakura T, Old LJ, Obata Y 1999 Absence of tumor necrosis factor rescues RelAdeficient mice from embryonic lethality. Proc Natl Acad Sci USA 96:2994 –2999 Rosenfeld ME, Prichard L, Shiojiri N, Fausto N 2000 Prevention of hepatic apoptosis and embryonic lethality in RelA/TNFR-1 double knockout mice. Am J Pathol 156:997–1007 Alcamo E, Mizgerd JP, Horwitz BH, Bronson R, Beg AA, Scott M, Doerschuk CM, Hynes RO, Baltimore D 2001 Targeted mutation of TNF receptor I rescues the RelA-deficient mouse and reveals a critical role for NF-␬B in leukocyte recruitment. J Immunol 167: 1592–1600 Bellas RE, FitzGerald MJ, Fausto N, Sonenshein GE 1997 Inhibition of NF-␬B activity induces apoptosis in murine hepatocytes. Am J Pathol 151:891– 896 Xu Y, Bialik S, Jones BE, Iimuro Y, Kitsis RN, Srinivasan A, Brenner DA, Czaja MJ 1998 NF-␬B inactivation converts a hepatocyte cell line TNF-␣ response from proliferation to apoptosis. Am J Physiol 275:C1058 –C1066 Nagaki M, Naiki T, Brenner DA, Osawa Y, Imose M, Hayashi H, Banno Y, Nakashima S, Moriwaki H 2000 Tumor necrosis factor ␣ prevents tumor necrosis factor receptor-mediated mouse hepatocyte apoptosis, but not fas-mediated apoptosis: role of nuclear factor-␬B. Hepatology 32:1272–1279 Chaisson ML, Brooling JT, Ladiges W, Tsai S, Fausto N 2002 Hepatocyte-specific inhibition of NF-␬B leads to apoptosis after TNF treatment, but not after partial hepatectomy. J Clin Invest 110:193–202 Luedde T, Assmus U, Wustefeld T, Meyer zu Vilsendorf A, Roskams T, Schmidt-Supprian M, Rajewsky K, Brenner DA, Manns MP, Pasparakis M, Trautwein C 2005 Deletion of IKK2 in hepatocytes does not sensitize these cells to TNF-induced apoptosis but protects from ischemia/reperfusion injury. J Clin Invest 115: 849 – 859 Decker K, Keppler D 1974 Galactosamine hepatitis: key role of the nucleotide deficiency period in the pathogenesis of cell injury and cell death. Rev Physiol Biochem Pharmacol 71:77–106 Nagaki M, Sugiyama A, Osawa Y, Naiki T, Nakashima S, Nozawa Y, Moriwaki H 1999 Lethal hepatic apoptosis mediated by tumor necrosis factor receptor, unlike Fas-mediated apoptosis, requires hepatocyte sensitization in mice. J Hepatol 31:997–1005 Rothe J, Lesslauer W, Lotscher H, Lang Y, Koebel P, Kontgen F, Althage A, Zinkernagel R, Steinmetz M, Bluethmann H 1993 Mice lacking the tumour necrosis factor receptor 1 are resistant to TNFmediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature 364:798 – 802 Leist M, Gantner F, Jilg S, Wendel A 1995 Activation of the 55 kDa TNF receptor is necessary and sufficient for TNF-induced liver failure, hepatocyte apoptosis, and nitrite release. J Immunol 154: 1307–1316 Gantner F, Leist M, Lohse AW, Germann PG, Tiegs G 1995 Concanavalin A-induced T-cell-mediated hepatic injury in mice: the role of tumor necrosis factor. Hepatology 21:190 –198 Libert C, Van Bladel S, Brouckaert P, Shaw A, Fiers W 1991 Involvement of the liver, but not of IL-6, in IL-1-induced desensi-

384

86.

87.

88. 89.

90.

91.

92.

93.

94. 95.

96. 97.

98.

99.

100. 101. 102. 103.

104. 105.

Endocrine Reviews, June 2007, 28(4):365–386 tization to the lethal effects of tumor necrosis factor. J Immunol 146:2625–2632 Lavon I, Goldberg I, Amit S, Landsman L, Jung S, Tsuberi BZ, Barshack I, Kopolovic J, Galun E, Bujard H, Ben-Neriah Y 2000 High susceptibility to bacterial infection, but no liver dysfunction, in mice compromised for hepatocyte NF-␬B activation. Nat Med 6:573–577 Luedde T, Beraza N, Kotsikoris V, van Loo G, Nenci A, De Vos R, Roskams T, Trautwein C, Pasparakis M 2007 Deletion of NEMO/IKK␥ in liver parenchymal cells causes steatohepatitis and hepatocellular carcinoma. Cancer Cell 11:119 –132 Maeda S, Chang L, Li ZW, Luo JL, Leffert H, Karin M 2003 IKK␤ is required for prevention of apoptosis mediated by cell-bound but not by circulating TNF␣. Immunity 19:725–737 Ogushi I, Iimuro Y, Seki E, Son G, Hirano T, Hada T, Tsutsui H, Nakanishi K, Morishita R, Kaneda Y, Fujimoto J 2003 Nuclear factor ␬B decoy oligodeoxynucleotides prevent endotoxin-induced fatal liver failure in a murine model. Hepatology 38:335–344 Wheeler MD, Yamashina S, Froh M, Rusyn I, Thurman RG 2001 Adenoviral gene delivery can inactivate Kupffer cells: role of oxidants in NF-␬B activation and cytokine production. J Leukoc Biol 69:622– 630 Uesugi T, Froh M, Arteel GE, Bradford BU, Gabele E, Wheeler MD, Thurman RG 2001 Delivery of I␬B superrepressor gene with adenovirus reduces early alcohol-induced liver injury in rats. Hepatology 34:1149 –1157 Suetsugu H, Iimuro Y, Uehara T, Nishio T, Harada N, Yoshida M, Hatano E, Son G, Fujimoto J, Yamaoka Y 2005 Nuclear factor ␬B inactivation in the rat liver ameliorates short term total warm ischaemia/reperfusion injury. Gut 54:835– 842 Fan C, Li Q, Zhang Y, Liu X, Luo M, Abbott D, Zhou W, Engelhardt JF 2004 I␬B␣ and I␬B␤ possess injury context-specific functions that uniquely influence hepatic NF-␬B induction and inflammation. J Clin Invest 113:746 –755 Cheng JD, Ryseck RP, Attar RM, Dambach D, Bravo R 1998 Functional redundancy of the nuclear factor ␬B inhibitors I␬B␣ and I␬B␤. J Exp Med 188:1055–1062 Zwacka RM, Zhang Y, Zhou W, Halldorson J, Engelhardt JF 1998 Ischemia/reperfusion injury in the liver of BALB/c mice activates AP-1 and nuclear factor ␬B independently of I␬B degradation. Hepatology 28:1022–1030 Michalopoulos GK, DeFrances MC 1997 Liver regeneration. Science 276:60 – 66 Cressman DE, Greenbaum LE, Haber BA, Taub R 1994 Rapid activation of post-hepatectomy factor/nuclear factor ␬B in hepatocytes, a primary response in the regenerating liver. J Biol Chem 269:30429 –30435 Iimuro Y, Nishiura T, Hellerbrand C, Behrns KE, Schoonhoven R, Grisham JW, Brenner DA 1998 NF␬B prevents apoptosis and liver dysfunction during liver regeneration. J Clin Invest 101:802– 811 Plumpe J, Malek NP, Bock CT, Rakemann T, Manns MP, Trautwein C 2000 NF-␬B determines between apoptosis and proliferation in hepatocytes during liver regeneration. Am J Physiol Gastrointest Liver Physiol 278:G173–G183 Kirillova I, Chaisson M, Fausto N 1999 Tumor necrosis factor induces DNA replication in hepatic cells through nuclear factor ␬B activation. Cell Growth Differ 10:819 – 828 Cressman DE, Greenbaum LE, DeAngelis RA, Ciliberto G, Furth EE, Poli V, Taub R 1996 Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Science 274:1379 –1383 Yang L, Magness ST, Bataller R, Rippe RA, Brenner DA 2005 NF-␬B activation in Kupffer cells after partial hepatectomy. Am J Physiol Gastrointest Liver Physiol 289:G530 –G538 Hellerbrand C, Jobin C, Iimuro Y, Licato L, Sartor RB, Brenner DA 1998 Inhibition of NF␬B in activated rat hepatic stellate cells by proteasome inhibitors and an I␬B super-repressor. Hepatology 27: 1285–1295 Hanahan D, Weinberg RA 2000 The hallmarks of cancer. Cell 100:57–70 Bond M, Fabunmi RP, Baker AH, Newby AC 1998 Synergistic upregulation of metalloproteinase-9 by growth factors and inflam-


106. 107.

108. 109.

110. 111. 112. 113.

114.

115.

116.

117.

118. 119.

120. 121.

122.

123.

124. 125. 126.

matory cytokines: an absolute requirement for transcription factor NF-␬B. FEBS Lett 435:29 –34 Koch AE, Polverini PJ, Kunkel SL, Harlow LA, DiPietro LA, Elner VM, Elner SG, Strieter RM 1992 Interleukin-8 as a macrophagederived mediator of angiogenesis. Science 258:1798 –1801 Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S, Kasem S, Gutkovich-Pyest E, Urieli-Shoval S, Galun E, Ben-Neriah Y 2004 NF-␬B functions as a tumour promoter in inflammation-associated cancer. Nature 431:461– 466 Maeda S, Kamata H, Luo JL, Leffert H, Karin M 2005 IKK␤ couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis. Cell 121:977–990 Sakurai T, Maeda S, Chang L, Karin M 2006 Loss of hepatic NF-␬B activity enhances chemical hepatocarcinogenesis through sustained c-Jun N-terminal kinase 1 activation. Proc Natl Acad Sci USA 103:10544 –10551 Lieber CS 2004 Alcoholic fatty liver: its pathogenesis and mechanism of progression to inflammation and fibrosis. Alcohol 34:9 –19 Farrell GC, Larter CZ 2006 Nonalcoholic fatty liver disease: from steatosis to cirrhosis. Hepatology 43:S99 –S112 Hotamisligil GS, Shargill NS, Spiegelman BM 1993 Adipose expression of tumor necrosis factor-␣: direct role in obesity-linked insulin resistance. Science 259:87–91 Feinstein R, Kanety H, Papa MZ, Lunenfeld B, Karasik A 1993 Tumor necrosis factor-␣ suppresses insulin-induced tyrosine phosphorylation of insulin receptor and its substrates. J Biol Chem 268:26055–26058 Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M, Shoelson SE 2001 Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikk␤. Science 293:1673–1677 Kim JK, Kim YJ, Fillmore JJ, Chen Y, Moore I, Lee J, Yuan M, Li ZW, Karin M, Perret P, Shoelson SE, Shulman GI 2001 Prevention of fat-induced insulin resistance by salicylate. J Clin Invest 108: 437– 446 Hundal RS, Petersen KF, Mayerson AB, Randhawa PS, Inzucchi S, Shoelson SE, Shulman GI 2002 Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes. J Clin Invest 109:1321–1326 Rohl M, Pasparakis M, Baudler S, Baumgartl J, Gautam D, Huth M, De Lorenzi R, Krone W, Rajewsky K, Bruning JC 2004 Conditional disruption of I␬B kinase 2 fails to prevent obesity-induced insulin resistance. J Clin Invest 113:474 – 481 Cai D, Yuan M, Frantz DF, Melendez PA, Hansen L, Lee J, Shoelson SE 2005 Local and systemic insulin resistance resulting from hepatic activation of IKK-␤ and NF-␬B. Nat Med 11:183–190 Arkan MC, Hevener AL, Greten FR, Maeda S, Li ZW, Long JM, Wynshaw-Boris A, Poli G, Olefsky J, Karin M 2005 IKK-␤ links inflammation to obesity-induced insulin resistance. Nat Med 11: 191–198 Naveau S, Giraud V, Borotto E, Aubert A, Capron F, Chaput JC 1997 Excess weight risk factor for alcoholic liver disease. Hepatology 25:108 –111 Robin MA, Demeilliers C, Sutton A, Paradis V, Maisonneuve C, Dubois S, Poirel O, Letteron P, Pessayre D, Fromenty B 2005 Alcohol increases tumor necrosis factor ␣ and decreases nuclear factor-␬B to activate hepatic apoptosis in genetically obese mice. Hepatology 42:1280 –1290 DeAngelis RA, Markiewski MM, Taub R, Lambris JD 2005 A high-fat diet impairs liver regeneration in C57BL/6 mice through overexpression of the NF-␬B inhibitor, I␬B␣. Hepatology 42:1148 – 1157 Guebre-Xabier M, Yang S, Lin HZ, Schwenk R, Krzych U, Diehl AM 2000 Altered hepatic lymphocyte subpopulations in obesityrelated murine fatty livers: potential mechanism for sensitization to liver damage. Hepatology 31:633– 640 Li Z, Soloski MJ, Diehl AM 2005 Dietary factors alter hepatic innate immune system in mice with nonalcoholic fatty liver disease. Hepatology 42:880 – 885 Li Z, Lin H, Yang S, Diehl AM 2002 Murine leptin deficiency alters Kupffer cell production of cytokines that regulate the innate immune system. Gastroenterology 123:1304 –1310 Yang SQ, Lin HZ, Lane MD, Clemens M, Diehl AM 1997 Obesity


127.

128. 129. 130.

131.

132.

133.

134.

135.

136.

137.

138.

139.

140.

141.

142.

143.

144.

increases sensitivity to endotoxin liver injury: implications for the pathogenesis of steatohepatitis. Proc Natl Acad Sci USA 94:2557– 2562 Faggioni R, Jones-Carson J, Reed DA, Dinarello CA, Feingold KR, Grunfeld C, Fantuzzi G 2000 Leptin-deficient (ob/ob) mice are protected from T cell-mediated hepatotoxicity: role of tumor necrosis factor ␣ and IL-18. Proc Natl Acad Sci USA 97:2367–2372 Hentze MW, Muckenthaler MU, Andrews NC 2004 Balancing acts: molecular control of mammalian iron metabolism. Cell 117: 285–297 Pietrangelo A 2006 Hereditary hemochromatosis. Biochim Biophys Acta 1763:700 –710 Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, Dormishian F, Domingo Jr R, Ellis MC, Fullan A, Hinton LM, Jones NL, Kimmel BE, Kronmal GS, Lauer P, Lee VK, Loeb DB, Mapa FA, McClelland E, Meyer NC, Mintier GA, Moeller N, Moore T, Morikang E, Prass CE, Quintana L, Starnes SM, Schatzman RC, Brunke KJ, Drayna DT, Risch NJ, Bacon BR, Wolff RK 1996 A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 13:399 – 408 Ahmad KA, Ahmann JR, Migas MC, Waheed A, Britton RS, Bacon BR, Sly WS, Fleming RE 2002 Decreased liver hepcidin expression in the Hfe knockout mouse. Blood Cells Mol Dis 29: 361–366 Krause A, Neitz S, Magert HJ, Schulz A, Forssmann WG, SchulzKnappe P, Adermann K 2000 LEAP-1, a novel highly disulfidebonded human peptide, exhibits antimicrobial activity. FEBS Lett 480:147–150 Park K, Verchere CB 2001 Identification of a heparin binding domain in the N-terminal cleavage site of pro-islet amyloid polypeptide. Implications for islet amyloid formation. J Biol Chem 276:16611–16616 Pigeon C, Ilyin G, Courselaud B, Leroyer P, Turlin B, Brissot P, Loreal O 2001 A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem 276:7811–7819 Muckenthaler M, Richter A, Gunkel N, Riedel D, PolycarpouSchwarz M, Hentze S, Falkenhahn M, Stremmel W, Ansorge W, Hentze MW 2003 Relationships and distinctions in iron-regulatory networks responding to interrelated signals. Blood 101:3690 –3698 Nicolas G, Chauvet C, Viatte L, Danan JL, Bigard X, Devaux I, Beaumont C, Kahn A, Vaulont S 2002 The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J Clin Invest 110:1037–1044 Nicolas G, Bennoun M, Devaux I, Beaumont C, Grandchamp B, Kahn A, Vaulont S 2001 Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice. Proc Natl Acad Sci USA 98:8780 – 8785 Nicolas G, Bennoun M, Porteu A, Mativet S, Beaumont C, Grandchamp B, Sirito M, Sawadogo M, Kahn A, Vaulont S 2002 Severe iron deficiency anemia in transgenic mice expressing liver hepcidin. Proc Natl Acad Sci USA 99:4596 – 4601 Nemeth E, Rivera S, Gabayan V, Keller C, Taudorf S, Pedersen BK, Ganz T 2004 IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J Clin Invest 113:1271–1276 Lee P, Peng H, Gelbart T, Beutler E 2004 The IL-6- and lipopolysaccharide-induced transcription of hepcidin in HFE-, transferrin receptor 2-, and ␤ 2-microglobulin-deficient hepatocytes. Proc Natl Acad Sci USA 101:9263–9265 Montosi G, Corradini E, Garuti C, Barelli S, Recalcati S, Cairo G, Valli L, Pignatti E, Vecchi C, Ferrara F, Pietrangelo A 2005 Kupffer cells and macrophages are not required for hepatic hepcidin activation during iron overload. Hepatology 41:545–552 Gordeuk VR, Ballou S, Lozanski G, Brittenham GM 1992 Decreased concentrations of tumor necrosis factor-␣ in supernatants of monocytes from homozygotes for hereditary hemochromatosis. Blood 79:1855–1860 Hirayama M, Kohgo Y, Kondo H, Shintani N, Fujikawa K, Sasaki K, Kato J, Niitsu Y 1993 Regulation of iron metabolism in HepG2 cells: a possible role for cytokines in the hepatic deposition of iron. Hepatology 18:874 – 880 Tran TN, Eubanks SK, Schaffer KJ, Zhou CY, Linder MC 1997


145. 146. 147. 148. 149.

150. 151.

152.

153. 154. 155.

156.

157. 158.

159.

160.

161. 162. 163.

164.

Secretion of ferritin by rat hepatoma cells and its regulation by inflammatory cytokines and iron. Blood 90:4979 – 4986 Cornejo P, Varela P, Videla LA, Fernandez V 2005 Chronic iron overload enhances inducible nitric oxide synthase expression in rat liver. Nitric Oxide 13:54 – 61 Xiong S, She H, Sung CK, Tsukamoto H 2003 Iron-dependent activation of NF-␬B in Kupffer cells: a priming mechanism for alcoholic liver disease. Alcohol 30:107–113 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 –5305 Chen C, Edelstein LC, Gelinas C 2000 The Rel/NF-␬B family directly activates expression of the apoptosis inhibitor Bcl-x(L). Mol Cell Biol 20:2687–2695 Zong WX, Edelstein LC, Chen C, Bash J, Gelinas C 1999 The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-␬B that blocks TNF␣-induced apoptosis. Genes Dev 13:382–387 Opipari Jr AW, Hu HM, Yabkowitz R, Dixit VM 1992 The A20 zinc finger protein protects cells from tumor necrosis factor cytotoxicity. J Biol Chem 267:12424 –12427 Stehlik C, de Martin R, Kumabashiri I, Schmid JA, Binder BR, Lipp J 1998 Nuclear factor (NF)-␬B-regulated X-chromosomelinked iap gene expression protects endothelial cells from tumor necrosis factor ␣-induced apoptosis. J Exp Med 188:211–216 Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin Jr AS 1998 NF-␬B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 281: 1680 –1683 Werner AB, de Vries E, Tait SW, Bontjer I, Borst J 2002 Bcl-2 family member Bfl-1/A1 sequesters truncated bid to inhibit its collaboration with pro-apoptotic Bak or Bax. J Biol Chem 277:22781–22788 Roy N, Deveraux QL, Takahashi R, Salvesen GS, Reed JC 1997 The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases. EMBO J 16:6914 – 6925 Deveraux QL, Roy N, Stennicke HR, Van Arsdale T, Zhou Q, Srinivasula SM, Alnemri ES, Salvesen GS, Reed JC 1998 IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J 17:2215–2223 de la Coste A, Fabre M, McDonell N, Porteu A, Gilgenkrantz H, Perret C, Kahn A, Mignon A 1999 Differential protective effects of Bcl-xL and Bcl-2 on apoptotic liver injury in transgenic mice. Am J Physiol 277:G702–G708 Duriez PJ, Wong F, Dorovini-Zis K, Shahidi R, Karsan A 2000 A1 functions at the mitochondria to delay endothelial apoptosis in response to tumor necrosis factor. J Biol Chem 275:18099 –18107 Chu ZL, McKinsey TA, Liu L, Gentry JJ, Malim MH, Ballard DW 1997 Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-␬B control. Proc Natl Acad Sci USA 94:10057–10062 Zapata JM, Krajewska M, Krajewski S, Kitada S, Welsh K, Monks A, McCloskey N, Gordon J, Kipps TJ, Gascoyne RD, Shabaik A, Reed JC 2000 TNFR-associated factor family protein expression in normal tissues and lymphoid malignancies. J Immunol 165:5084 – 5096 Javelaud D, Besancon F 2001 NF-␬B activation results in rapid inactivation of JNK in TNF ␣-treated Ewing sarcoma cells: a mechanism for the anti-apoptotic effect of NF-␬B. Oncogene 20:4365– 4372 De Smaele E, Zazzeroni F, Papa S, Nguyen DU, Jin R, Jones J, Cong R, Franzoso G 2001 Induction of gadd45␤ by NF-␬B downregulates pro-apoptotic JNK signalling. Nature 414:308 –313 Tang G, Minemoto Y, Dibling B, Purcell NH, Li Z, Karin M, Lin A 2001 Inhibition of JNK activation through NF-␬B target genes. Nature 414:313–317 Tang F, Tang G, Xiang J, Dai Q, Rosner MR, Lin A 2002 The absence of NF-␬B-mediated inhibition of c-Jun N-terminal kinase activation contributes to tumor necrosis factor ␣-induced apoptosis. Mol Cell Biol 22:8571– 8579 Chang L, Kamata H, Solinas G, Luo JL, Maeda S, Venuprasad K, Liu YC, Karin M 2006 The E3 ubiquitin ligase itch couples JNK activation to TNF␣-induced cell death by inducing c-FLIP(L) turnover. Cell 124:601– 613

386


165. Wang Y, Singh R, Lefkowitch JH, Rigoli RM, Czaja MJ 2006 Tumor necrosis factor-induced toxic liver injury results from JNK2dependent activation of caspase-8 and the mitochondrial death pathway. J Biol Chem 281:15258 –15267 166. Liu H, Lo CR, Czaja MJ 2002 NF-␬B inhibition sensitizes hepatocytes to TNF-induced apoptosis through a sustained activation of JNK and c-Jun. Hepatology 35:772–778 167. Schwabe RF, Uchinami H, Qian T, Bennett BL, Lemasters JJ, Brenner DA 2004 Differential requirement for c-Jun NH2-terminal kinase in TNF␣- and Fas-mediated apoptosis in hepatocytes. FASEB J 18:720 –722 168. Marderstein EL, Bucher B, Guo Z, Feng X, Reid K, Geller DA 2003 Protection of rat hepatocytes from apoptosis by inhibition of c-Jun N-terminal kinase. Surgery 134:280 –284 169. Bradham CA, Stachlewitz RF, Gao W, Qian T, Jayadev S, Jenkins G, Hannun Y, Lemasters JJ, Thurman RG, Brenner DA 1997 Reperfusion after liver transplantation in rats differentially activates the mitogen-activated protein kinases. Hepatology 25:1128 – 1135 170. Uehara T, Xi Peng X, Bennett B, Satoh Y, Friedman G, Currin R, Brenner DA, Lemasters J 2004 c-Jun N-terminal kinase mediates hepatic injury after rat liver transplantation. Transplantation 78: 324 –332 171. Uehara T, Bennett B, Sakata ST, Satoh Y, Bilter GK, Westwick JK, Brenner DA 2005 JNK mediates hepatic ischemia reperfusion injury. J Hepatol 42:850 – 859 172. Trautwein C, Rakemann T, Brenner DA, Streetz K, Licato L, Manns MP, Tiegs G 1998 Concanavalin A-induced liver cell damage: activation of intracellular pathways triggered by tumor necrosis factor in mice. Gastroenterology 114:1035–1045 173. Streetz K, Fregien B, Plumpe J, Korber K, Kubicka S, Sass G, Bischoff SC, Manns MP, Tiegs G, Trautwein C 2001 Dissection of the intracellular pathways in hepatocytes suggests a role for Jun kinase and IFN regulatory factor-1 in Con A-induced liver failure. J Immunol 167:514 –523 174. Lee EG, Boone DL, Chai S, Libby SL, Chien M, Lodolce JP, Ma A 2000 Failure to regulate TNF-induced NF-␬B and cell death responses in A20-deficient mice. Science 289:2350 –2354 175. Arvelo MB, Cooper JT, Longo C, Daniel S, Grey ST, Mahiou J, Czismadia E, Abu-Jawdeh G, Ferran C 2002 A20 protects mice from D-galactosamine/lipopolysaccharide acute toxic lethal hepatitis. Hepatology 35:535–543 176. Wullaert A, Wielockx B, Van Huffel S, Bogaert V, De Geest B, Papeleu P, Schotte P, El Bakkouri K, Heyninck K, Libert C, Beyaert R 2005 Adenoviral gene transfer of ABIN-1 protects mice from TNF/galactosamine-induced acute liver failure and lethality. Hepatology 42:381–389 177. Wullaert A, Verstrepen L, Van Huffel S, Adib-Conquy M, Cornelis S, Kreike M, Haegman M, El Bakkouri K, Sanders M, Verhelst K, Carpentier I, Cavaillon JM, Heyninck K, Beyaert R 2007 LIND/ABIN-3 is a novel lipopolysaccharide-inducible inhibitor of NF-␬B activation. J Biol Chem 282:81–90 178. Sakon S, Xue X, Takekawa M, Sasazuki T, Okazaki T, Kojima Y, Piao JH, Yagita H, Okumura K, Doi T, Nakano H 2003 NF-␬B inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and necrotic cell death. EMBO J 22:3898 –3909 179. Kamata H, Honda S, Maeda S, Chang L, Hirata H, Karin M 2005 Reactive oxygen species promote TNF␣-induced death and sus-


180.

181.

182.

183.

184. 185.

186.

187.

188.

189. 190.

191.

192.

193.

tained JNK activation by inhibiting MAP kinase phosphatases. Cell 120:649 – 661 Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K, Sawada Y, Kawabata M, Miyazono K, Ichijo H 1998 Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO J 17:2596 –2606 Tobiume K, Matsuzawa A, Takahashi T, Nishitoh H, Morita K, Takeda K, Minowa O, Miyazono K, Noda T, Ichijo H 2001 ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep 2:222–228 Gilot D, Loyer P, Corlu A, Glaise D, Lagadic-Gossmann D, Atfi A, Morel F, Ichijo H, Guguen-Guillouzo C 2002 Liver protection from apoptosis requires both blockage of initiator caspase activities and inhibition of ASK1/JNK pathway via glutathione S-transferase regulation. J Biol Chem 277:49220 – 49229 Sass G, Shembade ND, Tiegs G 2005 Tumour necrosis factor ␣ (TNF)-TNF receptor 1-inducible cytoprotective proteins in the mouse liver: relevance of suppressors of cytokine signalling. Biochem J 385:537–544 He Y, Zhang W, Zhang R, Zhang H, Min W 2006 Socs1 inhibits TNF-induced activation of ASK1-JNK inflammatory signaling by mediating ASK1 degradation. J Biol Chem 281:5559 –5566 Tsutsui T, Koide H, Fukahori H, Isoda K, Higashiyama S, Maeda I, Tashiro F, Yamato E, Miyazaki J, Yodoi J, Kawase M, Yagi K 2003 Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis. Biochem Biophys Res Commun 307:765–770 Chen LW, Egan L, Li ZW, Greten FR, Kagnoff MF, Karin M 2003 The two faces of IKK and NF-␬B inhibition: prevention of systemic inflammation but increased local injury following intestinal ischemia-reperfusion. Nat Med 9:575–581 Vercammen D, Beyaert R, Denecker G, Goossens V, Van Loo G, Declercq W, Grooten J, Fiers W, Vandenabeele P 1998 Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J Exp Med 187:1477–1485 Cauwels A, Janssen B, Waeytens A, Cuvelier C, Brouckaert P 2003 Caspase inhibition causes hyperacute tumor necrosis factor-induced shock via oxidative stress and phospholipase A2. Nat Immunol 4:387–393 Hirosumi J, Tuncman G, Chang L, Gorgun CZ, Uysal KT, Maeda K, Karin M, Hotamisligil GS 2002 A central role for JNK in obesity and insulin resistance. Nature 420:333–336 Tuncman G, Hirosumi J, Solinas G, Chang L, Karin M, Hotamisligil GS 2006 Functional in vivo interactions between JNK1 and JNK2 isoforms in obesity and insulin resistance. Proc Natl Acad Sci USA 103:10741–10746 Solinas G, Naugler W, Galimi F, Lee MS, Karin M 2006 Saturated fatty acids inhibit induction of insulin gene transcription by JNKmediated phosphorylation of insulin-receptor substrates. Proc Natl Acad Sci USA 103:16454 –16459 Longo CR, Patel VI, Shrikhande GV, Scali ST, Csizmadia E, Daniel S, Sun DW, Grey ST, Arvelo MB, Ferran C 2005 A20 protects mice from lethal radical hepatectomy by promoting hepatocyte proliferation via a p21waf1-dependent mechanism. Hepatology 42:156 –164 Lademann U, Kallunki T, Jaattela M 2001 A20 zinc finger protein inhibits TNF-induced apoptosis and stress response early in the signaling cascades and independently of binding to TRAF2 or 14-3-3 proteins. Cell Death Differ 8:265–272

Endocrine Reviews is published by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.