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IL-6/gp130/Stat3

Rebecca Taub

7

7.1

Introduction

The liver has an important role as the major detoxi- fying organ of the body and the first in line of de- fense against ingested toxins and many infectious agents. Consequently, the liver has evolutionarily conserved several adaptive responses including the ability to regenerate and protect itself against injury [27–29, 45, 60, 92, 93]. Within the liver the inter- leukin-6 (IL-6) system has been shown to be criti- cal for the acute phase response, proliferation and protection against injury during liver regeneration, and hepatoprotection after toxic or apoptotic dam- age. If Stat3 appears to be the dominant signal in the IL-6/glycoprotein 130 (gp130)-mediated acute phase response and provides a basis for many of the hepatoprotective properties of IL-6/gp130 signaling [92, 93], Stat3 does not account for the full prolif- erative effect of IL-6/gp130, and it appears that the mitogen-activated protein kinase (MAPK) signaling pathway downstream of IL-6/gp130 also contributes to the proliferative effects. The exact relationship and overlap between the hepatoprotective, antiap- optotic effects of IL-6/gp130 and the proliferative actions have not been clearly elucidated, but it is clear that both hepatoprotection and proliferative activities must coexist in order for the liver to re- cover from a variety of insults including infection, toxic or ischemic damage, and resection.

7.2

IL-6 Cytokines/gp130 Pathway

7.2.1

Interaction of IL-6 Cytokines with the gp130 Receptor Complex

The IL-6 cytokines form a subfamily of helix bundle cytokines, which include leukemia inhibitory factor

(LIF), ciliary neurotrophic factor (CNTF), oncosta- tin M (OSM), IL-11, cardiotropin-1 (CT-1), and cardi- otropin-like cytokine (CTC) [38] and activate Stat3 DNA-binding activity via interactions with gp130 receptor (Fig. 7.1). The topography of these helices is different for each cytokine, resulting in the spe- cificity with which a cytokine interacts with gp130 and its cognate receptor. The receptors, IL-6R and gp130, have extensive extracellular regions contain- ing Ig-like and fibronectin type III-like protein se- quences that include the cytokine-binding domain (CBD) (Fig. 7.2). Of the IL-6-like cytokines, only IL- 6 and IL-11 signal via a gp130 homodimer that is ad- ditionally bound to either an IL-6 specific receptor

Fig. 7.1. Model for IL-6, IL-6/Stat3 signaling pathway. IL-6 binds to its receptor, IL-6R, which binds to the gp130 receptor, result- ing in the activation of janus kinase (JAK). This leads to activa- tion of the MAPK pathway and activation of Stat3 by tyrosine (Y) phosphorylation. Dimerized Stat3 is able to translocate into the nucleus and activate gene transcription. In the liver, this proc- ess promotes liver regeneration, the acute phase response, and hepatoprotection against Fas and toxic damage. In Stat3 knock- out mice the Stat3 pathway is blocked, but the MAPK pathway is intact

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(gp80, IL-6R) or an IL-11 specific receptor (IL-11R).

The remainder of IL-6-like cytokines interact with a receptor containing one subunit of gp130 and either an LIF receptor (LIF, CT-1, OSM, CNTF, CLC) or an OSM receptor. Although gp130 is ubiquitous, cell type specificity of IL-6 cytokine family members is achieved because IL-6R (for example) is required for downstream signaling, and IL-6R expression is cell type limited.

7.2.2

Activation of JAK

The resultant receptor trimer undergoes a confor- mational change that allows for the interaction and activation of a Janus kinase (JAK), an intracellular tyrosine kinase. Gp130 may interact with several members of the JAK family including JAK1, JAK2, and TYK2. Of these, JAK1 appears to be most criti- cal for IL-6 signaling because in cells without JAK1, IL-6 signaling is greatly diminished [38, 52].

JAKs bind to the membrane proximal region of gp130, via the FERM domain (four point one, ezrin redixin and moesin), the interaction of JAK1 with gp130 being particularly strong. Activation of JAK results in tyrosine phosphorylation of gp130 at several sites, its own autophosphorylation, and ty- rosine phosphorylation of gp130-associated Stat3 and SHP2 (SH2 domain containing tyrosine phos- phatase) leading to signal transduction via the Stat3

and MAPK signal transduction pathways, respec- tively. SHP2 interacts specifically with tyrosine 759 of gp130 via its SH2 domain while the SH2 domain of Stat3 interacts better with other tyrosine residues (amino acids 767, 814, 905, 915).

7.2.3

Stat3 Signaling

Stat3 was first described as a DNA-binding activity from IL-6-stimulated hepatocytes that interacted with a palindromic sequence within the promoter region of several acute phase genes [38, 40, 41, 52, 87, 91]. The Stat family of transcription factors contains seven mammalian members, designated Stat1, 2, 3, 4, 5a, 5b, and 6 [52]. Stat3 is similar to other Stat pro- teins, having a conserved amino-terminus involved in tetramerization, a DNA-binding domain with a sequence specificity for a palindromic sequence similar to that of Stat1, an SH2 domain involved in receptor recruitment as well as Stat dimerization, and a carboxy-terminal transactivation domain.

Stat3 is activated by tyrosine phosphorylation at a single site (Y705) and by serine phosphorylation at a MAPK consensus site within its transactivation do- main. Tyrosine phosphorylation mediated by JAK is required for Stat3 dimerization, nuclear transloca- tion, and DNA binding (Fig. 7.1). Shuttling into and out of the nucleus is a complex process for this rela- tively large protein (90 kDa) and appears to require import and export proteins [38].

7.2.4

MAPK Signaling

Stimulation of gp130 leads not only to Stat signaling but to activation of the MAPK cascade. The current evidence suggests that SHP2 interacts with gp130 at Tyr 759 and undergoes tyrosine phosphorylation and activation by JAK1 leading to recruitment of Grb2-SOS (growth factor receptor bound protein/

son of sevenless). Grb2-SOS activates Ras, which in turn leads to Raf-MEK-MAPK (particularly extra- cellular signal-regulated kinase [ERK]1/2) activa- tion. MAPK signaling is a critical component of the proliferative response of cells including hepatocytes [89]. Recent evidence suggests that IL-6 signaling may also directly activate PI3 kinase, protein kinase B and AKT, other kinases involved in cell survival [38].

Fig. 7.2. Structural organization of various IL-6-type cytokine signaling components. Relevant tyrosine (Y) and serine (S) resi- dues of gp130, JAK and STAT proteins that become phosphor- ylated are indicated. CBM cytokine-binding module, SH2 src ho- mology-2, FERM 4.1/ezrin/redixin/moesin, FNIII fibronectin type III, KIR kinase inhibitory region, TM transmembrane

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7.2.5

Turning off the Signal;

SOCS and Other Negative Regulators

SHP2 is a phosphatase that interacts with JAK and mediates MAPK activation but also may terminate the signal by dephosphorylating JAK, gp130 and Stat3 once the SHP2 phosphatase becomes active.

Other protein tyrosine phosphatases such as T cell protein tyrosine phosphatase (Tc-PTPase) may be important as well (Fig. 7.3). mRNA and protein turnover of cytokines may play a role as cytokine mRNAs generally are relatively short lived and their half lives are regulated by mRNA stabilizing mol- ecules.

Specific inhibitors for Stats include PIAS (pro- tein inhibitor of activated Stats) proteins that spe- cifically bind to phosphorylated Stat proteins inhib- iting their transactivation potential. PIAS is a potent inhibitor of Stat3 DNA binding [15]; however, the level of PIAS in the liver under various conditions has not been examined.

Other specific inhibitors of IL-6 signaling are the suppressors of cytokine signaling (SOCS) fam- ily proteins (CIS, SOCS 1–3). SOCS proteins contain SH2 domains allowing them to interact with and block the kinase activity of JAKs. SOCS-3 may also bind specifically to gp130 tyrosine residues, block- ing gp130 signaling. SOCS-3 protein levels are dy- namically upregulated by IL-6 within the liver and could critically downregulate the IL-6 signal [14].

Interestingly, SOCS have the potential to inhibit any parallel JAK signaling in a given cell and would not necessarily be specific for terminating the gp130/

Stat3/IL-6 signals.

7.3

Role of IL-6/gp130/Stat3 in the Acute Phase Response

The innate immune system provides an organism with an immediate defense against invading organ- isms or other injuries [73, 104]. The systemic in- flammatory component of innate immunity or the acute phase response is a physiologic reaction to perturbations caused by infections or other injury and is largely mediated by hepatocytes that respond to IL-6 and other cytokines (tumor necrosis factor [TNF- α] and IL-1) released by Kupffer cells in reac- tion to lipopolysaccharides from the surface of in- vading microorganisms.

Acute phase proteins fall within three classes: (1) anti-infectious agents such as complement, C-reac- tive protein (CRP), and serum amyloid P (SAP); (2) metabolic regulators that increase glucose and fatty acids; and (3) procoagulation factors. In response to acute phase induction, alterations in hepatic gene expression are robust and gene array studies have demonstrated that as many of 7% of hepatic genes are transiently either up- or downregulated [104].

Acute phase gene promoters contain several regula- tory elements in addition to APRE (Stat3 DNA ele- ments) including C/enhancer-binding protein (EBP) binding sites, and are regulated by the combination of IL-6 and other cytokines.

The use of gene knockout mice for IL-6, gp130 (conditional knockout), Stat3 (conditional knock- out) has unequivocally established the importance of these molecules in mediating acute phase gene induction [3–5, 25, 46, 90, 99]. IL-6

–/–

mice are vi- able and show a defective acute phase response, par-

Fig. 7.3. Signals that turn off IL-6/gp130 pathways

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ticularly after stimulation with a local stimulation as opposed to systemic lipopolysaccharide (LPS).

Likewise, gp130-deleted livers show increased in- jury in response to LPS and defective expression of acute phase genes [99]. The ablation of Stat3 within the liver shows an even more defective acute phase response than IL-6

–/–

suggesting that IL-6-related cytokines may be in part responsible for the upregu- lation of Stat3 in response to acute phase reactants [5].

7.4

The Role of IL-6/gp130/Stat3 in Liver Regeneration

The liver has unusual properties of regeneration af- ter partial hepatectomy or toxic injury. The word “re- generation” is a misnomer because the lobes of the liver that are removed do not grow back, unlike the regeneration of limbs in amphibian models. Instead there is a hyperplastic response that involves the replication of virtually all of the cells in the remnant liver. Two-thirds partial hepatectomy in rodents is the model most commonly used to demonstrate the regenerative capacity of the liver; the remnant liver enlarges until the mass of the liver is restored within approximately 1 week, whereupon the process stops.

The process is compensatory because the size of the resultant liver is determined by the demands of the organism. This response involves the replication of greater than 90% of the differentiated functional cells within the liver and is not normally a stem-cell- mediated process [27–29, 39, 45, 60, 92, 93]. Animal models in which hepatocytes are directly damaged and undergo necrosis activate similar growth fac- tor and cytokine pathways as partial hepatectomy:

the undamaged hepatocytes and in some instances liver progenitor cells replicate [20, 26]. In fact, liver regeneration occurs most frequently in settings of liver damage by toxins, drugs, ischemia or hepatitis.

The medical implications for humans are protean because recovery from liver surgery, toxic liver dam- age and hepatitis require similar types of hepatic re- generation, the major difference from hepatectomy being that these insults cause hepatic necrosis and apoptosis.

Many growth factors including hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factors (TGF), insu- lin, glucagon, and cytokines, TNF- α, and IL-6 have been implicated in regulating this process. Both cytokine-dependent and independent pathways are critical for liver regeneration (Fig. 7.4) and must be carefully orchestrated [27–29, 45, 57, 60, 92, 93].

Cell division is rarely seen in hepatocytes in the normal adult liver, which are considered to be in the G

0

phase of the cell cycle. It has been shown that fol- lowing partial hepatectomy, approximately 95% of hepatic cells that are normally quiescent rapidly re- enter the cell cycle. In the rat liver the rate of DNA synthesis in hepatocytes (S phase) begins to increase after about 12 h and becomes maximal at about 24 h. The non-parenchymal liver cells, which consti- tute approximately 40% of liver cells, undergo DNA replication later: Kupffer cells 48 h, endothelial cells 96 h, biliary epithelial cells 48 h. Most of the increase in liver mass is seen by 3 days and mass restoration is complete in 5–7 days [33]. In the mouse liver the peak in DNA synthesis is later (36–40 h) and some- what variable between strains. The onset of DNA synthesis is well synchronized and proceeds from a portal to central direction in hepatocytes. Liver re- generation following massive hepatocyte necrosis or apoptosis induced by hepatic toxins such as carbon tetrachloride (CCl

4

) or fas ligand also involves pro- liferation of hepatocytes, but the cell cycle response is not as synchronized [45, 92]. Studies have shown that hepatocytes have incredible replicative func- tion. In hepatocyte transplantation studies, only a few hepatocytes are required to restore completely the liver mass [69, 77].

Fig. 7.4. Model for growth factor pathways postulated to regu- late liver regeneration after injury or partial hepatectomy. Pro- posed model of growth factor and cytokine activation of tran- scriptional cascade and DNA synthesis during liver regeneration.

Following partial hepatectomy, either gut-derived cytokines or de novo released cytokines activate hepatic non-parenchymal cells, resulting in increased production of TNF-α and IL-6. Other growth factors and accessory factors are released from other or- gans as shown

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7.4.1

Genetic Models that Provide Insight into the Regulatory Pathways of Liver Regeneration

Experiments performed by Nancy Bucher [62] pro- vided the first evidence that circulating growth factors present in the serum of hepatectomized rats induce hepatocyte replication in parabiosed unhepatectomized animals. Early work focused on studies in isolated hepatocytes led to the identifica- tion of several potential hepatocyte mitogens and growth factors such as HGF, TGF- α and TGF-β, an anti-proliferative factor, that might have important roles in regulating hepatocyte growth during liver regeneration. A large number of immediate early genes that were activated at the transition between G

0

and G

1

were identified (more than 100) and the advent of microarrays has expanded this list even more [6, 35, 44, 86]. Profiles of gene expression in- dicate that some genes show transient upregulation, but others, particularly those involved with protein synthesis and cell growth, are elevated throughout the major proliferative response of the liver. Specif- ic transcription factors such as NF- κB, Stat3, AP-1 and others are rapidly activated in remnant hepa- tocytes within minutes after a partial hepatectomy [93]. Intracellular signaling pathways are rapidly activated in a similar time frame including MAPK, c-Jun N-terminal kinase (JNK), and tyrosine phos- phorylation of specific receptors. However, which of the many signals and induced gene products were actually critical for liver regeneration was not ap- parent from the large array of activated gene prod- ucts that were identified, and knockout mice studies have been very helpful in defining specific roles and linking pathways [28, 84, 92]. With few exceptions, pathways are sufficiently redundant that the elimi- nation of any one gene product delays rather than completely impairs the restoration of hepatic mass.

7.4.2

IL-6-Dependent Pathways in Liver Regeneration

Knockout mouse studies established that normal liver regeneration after partial hepatectomy requires the IL-6 cytokine [18, 76] (see model, Figs. 7.4, 7.5).

Following partial hepatectomy, IL-6

–/–

livers have impaired liver regeneration characterized by liver necrosis and failure, a blunted DNA synthetic re- sponse in hepatocytes, and discrete G

1

phase abnor- malities including absence of Stat3 activation, and selective abnormalities in gene expression. Treat-

ment of IL-6

–/–

mice with a single preoperative dose of IL-6 returns Stat3 binding, gene expression, and hepatocyte proliferation to near normal and pre- vents liver damage, establishing IL-6 as a critical component of the regenerative response following partial hepatectomy. Most of the actions mediated by IL-6 occur within the first few hours posthepa- tectomy, and lead to progression through the early to mid phases of G

1

and ultimately to DNA synthesis and mitosis. Subsequent studies demonstrated that IL-6 is also critical for the acute phase response and reducing apopotosis after partial hepatectomy and some studies questioned a major role of IL-6 in the mitogenic response [9, 99, 100] as discussed below.

Using TNF receptor (TNF-R1) knockout mice [103], it was shown that TNF- α signaling is also required for a normal proliferative response post- hepatectomy, and this effect appears to be largely mediated by the ability of TNF- α to induce IL-6, because treatment of TNF-R1 knockout animals with IL-6 corrects the DNA synthetic defect in TNF- R1 knockout mice. Bone marrow transplantation studies demonstrated that most IL-6 is produced by macrophages or Kupffer cells within the liver [2]. The innate immune system is at least in part re- sponsible for the production of TNF- α and IL-6 by Kupffer cells. LPS, a component of innate immunity that interacts with the LPS receptor on Kupffer cells, has been shown to be important in triggering liver regeneration posthepatectomy and in liver injury models [16]. Moreover, studies indicate that mem- bers of the complement cascade C3a and C5a inter- act with their receptors on Kupffer cells to stimulate IL-6 and TNF- α release, and intercellular adhesion molecule (ICAM) appears to be necessary within this pathway. Defects in liver regeneration are ob- served in C3a- and C5a-deficient animals and these defects are correlated with reductions in IL-6, TNF- α, Stat3 and NF-κB [58, 81, 85].

Fig. 7.5. Growth factor and cytokine-regulated pathways acti- vated during liver regeneration showing key pathways and con- vergence of the signals that result in the activation of cell cycle regulatory molecules

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Thirty-six per cent of the immediate early genes activated during liver regeneration are regulated in part by IL-6 [54]. IL-6 treatment of IL-6

–/–

mice in the absence of hepatectomy induces a much smaller set of genes in the liver, suggesting that IL-6 cooper- ates with other hepatectomy-induced factors to acti- vate the large number of genes. Recently, a novel tar- get of IL-6 signaling was identified, stem cell factor (SCF), a growth factor that is activated by proteolytic cleavage of pro-SCF [74]. The mechanism by which IL-6 activates SCF, a ligand for the c-Kit tyrosine ki- nase receptor, is unknown. However, regenerative changes in IL-6

–/–

mice are corrected by treatment with SCF. SCF is able directly to activate Stat3 in- dependent of IL-6 signaling, and is also known to activate the MAPK signal transduction pathway.

IL-6 activates two major pathways during liver regeneration through the gp130 receptor/IL-6 re- ceptor, Stat3 and MAPK [92]. Knockout of Stat3 re- sults in embryonic lethality. To test the contribution of Stat3 to the growth response mediated by IL-6, liver-specific Stat3 knockouts were utilized (Alb

+

Stat3

fl/fl

). Hepatocyte DNA synthesis in Alb

+

Stat3

fl/

fl

livers was impaired posthepatectomy and abnor- malities in immediate-early gene activation largely correlated with but not identical to those seen in IL- 6

–/–

livers were observed [53]. Major effects on two known Stat3 targets, JunB and c-Fos, were observed.

Like IL-6

–/–

livers, G

1

phase cyclins including cyclin D1 and cyclin E had lower expression levels in Alb

+

Stat3

fl/fl

livers, indicating an abnormal G

1

to S phase transition. Alb

+

Stat3

fl/fl

livers show a 70% reduction in BrdU-labeled hepatocytes only at the peak DNA synthetic time point of 40 h posthepatectomy [53].

Therefore, Stat3 accounts for part of the cell cycle and DNA synthetic response of the hepatocytes dur- ing liver regeneration, which cannot be compensated for by induction of STAT1. Normal activation of the MAPK pathway in Alb

+

Stat3

fl/fl

livers reinforces the fact that at least part of the effect of IL-6 on hepato- cyte proliferation is not mediated by Stat3.

Conditional knockout of gp130 within the liver had less impact on hepatocyte DNA synthesis post- hepatectomy than would have been expected based on data with IL-6 and Stat3 knockout mice; however, defects in cyclin E and A expression were observed in gp130-deleted livers posthepatectomy, indicating that gp130 is important for normal cell cycle pro- gression [99].

Another study [9] indicated that IL-6 was pri- marily required for survival of the liver after par- tial hepatectomy; however, even in these studies mitogenesis was impaired in IL-6

–/–

livers at the early timepoints, consistent with Cressman et al.

[18]. Studies in which IL-6 has been overexpressed in the liver also support IL-6 as a major regulator of

hepatocyte proliferation, although the levels of IL-6 in these studies are elevated beyond the physiologic range. Studies in which soluble receptor IL-6R was also overexpressed support the importance of this molecule in hepatic growth mediated by IL-6 [31, 56, 70]. Delivery of high levels of IL-6 over a 2-week time period, even in the absence of excess IL-6R, re- sulted in massive hepatocyte proliferation, suggest- ing that the need for additional growth factors may be overcome by high levels of IL-6 [105]. Taken as a whole, it is agreed that IL-6 has pleiotrophic effects in the liver and is required for a normal integrated response to partial hepatectomy, which requires the acute phase response, suppression of apoptosis and induction of hepatocyte proliferation [106].

7.4.3

The Interface

Between Growth Factor-Regulated and Cytokine-Regulated Signals

Based on the weight of data, under physiologic con- ditions that occur in vivo, the two major pathways

“cytokine regulated” and “growth factor regulated”

[11, 67, 78, 95] depicted in Fig. 7.5 provide the criti- cal signals leading to liver regeneration [27–29, 60, 92]. Attempts to link these two pathways and estab- lish co-regulation and crosstalk are still in the early stages. However there are a few points of intersec- tion that allow hypotheses as to how the combina- tion of cytokine and growth factor signals leads to robust liver regeneration and repair from injury. For example, both IL-6 (and TNF- α) and HGF are able to activate JNK and MAPK/ERK, critical regulators of Jun activation, cell proliferation and cyclin D1 upregulation [18, 23, 53]. Cyclin D1 has been shown to be an important checkpoint protein in hepatic growth. Both IL-6/TNF and HGF upregulate AP-1 activity, which is comprised of a variety of leucine zipper transcription factor hetero- and homerdimers in addition to the classical c-Jun/c-Fos heterodimer (e.g. JunB, JunD, Fra, FosB, etc.). AP-1 activity is im- portant in activating a number of proteins involved in the growth response, and as described below is able to cooperate with Stat3 in amplifying the ex- pression of genes in the liver, an adaptive response during liver regeneration. Another possible point of intersection between HGF and IL-6 signals could be in the regulation of the insulin-like growth factor- binding protein-1 (IGFBP-1) gene, a pro-mitogenic and hepatoprotective protein that is upregulated by IL-6 in vivo [51], and potentially by HGF based on in vitro studies [98].

IGFBP-1 is one of the most rapidly and highly

induced genes and proteins in regenerating liver,

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a secreted protein that may modulate cell growth through IGF pathways or signal via IGF-independ- ent mechanisms involving activation or suppression of integrin signaling [49, 51] (Fig. 7.5). IGFBP-1 gene expression is induced more than 100-fold within 2 h after hepatectomy primarily in hepatocytes, and has a second peak of expression that correlates with the second round of hepatocyte DNA synthesis at 36 h posthepatectomy. Its transcription is in part regu- lated by IL-6 accounting for about 50% of IGFBP-1 gene and protein induction after partial hepatec- tomy. Although IGFBP-1

–/–

mice have normal devel- opment, they have abnormal liver regeneration af- ter partial hepatectomy [49], characterized by liver necrosis and reduced and delayed hepatocyte DNA synthesis. The abnormal regenerative response is associated with blunted activation of MAPK/ERK and a reduced induction of C/EBP- β protein expres- sion posthepatectomy. Expression of S phase cyc- lins, cyclin A and cyclin B1, is delayed and reduced in IGFBP-1

–/–

livers whereas, unlike IL-6

–/–

mice, cy- clin D1 expression is relatively normal. Treatment of IGFBP-1

–/–

mice with a preoperative dose of IGFBP- 1 induces MAPK/ERK activation and C/EBP- β ex- pression, suggesting that IGFBP-1 may support liver regeneration at least in part via its effect on MAPK/

ERK and C/EBP- β activities. Because IGF receptors have not been shown to play an important role in regulating hepatocyte growth, the hypothesis is that IGFBP-1 stimulates hepatocyte growth through IGF- independent pathways.

Both C/EBP- β (a leucine zipper transcription factor protein linked to cytokine-regulated path- ways) and IL-6 are involved in cytokine activa- tion pathways during the acute phase response in the liver [1, 4, 25, 90]. However, studies of C/EBP- β

–/–

and IL-6

–/–

animals indicated no positive cor- relation between IL-6 and C/EBP- β levels. In fact, IL-6 levels are elevated in C/EBP- β

–/–

animals and further treatment with IL-6 worsens their outcome after partial hepatectomy [32]. As in IL-6

–/–

livers, regeneration is impaired in C/EBP- β

–/–

livers but the genes and pathways affected are distinct from those regulated by IL-6 [32]. The mechanism as to how C/

EBP- β is activated and induced during liver regen- eration remains unknown, but the link in its activ- ity to IGFBP-1 expression is intriguing and places C/

EBP- β in a position within the regeneration pathway where it may be regulated by both growth factor and cytokine signals (Fig. 7.5).

7.4.4

Role of IL-6/Stat3 in the Maintenance of Liver Function During Liver Regeneration/

Role of Stat3 as a Co-activator of Hepatic Gene Transcription

The liver is a vital organ and must continue to func- tion as its cells proliferate during liver regeneration.

In most cells, for example the hematopoietic line- ages, proliferation is not compatible with differenti- ated function. How the liver is able to function while undergoing regeneration is an important question in liver biology. By examining the immediate-early gene response in the regenerating liver, it was possi- ble to find clues as to how the liver is able to function as it regenerates. Although many immediate-early genes are expressed in common in the regenerating liver and other growing cells, approximately one third of the genes are expressed at a high level or specifically in growing liver cells. Both hepatocytes and the non-parenchymal cells of the remnant liver express most of these genes, but the expression of a subset of genes is limited to hepatocytes [35, 44, 86].

Several of these liver-restricted immediate-early genes encode proteins that are involved in the gluco- neogenic response of the liver.

The induction of gluconeogenic genes represents an adaptive response of the liver whereby the rem- nant liver that comprises only one third of the origi- nal mass compensates by producing enough glucose for the whole organism [34, 75]. Liver-specific tran- scription factors have an important role in deter- mining liver-specific functions including the level of glucose production. They act by regulating the expression of genes encoding liver-specific enzymes (e.g. metabolic enzymes) and liver-specific secreted proteins (e.g. albumin). The adaptive response of the liver during regeneration that allows for mainte- nance of metabolic homeostasis is accomplished by an interplay between growth-induced transcription factors and pre-existing liver-specific transcription

Fig. 7.6. Proposed model of cooperative interaction between a growth-induced (c-Fos, c-Jun, Stat3) and constitutive hepatic factor (HNF-1) to upregulate the level of a liver-specific target gene (IGFBP-1) posthepatectomy

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factors that regulate the differentiated functions of the hepatocyte.

Normally in the liver the IGFBP-1, glucose-6- phosphatase, and alpha-fibrinogen and many other liver-restricted genes are regulated in the basal state by hepatic nuclear factor-1, HNF-1, a homeodo- main containing hepatic transcription factor. The level of HNF-1 does not change appreciably during liver regeneration. However, upregulation of HNF- 1 transcriptional activity occurs. This upregulation is accomplished by physical interactions between growth-induced transcription factors, Stat3 and AP-1 (c-Fos, c-Jun) and the resident hepatic tran- scription factor HNF-1 (Fig. 7.6) [51]. Two classes of transcription factors, growth induced (Stat3 and AP-1) and tissue specific (HNF-1), can interact as an adaptive response to liver injury to amplify expres- sion of hepatic genes important for the homeostatic response during organ repair. These data support the finding that Stat3 is able to function as a "co-ac- tivator" of gene transcription even in the absence of specific DNA binding [52]. The interaction between growth and resident hepatic transcription factors provides one mechanism by which the liver is able to maintain metabolic function despite the loss of two thirds of the functional mass.

7.4.5

The Re-establishment of Liver Size and Termination of Proliferation Posthepatectomy

The size of the liver is highly regulated and is con- trolled by the functional needs of the organism. The signals that determine the size of the liver are not well understood. However, there is evidence for dys- regulation of specific pathways that can alter hepat- ic mass. For example deletion of the cyclin inhibitor p27 results in increased organ size, whereas deletion of cyclin D1 results in reduced size [37, 55, 65]. Po- tentially TOR may be proven to be a master-regula- tor that determines the size of the liver by integrat- ing signals from nutrients and mitogens [65]. The pathway as depicted in Figs. 7.3 and 7.5 has several

"checkpoints" that indicate pathways of feedback inhibition of specific growth factor and cytokine pathways.

It has been shown that IL-6 signaling in the liver causes rapid and dramatic upregulation of SOCS-3 but not SOCS-1, 2, CIS, which correlates with the subsequent downregulation of phosphorylated Stat3, thereby terminating the IL-6 signal [14]. This find- ing may explain why hyperexpression of IL-6 may at times inhibit cell growth and cause injury [100]. The dramatic upregulation of SOC-3 and other cytokine

inhibitors by IL-6 may block its own signaling of the signaling of other cytokines. SOCS is an important mechanism that prevents uncontrolled cytokine signaling.

7.5

Role of IL-6/gp130 in Liver Injury

7.5.1

Acute Liver Injury

Growth factor/cytokines, such as HGF, IGFBP-1 and IL-6, promote hepatic survival by stimulating liver regeneration and providing hepatoprotection in a variety of liver injury models including Fas-medi- ated injury, toxic damage caused by hepatotoxins (such as CCl

4

) and ischemic liver injury [48, 50, 72, 94]. The most complete analyses of hepatoprotection have been performed using IL-6-deficient animals, and the role of IL-6 in protecting against injury and apoptosis has been relatively defined [9, 13, 22, 24, 31, 48, 61, 99]. Following acute CCl

4

treatment, IL- 6

–/–

mice develop increased hepatocellular injury and defective regeneration with significant blunting of Stat3 and NF- κB activation and reduced hepa- tocyte DNA synthetic and mitotic responses [48].

After CCl

4

treatment, unlike partial hepatectomy, increased hepatocyte apoptosis is noted in IL-6

–/–

livers. Pretreatment with IL-6 prior to CCl

4

reduces acute CCl4 injury and apoptosis, and accelerates re- generation in both IL-6

+/+

and IL-6

–/–

livers.

Several studies have examined the role of IL-6 in

liver injury following ischemia reperfusion. In one

study both hepatectomy and ischemia were simulta-

neously applied. It was found that hepatic ischemia

in combination with hepatectomy significantly re-

duced the mitogenic response in mouse livers [13,

80]. Treatment with IL-6 improves the mitogenic

response in the face of ischemic injury back to the

normal level. In warm/ischemia-reperfusion mod-

els, IL-6 is protective against ischemic injury and

IL-6

–/–

livers show increased injury. In this model

TNF- α results in injury, because antibodies to TNF-

α are able to reduce ischemic injury to a similar de-

gree as administration of IL-6. A program of gene

expression, activation of STAT3, NF- κB, and a high

degree of hepatocyte proliferation similar to that

in the hepatectomy model have been observed in

transplanted rodent livers subjected to prolonged

warm ischemia [22], indicating that IL-6-dependent

hepatic regeneration occurs following ischemic liver

injury. In another model of liver injury associated

with high levels of TNF- α, the Con A T cell activa-

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tion liver model, antibodies to TNF- α reduced in- jury as did injection of recombinant IL-6 [61].

Fas ligation, a purer model of hepatocyte apop- tosis than CCl

4

toxicity, provides more insight into mechanisms by which IL-6 reduces hepatocyte ap- optosis. IL-6

–/–

mice are more susceptible to Fas- mediated death than wild-type animals [47]. The direct anti-apoptotic effects of IL-6 are demonstrat- ed in vitro as IL-6 decreases Fas-mediated apopto- sis in both IL-6

–/–

and IL-6

+/+

primary hepatocyte cultures, suggesting that IL-6

–/–

hepatocytes have a pre-existing defect in anti-apoptotic pathways.

After Fas activation, IL-6

–/–

livers have evidence of both proximal and distal alterations in the apop- totic pathways including elevated caspase-8 and -3 activation-associated fragments, and loss of cyto- chrome c staining. IL-6

–/–

livers have reduced pre- existing protein expression of the anti-apoptotic factors Bcl-2 and Bcl-xL as well as more rapid degra- dation of FLICE-inhibitory protein (FLIP) following Fas treatment, which appears to be post-transcrip- tionally regulated (Fig. 7.7). Stat3 is the major effec- tor of IL-6-mediated hepatoprotections; it blocks apoptotic injury in two ways: (i) induction of anti- caspase regulators; and (ii) reduction of oxidative injury via upregulation of an anti-oxidant protein, Ref-1 [36, 94].

IGFBP-1, a protein whose expression is regulated at least in part by IL-6, is also highly hepatoprotec- tive in CCl

4

and Fas injury models [50]. The mecha- nism by which IGFBP-1 protects the liver appears to be largely distinct from that of IL-6. After Fas treat- ment, IGFBP-1 deficiency is associated with mas- sive hepatocyte apoptosis and caspase activation that is corrected by pretreatment with IGFBP-1. In IGFBP-1-deficient livers, Fas treatment is accompa- nied by elevated activated matrix metalloprotein- ase-9 (MMP-9), a known target of fibronectin signal transduction and activator of TGF- β, a hepatocyte apoptogen that is dramatically elevated. Both MMP- 9 and TGF- β expression are suppressed by IGFBP-1 treatment.

7.5.2

Chronic Liver Injury

Data support a role for growth factors such as HGF [17, 59, 97, 101] and cytokines such as IL-6 in pro- tecting the liver against chronic liver injury. Re- petitive doses of CCl

4

in the presence or absence of phenobarbital result in increased injury and fibrosis in IL-6

–/–

compared with IL-6

+/+

livers. After acute and chronic injury, IL-6

–/–

livers demonstrate the protracted presence of alpha-smooth muscle actin associated with activated stellate cells, suggesting a

disturbed response in wound healing that progress- es to fibrosis [48]. Contradictory results were ob- tained using a chronic CCl

4

model in IL-6

–/–

mice incompletely backcrossed onto a Balb/c strain [64].

In this model, increased fibrosis was observed in IL-6

+/+

(pure Balb/c) as compared to the IL-6

–/–

(six generation Balb/c) livers. The degree of fibrosis was significantly lower than was observed in Kovalovich et al. (2000) [48]. Conceivably this difference could be due to different susceptibilities of various mouse strains.

In the bile duct ligation model, IL-6

–/–

mice de- velop more severe liver disease, reduced hepatocyte proliferation, reduced liver mass increase, more ad- vanced biliary fibrosis and a higher mortality rate [24]. Treatment with recombinant IL-6 for several weeks improved several parameters including liver mass restitution. The authors concluded that IL-6 had the dual effect of contributing to biliary tree in- tegrity and maintenance of hepatocyte mass during chronic injury. The absence of IL-6 led to increased fibrosis.

Normally, acute liver injury is followed by a wound-healing response that seeks to contain the injury, reconstitute lost liver cell mass, and restore the extracellular framework of the liver [21, 30].

Cytokines, which may be pro- or anti-fibrogenic, have been shown to play a major role in the wound-

Fig. 7.7. Proposed model for the actions of IL-6 and Stat3 that result in hepatoprotection against Fas activation. Fas ligand (FasL) interaction with its receptor activates the caspase cascade that is blocked by IL-6 and Stat3 through the upregulation of FLIP, Bcl-2, and Bcl-XL. Fas activation also generates an oxidative stimulus that is blocked by the upregulation of Ref-1. Bax; Bid;

Apaf1; Rac1; FADD (Fas-associated death domain). Bax and Bid are proapoptotic proteins, Apaf1 is a component of the apopto- some along with cytochrome c and caspase-9. Rac1 is a small GTP-binding protein that activates NAPDH oxidase

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healing response to liver injury. Though the precise mechanisms remain unclear, IL-6

–/–

livers have per- sistent stellate cell activation following acute injury.

Activation denotes a conversion from a resting, vita- min A rich, perisinusoidal cell to one that is prolif- erative, fibrogenic, and contractile. Following reso- lution of injury, it is postulated that activated stel- late cells may be eliminated by apoptosis, although reversion back to a resting state has not been fully explored [42]. Absence of IL-6 may cause a persist- ence of activated stellate cells that would otherwise be eliminated, leading to increased chronic liver in- jury and fibrosis.

Active matrix metalloproteinase-2 (MMP-2) has been shown to be elevated both in rat models of chronic liver injury and in human cirrhotic livers and thus, may have a profibrogenic role [63, 88]. An interplay between MMP-2 and IL-6 in modulating hepatic injury and fibrosis is a possible explanation for the fibrotic response to IL-6 deficiency. After acute and chronic CCl

4

treatment, increased MMP-2 is observed in areas of necrosis but staining is con- sistently higher in the IL-6

–/–

livers [7]. IL-6

–/–

livers have persistent injury, increased α-smooth muscle actin ( α-SMA) staining, and increased MMP-2 lev- els. In chronic CCl

4

injury, IL-6-deficient livers have increased MMP-2 staining and persistent α-SMA staining correlating with increased collagen type I staining. These findings suggest that upregulation of MMP-2 in the IL-6-deficient livers may be impor- tant for mediating increased injury, delayed heal- ing, and progression to fibrosis.

7.6

Conclusions

The importance of IL-6 in the liver in regulating the acute phase response has been known for many years. In the past decade it has become evident that IL-6/gp130/Stat3 pathways in the liver have impor- tant roles in the adaptive response of the liver to in- jury, whether mediated by toxins, resection or infec- tious agents. IL-6 is able to activate a diverse range of intracellular signaling that leads to both prolifera- tive and hepatoprotective responses. The Stat3 path- way is largely responsible for the hepatoprotective effects of IL-6, whereas the growth effects appear to result from the combination of MAPK pathway and Stat3 pathway activation, and potentially other less well-characterized (AKT, PI3 kinase) pathways.

The anti-apoptotic effects of IL-6/Stat3 are due to a dual effect of stabilizing anti-apoptotic proteins and reducing the level of reactive oxygen species. The interplay of growth factor and cytokine signals as

depicted in Fig. 7.5 is a hypothesis based on current data and requires further support and refinement.

It is not clear how the size of the liver is determined, and why liver "mitogens" are also hepatoprotec- tive in liver injury models. Understanding of the mechanisms by which IL-6 ameliorates liver injury, fibrosis, and hepatocyte apoptosis, and stimulates regeneration post hepatic transplant could have im- portant therapeutic implications.

Selected Reading

Heinrich PC, Behrmann I, Haan S et al. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J 2003;374:1–20.

Koniaris LG, McKillop IH, Schwartz SI et al. Liver regeneration. J Am Coll Surg 2003;197:634–659.

Levy DE, Lee CK. What does Stat3 do? J Clin Invest 2002;109:1143–

1148.

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