Nature and Function
of Hepatic Tumor Necrosis Factor- α Signaling
Jörn M. Schattenberg, Mark J. Czaja
10
10.1
Introduction
Tumor necrosis factor- α (TNF) is a proinflamma- tory cytokine that regulates hepatic regeneration, injury and inflammation through autocrine and paracrine effects. The range of TNF's biological ac- tivities identifies it as a critical regulator of hepatic pathophysiology. Crucial to understanding the dis- parate effects of this cytokine on the liver has been the delineation of the cell signaling pathways that initiate these pleiotropic cellular processes. In par- ticular, investigations have attempted to determine how one factor could promote both cell proliferation and death in hepatocytes under different physiolog- ic circumstances. With these studies has come an understanding of the complex events that determine whether a hepatocyte proliferates or undergoes a- poptosis following TNF stimulation. This chapter will focus initially on signaling events that follow TNF ligand–receptor interaction, and subsequently on the hepatic pathophysiologic states regulated by TNF signaling. While considerable progress has been made in defining TNF signaling pathways in cultured hepatocytes, the challenge remains to de- termine how these signal cascades regulate human liver diseases.
10.2
TNF and TNF Receptors:
the Molecules and their Structure
Tumor necrosis factor- α is a cytokine produced pri- marily by activated macrophages, although epithe- lial cells, adipocytes and endothelial cells can also be sources of this protein [55, 70]. TNF is produced as a 26-kDa type II transmembrane protein with an extracellular C-terminal domain for receptor interaction, a single transmembrane domain, and an intracellular N-terminal domain essential for cell signaling [105]. The membrane form of TNF is
a homotrimer [9]. A soluble, 17-kDa form (sTNF) is produced through proteolytic cleavage of the trans- membrane form by the metalloprotease TNF alpha converting enzyme (TACE), a member of the mam- malian adamalysin (ADAM) family [15, 77]. The cleavage of TNF by TACE can be inhibited by the tis- sue inhibitors of metalloproteases (TIMP), includ- ing TIMP-3 [5]. The bioactivity of membrane-bound TNF is higher than that of sTNF, and sTNF homo- trimers dissociate below nanomolar concentrations and lose their bioactivity [107].
TNF exerts its biological effects by binding to ei- ther TNF receptor type 1 (TNF-R1, p55/65, CD120a) or TNF receptor type 2 (TNF-R2, p75/80, CD120b).
These receptors belong to the TNF receptor super- family that shares unique protein–protein interac- tion domains, which determine their cell signaling functions. These proteins are type I transmembrane molecules with an extracellular N-terminus and an intracellular C-terminus [105]. They are character- ized by the presence of extracellular cysteine-rich domains (CRD) that are critical for ligand binding and receptor interactions [19]. While the membra- nous and soluble forms of TNF induce equivalent activation of TNF-R1, full activation of TNF-R2 re- quires membrane-bound TNF [38]. Crystallograph- ic studies have suggested that a trimeric TNF-R1 interacts with the homotrimeric ligand [9]. Recent studies have demonstrated that rather than recep- tor trimerization occurring after ligand binding, the TNF-R exists as a preformed multimer to which the ligand binds in its homotrimeric form [1, 105].
The extracellular region responsible for receptor interaction and aggregation has been termed the preligand-binding assembly domain (PLAD) [19].
This region is physically distinct from the ligand-
binding domains, but crucial for TNF signaling. The
widespread effects of TNF stimulation are directly
related to the ubiquitous presence of TNF-R1 on
all human cells, while TNF-R2 expression is gener-
ally restricted to immune-derived and endothelial
cells [107]. Both receptor types are expressed in the
liver, but it is unclear whether type II receptors are
expressed only on non-parenchymal cells such as Kupffer cells, or on hepatocytes as well.
The expression of TNF-R1 and TNF-R2 is regu- lated differentially and in a cell-type-dependent manner. While TNF-R1 is controlled by a constitu- tive promoter, TNF-R2 expression is highly induc- ible and varies widely among cell types [45]. The rel- ative contribution of the two TNF receptors to TNF signaling is tissue dependent. In most cell types, including hepatocytes, TNF-R1 is required for the induction of apoptosis, while the role of TNF-R2 is less defined. Interestingly, TNF-R2 possesses a low- er binding affinity and higher dissociation rate for TNF than TNF-R1. This suggests that TNF-R2 may transiently bind and then release TNF, serving to in- crease local concentrations of TNF that then act on TNF-R1 [39]. This phenomenon is known as ligand- passing and may allow TNF-R1 activation at much lower TNF concentrations [10, 98]. Alternatively, overexpression of TNF-R2 may inhibit TNF signal- ing by competing with TNF-R1 for ligand [98]. Thus the overall biological effect of TNF may depend in part on the relative ratio of the two receptors [36].
However, TNF-R1 is capable of transducing all of the biological effects attributed to TNF at a much lower receptor density than TNF-R2. Therefore TNF-R2 is currently thought to play an accessory role, en- hancing or synergizing with TNF-R1 [105]. As will be discussed subsequently, the biological effects of TNF in the liver have been largely attributed to sig- naling through TNF-R1.
TNF-R cleavage normally occurs and yields solu- ble receptor fragments that have been implicated as decoy receptors capable of neutralizing TNF activity [85]. However, the binding affinity of the soluble re- ceptors is low in comparison to the membrane form, making their function unclear. Increases in circu- lating levels of these receptors have been reported in human liver diseases [94, 106]. These circulating receptors may serve to bind and neutralize the ac- tivity of TNF produced as part of the accompanying liver disorder. Alternatively they may temporarily bind and later release TNF, prolonging its biologi- cal effects. Soluble receptors have been used experi- mentally to block the effects of TNF in the liver. The administration of an engineered dimeric soluble TNF-R has been successfully employed in the pre- vention of toxic liver injury in rats [28].
10.3
TNF Signaling Pathways
10.3.1
Cell Death Signaling
Binding between TNF and TNF-R occurs at the plasma membrane through interactions between the CRD of the receptor and the trimeric ligand.
Binding results in a conformational change in the receptor and translocation of the receptor–ligand complex to lipid-enriched membrane microdo- mains known as lipid rafts [62]. Following ligand–
receptor interaction, an early intracellular signaling complex is formed to which signaling molecules are recruited [7]. While the outcome of TNF bind- ing to its receptor can result in divergent cellular effects, these early events are common to all of the biological effects of TNF signaling [47]. The intrac- ellular domains of TNF-R1 and TNF-R2 are devoid of intrinsic kinase activity and therefore depend on homophilic protein–protein interactions between motifs of approximately 80 amino acids for the initi- ation of cell signaling [86]. Based on the existence of one of two distinct domains, the TNF-R superfamily members are divided into two subgroups, the death domain (DD)-containing receptors and TNF-R-as- sociated factor (TRAF) interacting receptors [13].
TNF-R1 belongs to the first group and the earliest adaptor molecule recruited to the intracellular DD of the TNF-R1 is the TNF-R-associated death do- main protein (TRADD) [48]. Association with the DD is normally inhibited by binding of the silencer of death domain protein (SODD), which masks this site. Dissociation of SODD from the receptor oc- curs following ligand binding [52]. At this level of the TNF signaling pathway, the survival and death signaling pathways bifurcate and recruit different downstream effector molecules [47]. The apoptotic cell death pathway is activated following recruit- ment of the Fas-associated death domain (FADD/
MORT1) protein to TRADD through interactions
between the DD in each protein [21]. While critical
for TNF death signaling, FADD was first described
as an adaptor molecule mediating Fas-dependent
apoptosis [20]. In contrast, TRADD is not required
for death by the Fas pathway, but is unique to TNF
signaling. Besides the C-terminal DD, FADD carries
a second domain, the death effector domain (DED),
in its N-terminal region [101]. This domain recruits
proteins from the caspase (cysteine aspartate pro-
tease) family of enzymes. These proteases are ca-
pable of cleaving substrates after a loosely specific
series of amino acids that contain aspartate in the
first position. Crucial to their catalytic activity is the presence of a cysteine residue in the active center of the molecule [100]. The family can be divided into upstream initiator caspases, such as caspase-8 and -10, and the downstream effector caspases, caspase- 3, -6 and -7. The effector caspases are responsible for the cleavage of proteins whose functional loss induces apoptosis [35]. Caspases are constitutively expressed as inactive zymogens or procaspases that require cleavage into smaller active subunits. Pro- caspases contain DEDs, and the DED of caspase-8 allows its recruitment to the DED of FADD. Upon colocalization with FADD, high, localized concen- trations of procaspase-8 undergo autoproteolytic cleavage, releasing activated caspase-8. This com- plex has been termed the death-inducing signaling complex (DISC), and the mode of activation is re- ferred to as the induced proximity model of activa- tion [58, 78]. The specific involvement of FADD in TNF signaling in the liver has been demonstrated by studies in which adenoviral inhibition of FADD function in vivo blocked caspase-3 activation and liver damage from galactosamine-induced liver in- jury [94].
The apparent contradiction between the rapid recruitment of signaling molecules to TNF-R1, and the long delay before TNF-induced death occurs, has been recently resolved by studies in non-hepatic cells. The complex colocalizing with procaspase-8 in cells following TNF stimulation is undetectable before 30–60 min and not fully formed before 4–8 h [54, 74, 120]. This complex is composed of TRADD, FADD, the serine-threonine kinase receptor inter- acting protein (RIP), and the TNF-R-associated fac- tor-2 (TRAF-2), but is devoid of TNF-R. In accord- ance with this lack of receptor, the complex is de- tectable only in the cytosol, and not in membrane- enriched fractions [42, 74]. Thus, TNF-induced caspase-8 activation appears to occur following the dissociation of TRADD from the TNF-R, although the events triggering TNF-R-TRADD dissociation are unknown. The delay in formation of this intra- cellular DISC complex presumably explains why TNF-induced apoptosis in hepatocytes occurs at a much slower rate than that induced by the Fas death receptor.
After DISC formation, TNF-induced hepatocyte death results from the mitochondrial death pathway in which caspase-8 activation leads to functional changes in the mitochondria such as the mitochon- drial permeability transition (MPT). As a result, mitochondrial proteins such as cytochrome c are released into the cytosol and activate downstream caspases. Thus, the inhibition of cytochrome c re- lease from mitochondria by the MPT inhibitor cy- closporin A prevents hepatocyte apoptosis at a point
downstream of FADD binding to the TNF-R, but up- stream of caspase-3 activation [17]. The mechanism of cytochrome c release involves cleavage of the Bcl-2 family member Bid by caspase-8 [11]. Trun- cated Bid (tBid) migrates to the mitochondria and triggers oligomerization of the pro-apoptotic Bcl-2 family members Bax and Bak. These molecules then insert into the mitochondrial membrane, resulting in release of mitochondrial proteins including cyto- chrome c [109]. This function of Bid has been dem- onstrated to mediate hepatocyte death from TNF.
Hepatocytes deficient for Bid have increased resist- ance to TNF-induced cell death associated with the prevention of mitochondrial depolarization and cy- tochrome c release [124]. In vivo, Bid-deficient mice were partially protected from a TNF-dependent model of toxic liver injury [125]. Following release into the cytosol, cytochrome c triggers formation of the apoptosome, a complex with apoptosis pro- tease activating factor-1 (APAF-1) and procaspase-9.
Caspase-9 becomes activated and in turn activates caspase-3, resulting in apoptosis [127]. Hepatocytes and other cell types that are dependent on this mi- tochondrial death pathway have been termed type II cells. In contrast, type I cells generate high levels of caspase-8 that directly activate caspase-3. Accord- ingly, expression of the anti-apoptotic factors Bcl-2 and Bcl-X
Lthat inhibit Bid and Bax activation, pre- vents apoptosis in type II, but not type I cells [90].
In support of the concept of the hepatocyte as a type II cell is that in vivo Bcl-2 or Bcl-X
Loverexpression is partially effective in preventing liver injury from TNF [29, 104].
The mechanisms by which mitochondria pro- mote hepatocyte death from TNF are likely to be even more complex than those outlined. For ex- ample, the release of mitochondrial proteins other than cytochrome c may be involved. The mitochon- drial protein SMAC/DIABLO has been implicated in TNF-induced apoptosis [32], but the involvement of this protein in hepatocyte death is unknown. An- other mechanism of mitochondrial death pathway activation in hepatocytes is through the lysosomal cysteine protease cathepsin B. Hepatocyte death from TNF in vitro and in vivo is dependent on re- lease of cathepsin B from acidic vesicles [40, 110].
The pro-apoptotic effect of cathepsin B is above the
level of mitochondrial cytochrome c release as this
process is blocked in TNF-treated cathepsin B null
hepatocytes [40]. Additional evidence of an effect of
this protease on mitochondria is that in a cell-free
system, cathepsin B induced mitochondrial cyto-
chrome c release [40]. How the action of cathepsin
B integrates with the other components of the hepa-
tocyte TNF–mitochondrial death pathway is not yet
known. However, recent data suggest that this effect
of cathepsin B is mediated through activation of caspase-2 (G.J. Gores, personal communication).
Although the mitochondrial death pathway (summarized in Fig. 10.1) is an important mecha- nism of hepatocyte death from TNF, it is clearly not the only form of TNF death signaling. The partial effects of Bid ablation [125], or Bcl-2/Bcl-X
Loverex- pression [29, 104], on TNF-dependent liver injury in vivo suggest that the mitochondrial death path- way may be less important in whole liver than in cultured hepatocytes. Alternatively, the mitochon- drial pathway may be a rapid form of TNF-induced death whose inhibition uncovers another, slower
death signaling pathway. Other evidence exists for alternative TNF signaling pathways. TNF-induced cell death independent of caspase activation oc- curs in a differentiated hepatocyte cell line and in an in vivo model of TNF-dependent hepatitis [53, 60]. TNF can also cause necrosis as well as apopto- sis, as demonstrated by the ability of cytochrome P450 2E1 overexpression to sensitize rat hepatocytes to TNF-induced necrosis [68]. If these pathways of caspase-independent cell death occur in vivo, their existence would affect the design of anti-TNF treat- ments for liver disease. It is possible that therapeutic agents aimed at blocking the mitochondrial apop-
Fig. 10.1. TNF-induced proliferative and cell death signaling pathways. Binding of TNF to the TNF-R1 induces the recruitment of proteins through homophilic interactions of conserved do- mains. Initially there is binding of the adaptor molecule TRADD to the TNF-R1. The survival pathway (left side of the figure) is ac- tivated following recruitment of TRAF-2 and RIP to TRADD. This complex activates IKK, which phosphorylates IκB. Phosphorylat- ed IκB undergoes proteasome-dependent degradation, releas- ing NF-κB heterodimers that translocate to the nucleus and ac- tivate genes that may be necessary for TNF-induced hepatocyte proliferation. In addition, NF-κB-regulated gene products act to block the TNF apoptotic death pathway (right side of the fig- ure). Signaling through TRAF-2 leads to activation of the JNK/c- Jun/AP-1 pathway. AP-1-dependent gene expression promotes
hepatocyte proliferation, but sustained AP-1 activation may al- ternatively serve to trigger the apoptotic death pathway. In the death pathway, the TRADD-TRAF-2-RIP complex dissociates from the TNF-R1 and recruits FADD and procaspase-8. This complex releases caspase-8 following its autolytic activation. Caspase-8 cleaves Bid, producing a truncated and active tBid. tBid activates the pro-apoptotic Bcl-2 family members Bax and Bak. Their oli- gomerization and integration into the mitochondrial membrane results in cytochrome c release. In a complex consisting of cyto- chrome c and APAF-1, procaspase-3 is recruited and activated, resulting in apoptosis. Additionally, caspase-8 activation causes release of the lysosomal enzyme cathepsin B, which acts in an undefined fashion to activate the mitochondrial death pathway
totic pathway or caspase activation will not be ef- fective because the hepatocyte is then shunted into an alternative, caspase-independent death signaling pathway.
10.3.2
Cell Survival Signaling Pathways
The ability of TNF to induce either cellular prolif- eration or death has led to the concept that TNF signaling acts as “a double-edged sword” [1]. The mechanism by which hepatocytes block the TNF death signaling pathway has recently been deline- ated. Investigations in cultured hepatocytes and animals have identified nuclear factor- κB (NF-κB) signaling as critical for the maintenance of hepato- cyte resistance to TNF killing. The NF- κB family of transcription factors consists of NF- κB1/p50, NF- κB2/p52, c-Rel, RelA/p65 and RelB. In addition, pre- cursor proteins of NF- κB1 (p105) and NF-κB2 (p100) exist and have been implicated in the regulation of NF- κB activation [75, 93]. The common feature of these NF- κB family members is the presence of the Rel homology domain (RHD), which is important for protein dimerization, DNA binding, nuclear lo- calization and interactions with inhibitory proteins [37].
NF- κB activation occurs in response to a number of inflammatory mediators including TNF. The ini- tial steps of TNF-dependent NF- κB activation are identical to the TNF-initiated apoptotic pathway described previously (Fig. 10.1). Following recep- tor–ligand interaction, recruited TRADD binds to TRAF family proteins [83]. This family consists of six distinct members, TRAF 1–6, all of which are capable of binding to the TRADD death domain by means of their highly conserved C-terminal TRAF- C domain [82]. TRAF-2, TRAF-5 and TRAF-6 have been implicated in the activation of NF- κB, and de- letion of Traf-2 and Traf-5 in mice blocks TNF-in- duced NF- κB activation [97]. These proteins trigger NF- κB activation through their effects on the IκB kinases (IKK). In resting cells, NF- κB is sequestered in the cytoplasm as an inactive heterodimeric com- plex bound to an inhibitory counterpart I κB. Phos- phorylation of I κB by IKK results in IκB dissociation from NF- κB, ubiquination and proteasome-depend- ent degradation [84]. As a result, the nuclear locali- zation signal of NF- κB is unmasked, leading to its translocation to the nucleus and activation of gene expression. The IKK complex consists of two cata- lytically active kinases, IKK α and IKKβ, the regu- latory kinase IKK γ/NEMO, and heat shock protein 90 (Hsp90) with its associated protein cdc37. TRAF proteins recruit the IKK complex to the membrane-
bound TNF-R complex, but a second receptor-bound protein, RIP, is also required for full IKK activation [33]. Rip-deficient cells are able to recruit IKK α and IKK β to the TNF-R complex following TNF binding, but recruitment of the regulatory subunit IKK γ/
NEMO is significantly reduced [34]. While identi- cal functions for these proteins in hepatocytes are likely, this remains to be established. It is known that NF- κB activation in hepatocytes is mediated by the actions of IKK β and not IKKα [91]. Additional regulation of the transcriptional activity of NF- κB can result from direct phosphorylation of its subu- nits [79]. Numerous kinases have been implicated in NF- κB phosphorylation, including mitogen-acti- vated protein kinases (MAPK), protein kinase C iso- forms, casein kinase II and the nuclear DNA repair enzyme poly(ADP-ribose) polymerase-1 (PARP-1) [64, 80, 108]. The contribution of phosphorylation to NF- κB activation in the liver is unknown.
The initial suggestion that NF- κB signaling was important in hepatic homeostasis was the finding that genetic ablation of the NF- κB p65 subunit re- sulted in embryonic lethality from hepatocellular apoptosis [12]. Subsequent studies demonstrated that the hepatic apoptosis in p65-NF- κB null mice was induced by TNF, because the mice were rescued from their lethal phenotype through simultaneous inactivation of TNF-R1 [4]. Knockouts of the Ikk β or Ikk γ/Nemo genes are also embryonic lethals, stress- ing the physiological importance of IKK-dependent I κB phosphorylation in hepatic NF-κB activation [37]. The initial confirmation that NF- κB activa- tion mediated hepatocyte resistance to TNF toxicity came from studies of NF- κB inactivation in cultured hepatocytes. In both primary rat hepatocytes and a non-transformed rat hepatocyte line, inhibition of NF- κB activity by adenoviral delivery of a phospho- rylation defective mutant I κB sensitized these cells to death from TNF [17, 112]. Despite virtual total in- hibition of NF- κB activity with this adenovirus, only partial cell death occurred, suggesting that other signaling pathways may compensate for the loss of NF- κB. Investigations in human hepatocytes have demonstrated the additional involvement of NF- κB- independent survival pathways mediated by sphin- gosine kinase and phosphatidylinositol 3-kinase/
Akt in hepatocyte resistance to TNF toxicity [81].
However, in mouse hepatocyte studies, Akt signal- ing promoted resistance to TNF through induction of NF- κB activation [44]. Thus, it remains unclear which signaling pathways in addition to NF- κB may affect hepatocyte resistance to TNF.
The mechanism by which NF- κB signaling
blocks the TNF death signaling pathway in hepa-
tocytes is unknown. NF- κB inactivation sensitizes
hepatocytes to TNF-induced death through the clas-
sical mitochondrial death pathway, as evidenced by mitochondrial changes, cytochrome c release, and resultant caspase-dependent apoptosis [17, 112].
The fact that hepatocyte resistance to TNF toxic- ity requires transcription and translation [63], and activation of the transcription factor NF- κB, sug- gests that NF- κB upregulates a gene(s) that protects against TNF cytotoxicity. However, the identity of this gene(s) remains unknown. Several TNF-regu- lated, NF- κB-dependent genes have been impli- cated in the protection of non-hepatic cells from TNF toxicity, but TNF protective genes may well be cell-type specific. In TNF-treated hepatocytes, the TNF-responsive, NF- κB-dependent gene inducible nitric oxide synthase (iNOS) has been demonstrated to be protective against TNF toxicity [43]. However, the protective effects of endogenous iNOS were only partial, suggesting that its induction cannot com- pletely explain NF- κB-mediated hepatocyte resist- ance to TNF toxicity.
Recent investigations have demonstrated that the protective effects of NF- κB activation may occur not from the induction of a gene with direct anti- apoptotic effects, but rather through crosstalk with the MAPK signaling pathway. An additional TNF signaling pathway is the TRAF-2-dependent activa- tion of the MAPK family member c-Jun N-terminal kinase (JNK) [69, 121]. Activation of JNK following recruitment of TRAF-2 to the receptor signaling complex involves a series of kinases that converge on the MAPK kinases MKK4 and MKK7, which then in turn phosphorylate and activate JNK [103]. Ac- tivated JNK phosphorylates substrates that include the activator protein-1 (AP-1) transcription factor subunit c-Jun, leading to increased AP-1 transcrip- tional activity [25]. Transient JNK/AP-1 activation occurs after TNF stimulation and may in part medi- ate the proliferative effects of TNF [92]. With NF- κB inhibition, TNF induces a prolonged activation of JNK and AP-1 in hepatocytes and non-hepatic cells in association with cell death [30, 69]. Death from TNF and NF- κB inactivation was blocked in a rat non-transformed cell line by inhibition of c-Jun function through adenoviral expression of the c-Jun dominant negative TAM67. Blocking c-Jun function with TAM67 prevented mitochondrial cytochrome c release and caspase activation, suggesting that an AP-1 gene product activated the TNF mitochondrial death pathway [69]. However, in studies in primary rat hepatocytes, although the chemical JNK inhibi- tor SP600125 blocked death from TNF and NF- κB inhibition, TAM67 expression had no effect on death in these cells (D.A. Brenner, personal communica- tion). The JNK inhibitor also blocked death at the level of the mitochondria, suggesting that JNK itself has direct pro-apoptotic effects on mitochondria.
Finally, in TNF-treated hepatoma cells, JNK exerted an anti-apoptotic effect independent of c-Jun/AP-1 activation when JNK was inhibited by a dominant negative protein to the upstream kinase transform- ing growth factor- β activated kinase (TAK) [67].
The divergent results in these studies may reflect cell type differences, or the fact that inhibition of the JNK/c-Jun/AP-1 pathway at different levels may have additional effects on other effector molecules and signaling pathways. Although in vivo correlates of the effects of JNK/AP-1 signaling on TNF-induced liver injury have been lacking, a recent in vivo study as discussed subsequently has shown that TNF-de- pendent liver injury from concanavalin A (Con A) is blocked in Jnk knockout mice.
The activation and involvement of other MAPK family members in hepatocyte TNF signaling have not been described. Although extracellular signal- regulated kinase 1/2 (ERK1/2) has been implicated as a cytoprotective pathway in several forms of cell death, ERK1/2 has not been shown to be regulated by TNF or to protect against TNF toxicity in hepato- cytes. TNF is known to activate p38 MAPK in a RIP- dependent manner in fibroblasts [61]. However, p38 MAPK activation is not seen in a rat hepatocyte cell line after TNF stimulation, and inhibition of this MAPK does not affect death from TNF in these cells (M.J. Czaja, unpublished observation).
10.4
Function of TNF Signaling in Hepatic Pathophysiology
10.4.1
Liver Regeneration
The liver has the unique capability to switch from
a quiescent to a proliferative state in response to a
loss of mass secondary to surgical reduction or cell
death. A critical function for TNF signaling in he-
patic regeneration has been demonstrated by inves-
tigations in both models of partial hepatectomy and
liver injury. Within an hour, TNF is produced in the
liver in response to a partial hepatectomy [70]. In
contrast to macrophage-dependent TNF generation
during liver injury, there is evidence that the sourc-
es of TNF production after partial hepatectomy are
biliary and endothelial cells [70]. Neutralizing anti-
TNF antibodies inhibit hepatocyte proliferation
following partial hepatectomy, clearly implicating
TNF as a direct or indirect hepatocyte mitogen in
this model [2]. Subsequent studies of partial hepa-
tectomy in Tnf-r1 knockout mice also demonstrated
reduced DNA synthesis and liver mass in these mice as compared to wild-type animals [116]. Inhibition of the biological activities of TNF led to reduced acti- vation of the downstream transcriptional regulators NF- κB, signal transducer and activator of transcrip- tion 3 (STAT3), and AP-1, but not of CCAAT/enhanc- er-binding protein (C/EBP), as measured by levels of DNA binding [116]. In contrast, posthepatectomy liver regeneration was normal in Tnf-r2 null mice [117]. Absence of the type 2 receptor had no effect on NF- κB and STAT3 binding or IL-6 production, but caused a delay in AP-1 and C/EBP binding [117].
These data suggest that TNF-induced proliferation occurs exclusively through signaling by the type 1 receptor, again demonstrating the predominant role for the TNF-R1 in hepatic TNF responses. Studies of carbon tetrachloride-induced liver regeneration also demonstrated that a lack of TNF-R1-mediated signaling decreased the regenerative response [115].
However, in all of these studies loss of TNF signal- ing merely delayed liver regeneration, but did not prevent the eventual return of the liver to a normal mass. Thus, TNF seems directly or indirectly to pro- mote the initiation of hepatocyte proliferation, but TNF signaling is not obligatory for liver regenera- tion to occur.
Subsequent investigations in Il-6 null mice sug- gested that this cytokine is critical for liver regen- eration and survival after partial hepatectomy [24], in part through the activation of STAT3 [66]. In another study, similar effects were seen, although some mice survived and had a normal regenerative response [16]. In carbon tetrachloride-treated mice lacking Tnf-r1, the reduction in liver regeneration resulting from the absence of TNF signaling was prevented by injection of IL-6 [115]. These investi- gations all suggested that the effects of TNF on liver regeneration may not be direct, but rather mediated through IL-6 signaling. A possible signaling cascade for liver regeneration is that TNF-induced activation of NF- κB leads to the production from Kupffer cells or other non-parenchymal cells of IL-6 that acti- vates STAT3 and other downstream signals required for hepatocyte proliferation. However, other studies in Il-6 null mice and in mice with ablation of the IL- 6 downstream signaling molecule glycoprotein 130 (gp130) have revealed no significant effect of the loss of IL-6 signaling on DNA synthesis after partial hepatectomy [89, 111]. Treatment with lipopolysac- charide (LPS) after partial hepatectomy did result in a reduction in DNA synthesis and survival in Il- 6 null mice associated with decreased Bcl-X
Llevels and increased hepatocyte apoptosis [111]. This work suggests that the function of IL-6 after partial hepa- tectomy is not to regulate DNA synthesis, but to up- regulate protective acute phase proteins. The direct
effects of TNF on hepatocyte proliferation after par- tial hepatectomy therefore need to be re-evaluated.
The importance of TNF-induced NF- κB acti- vation after partial hepatectomy was suggested by studies in which inhibition of NF- κB signaling by adenoviral expression of a phosphorylation-de- fective mutant I κB blocked liver regeneration and caused massive hepatic apoptosis [49]. DNA syn- thesis was unaffected by NF- κB inhibition, but cell cycle block in late S or G2 occurred along with ap- optosis. The p65 NF- κB subunit seems most critical for this NF- κB function as liver regeneration after partial hepatectomy was unaffected by the absence of its heterodimeric partner p50 [31]. A potential NF- κB-dependent gene responsible for protection against the cytotoxic effects of TNF after partial hepatectomy was identified in studies that revealed marked post-partial hepatectomy apoptosis in iNos knockout mice [87]. However, it is unclear whether NF- κB activation in hepatocytes mediated these ef- fects. Hepatocyte-specific ablation of NF- κB or its upstream kinase IKK β failed to affect post-partial hepatectomy-induced liver regeneration [18, 71].
One study also noted an absence of apoptosis, but the 45% inhibition of NF- κB activation achieved in this study may have been insufficient to block fully the protective effects of NF- κB [18]. The discordant results of global versus hepatocyte-specific NF- κB inhibition may reflect species differences, or the fact that additional effects on non-parenchymal cell NF- κB activity are required to trigger apopto- sis after partial hepatectomy. A correlation between the effects of the various forms of NF- κB inhibition employed in these investigations with post-partial hepatectomy IL-6 production is important, as find- ings of decreased IL-6 production only with inhibi- tion of non-parenchymal cell NF- κB activation may explain these contradictory results.
Less is known about the role of TNF signaling in chronic states of liver injury and regeneration as- sociated with elevated TNF levels such as alcoholic liver disease. Rats chronically fed alcohol have a decreased regenerative response after partial hepa- tectomy despite serum TNF and IL-6 levels similar to normal rats [3]. Ethanol-fed rats had decreased NF- κB and c-Jun activation following partial hepa- tectomy, suggesting an alcohol-induced impair- ment of these proliferative signaling pathways [118].
However, ethanol-fed rats were clearly responsive to
TNF as its inhibition reduced liver regeneration to a
greater extent in ethanol-fed animals than in con-
trol rats [3]. Thus, there is contradictory evidence
that ethanol-fed animals are both more dependent
on the regenerative stimulus of TNF, as well as re-
fractory to its effects on signaling pathways thought
to be involved in proliferative TNF signaling.
10.4.2
Toxin-Induced Liver Injury
Until recently, toxin-induced liver injury was thought to result from the direct biochemical effects of the toxin or its metabolite on the hepatocyte. It is now apparent that liver injury results in large part from the effects of inflammatory cell products on the hepatocyte and its cell signaling pathways. Most prominent among the products of inflammation that mediate toxin-induced liver injury is TNF. TNF is produced as part of the liver's response to hepa- totoxins such as carbon tetrachloride and galactos- amine [26]. It was first demonstrated in the carbon tetrachloride model that TNF induced most of the liver injury from this hepatotoxin. Neutralization of TNF by a dimeric soluble receptor markedly re- duced carbon tetrachloride-induced liver injury as measured by transaminases, histology and mortal- ity [28]. Decreased injury from carbon tetrachloride was also subsequently demonstrated in both Tnf and Tnf-r1 null mice [76]. The involvement of TNF in hepatotoxic liver injury has now been demonstrated for a number of toxins including ones relevant to human liver disease such as ethanol [122]. The simi- lar ability of agents that neutralize LPS to prevent liver injury from carbon tetrachloride and ethanol suggests that a common mechanism of toxic injury is through LPS-induced macrophage activation and the resultant production of TNF that directly causes hepatocyte injury and death.
The ability of TNF to act as a hepatocyte cytotox- in had to be reconciled with its beneficial mitogenic effects after partial hepatectomy. Another apparent contradiction in the concept of TNF as a mediator of liver injury was that although TNF was known to be cytotoxic to many transformed cells, normal cells were resistant to TNF toxicity. However, cells normally resistant to TNF, including hepatocytes, become sensitized to death from TNF by transcrip- tional or translational arrest [63]. These findings suggested that: (1) resistance to TNF requires tran- scriptional upregulation of a protective gene(s); (2) following partial hepatectomy, the remaining nor- mal cells are able to upregulate this protective gene, resulting in a non-toxic, proliferative TNF effect;
and (3) because hepatotoxins interfere with macro- molecular synthesis, they might block the protec- tive response and thereby sensitize hepatocytes to death from TNF. Alternatively, toxins might trigger TNF injury by somehow causing such a massive out- pouring of TNF that cellular protective mechanisms are overwhelmed and toxicity occurs despite their induction (Fig. 10.2). Although some toxins do aug- ment the induction of TNF by LPS [46], others act
by interfering with the ability of hepatocytes to up- regulate protective genes [27]. This later mechanism is the predominant one by which toxins sensitize hepatocytes to death from TNF.
These findings have led to an investigative focus on the identification of the protective factor(s) that mediates hepatocyte resistance to TNF toxicity. The ability of TNF to induce cellular oxidative stress ini- tially led to an examination of the potential role of antioxidant factors in hepatocyte TNF resistance.
Although the antioxidant enzyme manganese su- peroxide dismutase (MnSOD) had been implicated as the protective factor in non-hepatic cells, it was demonstrated that MnSOD was not the inducible pro- tective factor in hepatocytes because although TNF upregulated MnSOD gene expression, no increase in protein occurred [27]. Levels of the principle hepatic non-enzymatic antioxidant glutathione (GSH) are also not regulated at a transcriptional level by TNF [113]. However, depressed levels of GSH do worsen TNF-induced toxic liver injury in vivo [113]. Con- tradictory reports of glutathione depletion prevent- ing TNF-induced hepatocyte death are an artifact of the ability of sudden, profound decreases in GSH to block DISC formation [41]. Oxidative stress, even in the absence of antioxidant depletion, may also act to promote death from TNF. Chronic oxidative stress generated by overexpression of cytochrome P450
Fig. 10.2. Mechanisms by which TNF induces proliferation after partial hepatectomy but cell death after toxic liver injury. Fol- lowing partial hepatectomy (left figure), increased levels of TNF cause an upregulation of protective factors in the remaining hepatocytes. These cells are resistant to the toxic effects of TNF and undergo cell proliferation, leading to liver regeneration. In contrast, increases in TNF during toxic liver injury activate cell signaling events that result in cell death (right figure). Although hepatocellular protective factors are upregulated, the hepato- toxin may promote such a massive release of TNF that the defen- sive mechanisms are overwhelmed and cell death occurs. Alter- natively, the inhibitory effects of the toxin on macromolecular synthesis may block the generation of protective factors, and in their absence hepatocytes undergo cell death
2E1 sensitizes cultured hepatocytes to death from TNF [68], and may play a role in promoting TNF injury in steatohepatitis. Thus, it seems that while TNF does not directly regulate antioxidants, im- paired antioxidant defenses or increased oxidative stress may promote liver injury from TNF. However, the role of oxidative stress in TNF death signaling has become even more complex with the realization that antioxidants can act to promote death from TNF [8].
The previously discussed in vitro findings that NF- κB inactivation and resultant JNK/AP-1 over- activation sensitize hepatocytes to death from TNF suggest that these pathways may be involved in TNF-mediated injury from hepatotoxins. Evidence of in vivo impairment of NF- κB signaling or JNK/
AP-1 overactivation is currently lacking, however.
LPS administration to mice chronically fed etha- nol resulted in increased liver injury as compared to control-fed mice [59]. However, no alterations in LPS-induced NF- κB or JNK activation occurred in the ethanol-fed mice. Despite caspase-3 activation in LPS-treated control mice, caspase activation, cy- tochrome c release and DNA fragmentation were all absent in the ethanol-fed mice after LPS treatment [59]. Sensitization of hepatocytes to LPS injury by ethanol therefore did not occur through the mito- chondrial caspase-dependent pathway. Investiga- tions in the galactosamine/LPS model have dem- onstrated that this TNF-induced form of toxic liver injury is caspase dependent, but also may occur through a mechanism other than the mitochondrial death pathway [50]. Finally, while cotreatment with galactosamine and the immune modulator Con A results in a caspase-dependent death, injury from Con A alone, although TNF mediated, is caspase independent [60]. Thus, a failure of NF- κB-depend- ent gene upregulation resulting in activation of the caspase-dependent mitochondrial death pathway cannot be the mechanism of all TNF-induced liver injury.
Although whether NF- κB activation is inhibited by hepatotoxins remains unclear, recent studies have demonstrated that NF- κB activation in vivo attenu- ates toxic liver injury. With the use of a conditional Cre-lox system, selective hepatocyte ablation of Ikk β, the upstream activating kinase of NF-κB, led to increased liver injury from galactosamine/LPS as well as Con A [71]. Injury did not occur with LPS ad- ministration alone, suggesting that NF- κB inactiva- tion by itself was insufficient to sensitize the liver to injury from TNF. It remains possible, however, that injury would occur with a more robust induction of TNF. While JNK activation after galactosamine in- jury was not examined, in Con A injury increased JNK activation occurred in the absence of NF- κB
signaling, and injury was decreased in either Jnk1 or Jnk2 knockout animals [71]. These findings are con- sistent with those in cultured hepatocytes, and dem- onstrate both the ability of NF- κB to downregulate hepatic JNK activation in vivo, and the involvement of the JNK signaling pathway in hepatocyte injury in whole liver [69].
While it is possible that NF- κB activity is nec- essary solely to inhibit JNK activation, other NF- κB-dependent genes are likely to be involved in re- sistance against TNF toxicity. Although a number of potential, TNF-inducible, NF- κB-dependent protective genes have been identified in non-he- patic cells, their function in hepatocytes is largely unknown. One gene specifically studied in hepato- cytes is iNOS, which was previously discussed for its protective effects after partial hepatectomy. TNF induces hepatic iNOS in mice, and this upregulation is blocked by inhibition of NF- κB [43]. TNF by itself caused liver damage in iNos null mice, but the de- gree of injury suggested that loss of iNOS alone was not sufficient to fully sensitize hepatocytes to death from TNF [43]. Nitric oxide generated by iNOS may protect hepatocytes from TNF-induced toxic in- jury by the inactivation of caspases and inhibition of the mitochondrial death pathway [65]. A second, TNF-inducible protective factor, A20, has also been shown to block the lethality of galactosamine/LPS when overexpressed in mice [6]. However, it is not known if toxins prevent the normal TNF-induced upregulation of this factor. The effect of A20 overex- pression appears to be through the promotion of liv- er regeneration rather than the prevention of injury, suggesting that factors that selectively block TNF death signaling may offer an advantage over TNF- neutralizing agents by preserving the proliferative effects of TNF [6].
10.4.3
Ischemia-Reperfusion Injury
Hepatic ischemia-reperfusion (IR) injury occurs
following complete or partial interruption of he-
patic blood flow and the subsequent restoration of
blood supply and reoxygenation. During the initial
phase of IR injury, reduced oxygenation results in
a decrease in cellular pH from anaerobic glycolysis,
the onset of mitochondrial dysfunction and the gen-
eration of reactive oxygen species. The activation of
Kupffer cells leads to the release of TNF, chemokines
and activated complement that all serve to amplify
Kupffer cell activation, and recruit inflammatory
cells through increased endothelial expression of
cellular adhesion molecules [114, 123]. The involve-
ment of TNF in IR injury has been demonstrated by
a reduction in injury in Tnf-r1 knockout mice [88].
However, in contrast to toxin-induced liver injury, the hepatocellular damage during IR injury does not result from the direct cellular toxic effects of TNF. Rather TNF mediates injury through its abil- ity to recruit and activate inflammatory cells [23].
The resulting parenchymal and vascular-endothe- lial damage appears to be a necrotic rather than ap- optotic cell death [51].
The use of a brief, benign period of ischemia to
“precondition” the liver has been shown to protect the organ from a more lengthy and injurious sub- sequent period of IR. Preconditioning leads to in- creased NF- κB activation and decreased release of TNF during the subsequent episode of IR [99]. These observations are in agreement with the ability of low levels of TNF to induce hepatoprotective NF- κB and AP-1 signaling. Thus, stimulation of TNF protective pathways may be a strategy to prevent liver injury, and ischemic preconditioning in patients undergo- ing partial hepatectomy has been shown to result in decreased liver injury [22].
10.4.4
Viral Hepatitis
The pathogenesis of liver cell injury in acute and chronic viral hepatitis is poorly understood, but proinflammatory cytokines are thought to play a central role in modulating the cellular immune re- sponse, virus replication and liver cell injury. Infil- trating cytotoxic T-lymphocytes decrease hepatitis B virus (HBV) gene expression and replication in the liver of HBV transgenic mice through the secre- tion of TNF as well as interferon- α, -β and -γ [73].
TNF reduces the production and secretion of HBV DNA and induces apoptosis in an HBV transgenic hepatoma cell line [102, 119]. TNF also induces non- cytopathic HBV clearance from infected hepatocytes through the activation of IKK and NF- κB signaling, which triggers disruption of the viral capsid [14].
Central to the pro-apoptotic effects of TNF in infect- ed hepatocytes is HBV protein X (HBx). Expression of HBx increases the susceptibility of hepatocytes to cell death from TNF through activation of MAPK kinase 1 (MKK1) and n-Myc [95]. Hyperactivation of caspase-8 and -3 in response to TNF stimulation occurs from inactivation of the inhibitor protein of caspase-8, FLICE-inhibitory protein (FLIP) [57].
However, HBx protein has also been reported to ex- ert an anti-apoptotic effect through the activation of the NF- κB subunits RelA/p65 and c-Rel [96].
Similarly, opposing results have been reported in studies of hepatitis C virus (HCV) core protein in human hepatoma cell lines. Overexpression of HCV
core protein was demonstrated to inhibit TNF-in- duced apoptosis by inducing NF- κB activation [72].
In contrast, this protein has also been reported to enhance TNF-induced apoptosis without affecting NF- κB activation [126]. Thus, further studies are needed to resolve the apparently conflicting reports on the roles of TNF and NF- κB signaling in viral hepatitis.
10.5 Conclusions
The involvement of TNF in a number of pathophysi- ologic conditions is evidence for the importance of TNF signaling in the liver. While considerable progress in understanding hepatic TNF signaling pathways has been made, significant gaps in our knowledge still exist, particularly in the area of the final gene products that mediate the biological ef- fects of TNF. The ample evidence of alterations in the levels of TNF ligand and receptors in human studies [56, 94, 106] suggests that TNF signaling may affect the outcome of human liver disease.
While therapeutic efforts to block TNF function in these diseases may prove beneficial, neutralization of all TNF function may prove ineffective in treat- ing these diseases. An additional understanding of hepatic TNF signaling pathways may lead to agents that selectively target the beneficial or cytotoxic ef- fects of TNF and thus have added effectiveness in treating human liver disease.
Selected Reading
Ghosh S, Karin M. Missing pieces in the NF-κB puzzle. Cell 2002;109(Suppl):S81–S96. (This review paper discusses the NF-κB signaling pathways, their regulation and the IKK com- plexes in further detail.)
Wang H, Czura CJ, Tracey KJ. Tumor necrosis factor. In: Thom- son AW, Lotze MT, eds. The Cytokine Handbook, vol II (4th ed). Amsterdam: Academic Press, 2003:837–860. (This book chapter provides a comprehensive discussion of the effects of TNF signaling in both hepatic and extrahepatic tissues.) Suryprasad AG, Prindiville T. The biology of TNF blockade. Au-
toimmunity Reviews 2003;2:346–357. (This paper reviews the methods of anti-TNF therapy in autoimmune and chron- ic inflammatory diseases.)
Maeda S, Chang L, Li Z-W et al. IKKβ is required for prevention of apoptosis mediated by cell-bound but not circulating TNFα.
Immunity 2003;19:725–737. (This is an original research pa- per that details the effects of hepatocyte-specific IKKβ abla- tion on liver injury from TNF.)
References
1. Aggarwal BB. Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol 2003;3:745–756.
2. Akerman P, Cote P, Yang SQ et al. Antibodies to tumor necro- sis factor-alpha inhibit liver regeneration after partial hepa- tectomy. Am J Physiol 1992;263:G579–G585.
3. Akerman PA, Cote PM, Yang SQ et al. Long-term ethanol consumption alters the hepatic response to the regen- erative effects of tumor necrosis factor-alpha. Hepatology 1993;17:1066–1073.
4. Alcamo E, Mizgerd JP, Horwitz BH et al. Targeted mutation of TNF receptor I rescues the RelA-deficient mouse and reveals a critical role for NF-kappa B in leukocyte recruitment. J Im- munol 2001;167:1592–1600.
5. Amour A, Slocombe PM, Webster A et al. TNF-alpha con- verting enzyme (TACE) is inhibited by TIMP-3. FEBS Lett 1998;435:39–44.
6. Arvelo MB, Cooper JT, Longo C et al. A20 protects mice from D-galactosamine/lipopolysaccharide acute toxic lethal hep- atitis. Hepatology 2002;35:535–543.
7. Ashkenazi A, Dixit VM. Apoptosis control by death and de- coy receptors. Curr Opin Cell Biol 1999;11:255–260.
8. Bai J, Cederbaum AI. Overexpression of catalase in the mito- chondrial or cytosolic compartment increases sensitivity of HepG2 cells to tumor necrosis factor-alpha-induced apop- tosis. J Biol Chem 2000;275:19241–19249.
9. Banner DW, D'Arcy A, Janes W et al. Crystal structure of the soluble human 55 kd TNF receptor-human TNF beta complex: implications for TNF receptor activation. Cell 1993;73:431–445.
10. Barbara JA, Smith WB, Gamble JR et al. Dissociation of TNF- alpha cytotoxic and proinflammatory activities by p55 receptor- and p75 receptor-selective TNF-alpha mutants.
EMBO J 1994;13:843–850.
11. Barnhart BC, Alappat EC, Peter ME. The CD95 type I/type II model. Semin Immunol 2003;15:185–193.
12. Beg AA, Sha WC, Bronson RT et al. Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature 1995;376:167–170.
13. Bhardwaj A, Aggarwal BB. Receptor-mediated choreogra- phy of life and death. J Clin Immunol 2003;23:317–332.
14. Biermer M, Puro R, Schneider RJ. Tumor necrosis factor alpha inhibition of hepatitis B virus replication involves disruption of capsid integrity through activation of NF-kappaB. J Virol 2003;77:4033–4042.
15. Black RA, Rauch CT, Kozlosky CJ et al. A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 1997;385:729–733.
16. Blindenbacher A, Wang X, Langer I et al. Interleukin 6 is im- portant for survival after partial hepatectomy in mice. Hepa- tology 2003;38:674–682.
17. Bradham CA, Qian T, Streetz K et al. The mitochondrial per- meability transition is required for tumor necrosis factor al-
pha-mediated apoptosis and cytochrome c release. Mol Cell Biol 1998;18:6353–6364.
18. Chaisson ML, Brooling JT, Ladiges W et al. Hepatocyte-spe- cific inhibition of NF-kappaB leads to apoptosis after TNF treatment, but not after partial hepatectomy. J Clin Invest 2002;110:193–202.
19. Chan FK, Chun HJ, Zheng L et al. A domain in TNF receptors that mediates ligand-independent receptor assembly and signaling. Science 2000;288:2351–2354.
20. Chinnaiyan AM, O'Rourke K, Tewari M, Dixit VM. FADD, a nov- el death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 1995;81:505–512.
21. Chinnaiyan AM, Tepper CG, Seldin MF et al. FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis. J Biol Chem 1996;271:4961–4965.
22. Clavien PA, Yadav S, Sindram D, Bentley RC. Protective effects of ischemic preconditioning for liver resection performed under inflow occlusion in humans. Ann Surg 2000;232:155–
162.
23. Colletti LM, Kunkel SL, Walz A et al. The role of cytokine net- works in the local liver injury following hepatic ischemia/
reperfusion in the rat. Hepatology 1996;23:506–514.
24. Cressman DE, Greenbaum LE, DeAngelis RA et al. Liver fail- ure and defective hepatocyte regeneration in interleukin-6- deficient mice. Science 1996;274:1379–1383.
25. Czaja MJ. The future of GI and liver research: editorial per- spectives. III. JNK/AP-1 regulation of hepatocyte death. Am J Physiol Gastrointest Liver Physiol 2003;284:G875–G879.
26. Czaja MJ, Flanders KC, Biempica L et al. Expression of tumor necrosis factor-alpha and transforming growth factor-beta 1 in acute liver injury. Growth Factors 1989;1:219–226.
27. Czaja MJ, Schilsky ML, Xu Y et al. Induction of MnSOD gene expression in a hepatic model of TNF-alpha toxicity does not result in increased protein. Am J Physiol 1994;266:G737–
G744.
28. Czaja MJ, Xu J, Alt E. Prevention of carbon tetrachloride-in- duced rat liver injury by soluble tumor necrosis factor recep- tor. Gastroenterology 1995;108:1849–1854.
29. de la Coste A, Fabre M, McDonell N et al. Differential protec- tive effects of Bcl-xL and Bcl-2 on apoptotic liver injury in transgenic mice. Am J Physiol 1999;277:G702–G708.
30. De Smaele E, Zazzeroni F, Papa S et al. Induction of gadd45beta by NF-kappaB downregulates pro-apoptotic JNK signalling. Nature 2001;414:308–313.
31. DeAngelis RA, Kovalovich K, Cressman DE, Taub R. Normal liver regeneration in p50/nuclear factor kappaB1 knockout mice. Hepatology 2001;33:915–924.
32. Deng Y, Ren X, Yang L et al. A JNK-dependent pathway is required for TNFalpha-induced apoptosis. Cell 2003;115:61–
70.
33. Devin A, Cook A, Lin Y et al. The distinct roles of TRAF2 and RIP in IKK activation by TNF-R1: TRAF2 recruits IKK to TNF-R1 while RIP mediates IKK activation. Immunity 2000;12:419–
429.
34. Devin A, Lin Y, Yamaoka S et al. The alpha and beta subu- nits of IkappaB kinase (IKK) mediate TRAF2-dependent IKK recruitment to tumor necrosis factor (TNF) receptor 1 in re- sponse to TNF. Mol Cell Biol 2001;21:3986–3994.
35. Earnshaw WC, Martins LM, Kaufmann SH. Mammalian cas- pases: structure, activation, substrates, and functions dur- ing apoptosis. Annu Rev Biochem 1999;68:383–424.
36. Fotin-Mleczek M, Henkler F, Samel D et al. Apoptotic cross- talk of TNF receptors: TNF-R2 induces depletion of TRAF2 and IAP proteins and accelerates TNF-R1-dependent activa- tion of caspase-8. J Cell Sci 2002;115:2757–2770.
37. Ghosh S, Karin M. Missing pieces in the NF-kappaB puzzle.
Cell 2002;109 (Suppl):S81–S96.
38. Grell M, Douni E, Wajant H et al. The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor. Cell 1995;83:793–
802.
39. Grell M, Wajant H, Zimmermann G, Scheurich P. The type 1 receptor (CD120a) is the high-affinity receptor for soluble tumor necrosis factor. PNAS 1998;95:570–575.
40. Guicciardi ME, Deussing J, Miyoshi H et al. Cathepsin B con- tributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Invest 2000;106:1127–1137.
41. Haouzi D, Lekehal M, Tinel M et al. Prolonged, but not acute, glutathione depletion promotes Fas-mediated mitochon- drial permeability transition and apoptosis in mice. Hepatol- ogy 2001;33:1181–1188.
42. Harper N, Hughes M, MacFarlane M, Cohen GM. Fas-associat- ed death domain protein and caspase-8 are not recruited to the tumor necrosis factor receptor 1 signaling complex dur- ing tumor necrosis factor-induced apoptosis. J Biol Chem 2003;278:25534–25541.
43. Hatano E, Bennett BL, Manning AM et al. NF-kappaB stimu- lates inducible nitric oxide synthase to protect mouse hepa- tocytes from TNF-alpha- and Fas-mediated apoptosis. Gas- troenterology 2001;120:1251–1262.
44. Hatano E, Brenner DA. Akt protects mouse hepatocytes from TNF-alpha- and Fas-mediated apoptosis through NK- kappa B activation. Am J Physiol Gastrointest Liver Physiol 2001;281:G1357–G1368.
45. Hehlgans T, Seitz C, Lewis C, Mannel DN. Hypoxic upregula- tion of TNF receptor type 2 expression involves NF-IL-6 and is independent of HIF-1 or HIF-2. J Interferon Cytokine Res 2001;21:757–762.
46. Honchel R, Marsano L, Cohen D et al. Lead enhances lipopol- ysaccharide and tumor necrosis factor liver injury. J Lab Clin Med 1991;117:202–208.
47. Hsu H, Shu HB, Pan MG, Goeddel DV. TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 1996;84:299–308.
48. Hsu H, Xiong J, Goeddel DV. The TNF receptor 1-associated protein TRADD signals cell death and NF-kappa B activation.
Cell 1995;81:495–504.
49. Iimuro Y, Nishiura T, Hellerbrand C et al. NFkappaB prevents apoptosis and liver dysfunction during liver regeneration. J Clin Invest 1998;101:802–811.
50. Jaeschke H, Farhood A, Cai SX et al. Protection against TNF- induced liver parenchymal cell apoptosis during endotox- emia by a novel caspase inhibitor in mice. Toxicol Appl Phar- macol 2000;169:77–83.
51. Jaeschke H, Lemasters JJ. Apoptosis versus oncotic necrosis in hepatic ischemia/reperfusion injury. Gastroenterology 2003;125:1246–1257.
52. Jiang Y, Woronicz JD, Liu W, Goeddel DV. Prevention of con- stitutive TNF receptor 1 signaling by silencer of death do- mains. Science 1999;283:543–546.
53. Jones BE, Lo CR, Liu H et al. Hepatocytes sensitized to tu- mor necrosis factor-alpha cytotoxicity undergo apoptosis through caspase-dependent and caspase-independent pathways. J Biol Chem 2000;275:705–712.
54. Juo P, Kuo CJ, Yuan J, Blenis J. Essential requirement for cas- pase-8/FLICE in the initiation of the Fas-induced apoptotic cascade. Curr Biol 1998;8:1001–1008.
55. Kern PA, Ranganathan S, Li C et al. Adipose tissue tumor necrosis factor and interleukin-6 expression in human obes- ity and insulin resistance. Am J Physiol Endocrinol Metab 2001;280:E745–E751.
56. Khoruts A, Stahnke L, McClain CJ et al. Circulating tumor necrosis factor, interleukin-1 and interleukin-6 concentra- tions in chronic alcoholic patients. Hepatology 1991;13:267–
276.
57. Kim KH, Seong BL. Pro-apoptotic function of HBV X protein is mediated by interaction with c-FLIP and enhancement of death-inducing signal. EMBO J 2003;22:2104–2116.
58. Kischkel FC, Hellbardt S, Behrmann I et al. Cytotoxicity- dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor.
EMBO J 1995;14:5579–5588.
59. Koteish A, Yang S, Lin H et al. Chronic ethanol exposure po- tentiates lipopolysaccharide liver injury despite inhibiting Jun N-terminal kinase and caspase 3 activation. J Biol Chem 2002;277:13037–13044.
60. Kunstle G, Hentze H, Germann PG et al. Concanavalin A hepatotoxicity in mice: tumor necrosis factor-mediated or- gan failure independent of caspase-3-like protease activa- tion. Hepatology 1999;30:1241–1251.
61. Lee TH, Huang Q, Oikemus S et al. The death domain kinase RIP1 is essential for tumor necrosis factor alpha signal- ing to p38 mitogen-activated protein kinase. Mol Cell Biol 2003;23:8377–8385.
62. Legler DF, Micheau O, Doucey MA et al. Recruitment of TNF receptor 1 to lipid rafts is essential for TNFalpha-mediated NF-kappaB activation. Immunity 2003;18:655–664.
63. Leist M, Gantner F, Bohlinger I et al. Murine hepatocyte ap- optosis induced in vitro and in vivo by TNF-alpha requires transcriptional arrest. J Immunol 1994;153:1778–1788.
64. Leitges M, Sanz L, Martin P et al. Targeted disruption of the zetaPKC gene results in the impairment of the NF-kappaB pathway. Mol Cell 2001;8:771–780.
65. Li J, Bombeck CA, Yang S et al. Nitric oxide suppresses a- poptosis via interrupting caspase activation and mitochon- drial dysfunction in cultured hepatocytes. J Biol Chem 1999;274:17325–17333.
66. Li W, Liang X, Kellendonk C et al. STAT3 contributes to the mi- togenic response of hepatocytes during liver regeneration.
J Biol Chem 2002;277:28411–28417.
67. Liedtke C, Plumpe J, Kubicka S et al. Jun kinase modulates tumor necrosis factor-dependent apoptosis in liver cells.
Hepatology 2002;36:315–325.
68. Liu H, Jones BE, Bradham C, Czaja MJ. Increased cytochrome P-450 2E1 expression sensitizes hepatocytes to c-Jun-medi- ated cell death from TNF-alpha. Am J Physiol Gastrointest Liver Physiol 2002;282:G257–G266.
69. Liu H, Lo CR, Czaja MJ. NF-kappaB inhibition sensitizes hepa- tocytes to TNF-induced apoptosis through a sustained acti- vation of JNK and c-Jun. Hepatology 2002;35:772–778.
70. Loffreda S, Rai R, Yang SQ et al. Bile ducts and portal and cen- tral veins are major producers of tumor necrosis factor alpha in regenerating rat liver. Gastroenterology 1997;112:2089–
2098.
71. Maeda S, Chang L, Li ZW et al. IKKbeta is required for preven- tion of apoptosis mediated by cell-bound but not by circu- lating TNFalpha. Immunity 2003;19:725–737.
72. Marusawa H, Hijikata M, Chiba T, Shimotohno K. Hepatitis C virus core protein inhibits Fas- and tumor necrosis factor alpha-mediated apoptosis via NF-kappaB activation. J Virol 1999;73:4713–4720.
73. McClary H, Koch R, Chisari FV, Guidotti LG. Relative sensitiv- ity of hepatitis B virus and other hepatotropic viruses to the antiviral effects of cytokines. J Virol 2000;74:2255–2264.
74. Micheau O, Tschopp J. Induction of TNF receptor I-medi- ated apoptosis via two sequential signaling complexes. Cell 2003;114:181–190.
75. Moorthy AK, Ghosh G. p105-Ikappa Bgamma and prototypi- cal Ikappa Bs use a similar mechanism to bind but a different mechanism to regulate the subcellular localization of NF- kappa B. J Biol Chem 2003;278:556–566.
76. Morio LA, Chiu H, Sprowles KA et al. Distinct roles of tumor necrosis factor-alpha and nitric oxide in acute liver injury induced by carbon tetrachloride in mice. Toxicol Appl Phar- macol 2001;172:44–51.
77. Moss ML, Lambert MH. Shedding of membrane proteins by ADAM family proteases. Essays Biochem 2002;38:141–153.
78. Muzio M, Stockwell BR, Stennicke HR et al. An induced proximity model for caspase-8 activation. J Biol Chem 1998;273:2926–2930.
79. Neumann M, Grieshammer T, Chuvpilo S et al. RelA/p65 is a molecular target for the immunosuppressive action of pro- tein kinase A. EMBO J 1995;14:1991–2004.
80. Oliver FJ, Menissier-de Murcia J, Nacci C et al. Resistance to endotoxic shock as a consequence of defective NF-kappaB activation in poly (ADP-ribose) polymerase-1 deficient mice.
EMBO J 1999;18:4446–4454.
81. Osawa Y, Banno Y, Nagaki M et al. TNF-alpha-induced sphin- gosine 1-phosphate inhibits apoptosis through a phosphati-
dylinositol 3-kinase/Akt pathway in human hepatocytes. J Immunol 2001;167:173–180.
82. Park YC, Burkitt V, Villa AR et al. Structural basis for self-as- sociation and receptor recognition of human TRAF2. Nature 1999;398:533–538.
83. Park YC, Ye H, Hsia C et al. A novel mechanism of TRAF sig- naling revealed by structural and functional analyses of the TRADD-TRAF2 interaction. Cell 2000;101:777–787.
84. Perkins ND. The Rel/NF-kappa B family: friend and foe.
Trends Biochem Sci 2000;25:434–440.
85. Peschon JJ, Slack JL, Reddy P et al. An essential role for ec- todomain shedding in mammalian development. Science 1998;282:1281–1284.
86. Peter ME, Krammer PH. The CD95(APO-1/Fas) DISC and be- yond. Cell Death Differ 2003;10:26–35.
87. Rai RM, Lee FY, Rosen A et al. Impaired liver regeneration in inducible nitric oxide synthase deficient mice. Proc Natl Acad Sci USA 1998;95:13829–13834.
88. Rudiger HA, Clavien PA. Tumor necrosis factor alpha, but not Fas, mediates hepatocellular apoptosis in the murine ischemic liver. Gastroenterology 2002;122:202–210.
89. Sakamoto T, Liu Z, Murase N et al. Mitosis and apoptosis in the liver of interleukin-6-deficient mice after partial hepate- ctomy. Hepatology 1999;29:403–411.
90. Scaffidi C, Fulda S, Srinivasan A et al. Two CD95 (APO-1/Fas) signaling pathways. EMBO J 1998;17:1675–1687.
91. Schwabe RF, Bennett BL, Manning AM, Brenner DA. Differ- ential role of I kappa B kinase 1 and 2 in primary rat hepato- cytes. Hepatology 2001;33:81–90.
92. Schwabe RF, Bradham CA, Uehara T et al. c-Jun-N-terminal kinase drives cyclin D1 expression and proliferation during liver regeneration. Hepatology 2003;37:824–832.
93. Solan NJ, Miyoshi H, Carmona EM et al. RelB cellular regula- tion and transcriptional activity are regulated by p100. J Biol Chem 2002;277:1405–1418.
94. Streetz K, Leifeld L, Grundmann D et al. Tumor necrosis fac- tor alpha in the pathogenesis of human and murine fulmi- nant hepatic failure. Gastroenterology 2000;119:446–460.
95. Su F, Schneider RJ. Hepatitis B virus HBx protein sensitizes cells to apoptotic killing by tumor necrosis factor alpha. Proc Natl Acad Sci USA 1997;94:8744–8749.
96. Su F, Theodosis CN, Schneider RJ. Role of NF-kappaB and myc proteins in apoptosis induced by hepatitis B virus HBx protein. J Virol 2001;75:215–225.
97. Tada K, Okazaki T, Sakon S et al. Critical roles of TRAF2 and TRAF5 in tumor necrosis factor-induced NF-kappa B activation and protection from cell death. J Biol Chem 2001;276:36530–36534.
98. Tartaglia LA, Pennica D, Goeddel DV. Ligand passing: the 75-kDa tumor necrosis factor (TNF) receptor recruits TNF for signaling by the 55-kDa TNF receptor. J Biol Chem 1993;268:18542–18548.
99. Teoh N, Leclercq I, Pena AD, Farrell G. Low-dose TNF-al- pha protects against hepatic ischemia-reperfusion in- jury in mice: implications for preconditioning. Hepatology 2003;37:118–128.
100. Thornberry NA, Lazebnik Y. Caspases: enemies within. Sci- ence 1998;281:1312–1316.
101. Tibbetts MD, Zheng L, Lenardo MJ. The death effector do- main protein family: regulators of cellular homeostasis. Nat Immunol 2003;4:404–409.
102. Togashi H, Ohno S, Matsuo T et al. Interferon-gamma, tu- mor necrosis factor-alpha, and interleukin 1-beta suppress the replication of hepatitis B virus through oxidative stress.
Res Commun Mol Pathol Pharmacol 2000;107:407–417.
103. Tournier C, Dong C, Turner TK et al. MKK7 is an essential component of the JNK signal transduction pathway activat- ed by proinflammatory cytokines. Genes Dev 2001;15:1419–
1426.
104. Van Molle W, Denecker G, Rodriguez I et al. Activation of caspases in lethal experimental hepatitis and prevention by acute phase proteins. J Immunol 1999;163:5235–5241.
105. Vandenabeele P, Declercq R, Beyaert W, Fiers W. Two tumor necrosis factor receptors: structure and function. Trends Cell Biol 1988;5:392–399.
106. Volpes R, van den Oord JJ, De Vos R, Desmet VJ. Hepatic ex- pression of type A and type B receptors for tumor necrosis factor. J Hepatol 1992;14:361–369.
107. Wajant H, Pfizenmaier K, Scheurich P. Tumor necrosis factor signaling. Cell Death Differ 2003;10:45–65.
108. Wang D, Westerheide SD, Hanson JL, Baldwin AS, Jr. Tumor necrosis factor alpha-induced phosphorylation of RelA/
p65 on Ser529 is controlled by casein kinase II. J Biol Chem 2000;275:32592–32597.
109. Wei MC, Zong WX, Cheng EH et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 2001;292:727–730.
110. Werneburg NW, Guicciardi ME, Bronk SF, Gores GJ. Tumor necrosis factor-alpha-associated lysosomal permeabiliza- tion is cathepsin B dependent. Am J Physiol Gastrointest Liver Physiol 2002;283:G947–G956.
111. Wuestefeld T, Klein C, Streetz KL et al. Interleukin-6/glyco- protein 130-dependent pathways are protective during liver regeneration. J Biol Chem 2003;278:11281–11288.
112. Xu Y, Bialik S, Jones BE et al. NF-kappaB inactivation con- verts a hepatocyte cell line TNF-alpha response from prolif- eration to apoptosis. Am J Physiol 1998;275:C1058–C1066.
113. Xu Y, Jones BE, Neufeld DS, Czaja MJ. Glutathione modu- lates rat and mouse hepatocyte sensitivity to tumor necro- sis factor toxicity. Gastroenterology 1998;115:1229–1237.
114. Yadav SS, Howell DN, Gao W et al. L-selectin and ICAM-1 mediate reperfusion injury and neutrophil adhesion in the warm ischemic mouse liver. Am J Physiol 1998;275:G1341–
G1352.
115. Yamada Y, Fausto N. Deficient liver regeneration after car- bon tetrachloride injury in mice lacking type 1 but not type 2 tumor necrosis factor receptor. Am J Pathol 1998;152:1577–
1589.
116. Yamada Y, Kirillova I, Peschon JJ, Fausto N. Initiation of liver growth by tumor necrosis factor: deficient liver regenera- tion in mice lacking type I tumor necrosis factor receptor.
Proc Natl Acad Sci USA 1997;94:1441–1446.
117. Yamada Y, Webber EM, Kirillova I et al. Analysis of liver re- generation in mice lacking type 1 or type 2 tumor necrosis factor receptor: requirement for type 1 but not type 2 re- ceptor. Hepatology 1998;28:959–970.
118. Yang SQ, Lin HZ, Yin M et al. Effects of chronic ethanol con- sumption on cytokine regulation of liver regeneration. Am J Physiol 1998;275:G696–G704.
119. Yared G, Hussain KB, Nathani MG et al. Cytokine-mediated apoptosis and inhibition of virus production and anchor- age independent growth of viral transfected hepatoblas- toma cells. Cytokine 1998;10:586–595.
120. Yeh WC, Itie A, Elia AJ et al. Requirement for Casper (c-FLIP) in regulation of death receptor-induced apoptosis and em- bryonic development. Immunity 2000;12:633–642.
121. Yeh WC, Shahinian A, Speiser D et al. Early lethality, func- tional NF-kappaB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 1997;7:715–725.
122. Yin M, Wheeler MD, Kono H et al. Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice.
Gastroenterology 1999;117:942–952.
123. Young CS, Palma JM, Mosher BD et al. Hepatic ischemia/
reperfusion injury in P-selectin and intercellular adhesion molecule-1 double-mutant mice. Am Surg 2001;67:737–
744.
124. Zhao Y, Ding WX, Qian T et al. Bid activates multiple mi- tochondrial apoptotic mechanisms in primary hepato- cytes after death receptor engagement. Gastroenterology 2003;125:854–867.
125. Zhao Y, Li S, Childs EE et al. Activation of pro-death Bcl-2 family proteins and mitochondria apoptosis pathway in tu- mor necrosis factor-alpha-induced liver injury. J Biol Chem 2001;276:27432–27440.
126. Zhu N, Khoshnan A, Schneider R et al. Hepatitis C virus core protein binds to the cytoplasmic domain of tumor necrosis factor (TNF) receptor 1 and enhances TNF-induced apopto- sis. J Virol 1998;72:3691–3697.
127. Zou H, Li Y, Liu X, Wang X. An APAF-1.cytochrome c mul- timeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 1999;274:11549–11556.
