40

INTERLEUKIN-6 SIGNALING DURING THE ACUTE-PHASE RESPONSE

OF THE LIVER

JOHANNES G. BODEPETER C. HEINRICH

ACUTE-PHASE RESPONSE OF THE ORGANISM 565

ACUTE-PHASE RESPONSE OF THE LIVER AND INVOLVEMENT OF THE NEUROENDOCRINE AXIS 566

RELEVANCE OF THE ACUTE-PHASE RESPONSE IN RELATION TO METABOLIC FUNCTIONS 568

INTERLEUKIN-6–INDUCED ACUTE-PHASE PROTEIN SYNTHESIS IN HEPATOCYTES THROUGH THE JAK/STAT PATHWAY 568Interleukin-6–Type Cytokines and Their Receptors 568Interleukin-6 Signaling 569Janus Kinases 570STAT Family of Transcription Factors 572

NEGATIVE REGULATION OF INTERLEUKIN-6 SIGNALING 573Tyrosine Phosphatases 573

Recent reviews on the subject of cytokines and the acutephase response have been published (1–6) (see Chapter41). This chapter focuses on the acute-phase response ofthe liver with emphasis on interleukin-6 (IL-6) signaltransduction regulating acute-phase protein (APP)expression.

ACUTE-PHASE RESPONSE OF THEORGANISM

Neoplasm, tissue injury, infection, or inflammation areaccompanied by a number of changes within the organism,representing an immediate set of inflammatory reactionscounteracting these challenges, aiming at the isolation andneutralization of pathogens and the prevention of furtherpathogen entry. The resulting minimization of tissue dam-age and promotion of repair processes permits the homeo-static mechanisms of the organism to rapidly restore normalphysiologic function. The inflammatory cascade is initiatedthrough activated blood monocytes and tissue macrophagesat the sites of injury by the release of a set of primaryinflammatory mediators, such as histamine, leukotrienes,prostaglandins, and the proinflammatory cytokines IL-1β

J. G. Bode: Department of Internal Medicine, Division of Gastroenterol-ogy Hepatology and Infectiology, Laboratory of Experimental Hepatology,Heinrich-Heine University, Düsseldorf, 40225 Düsseldorf, Germany.

P. C. Heinrich: Department of Biochemistry, University Hospital of theRheinisch Westfälische Technische Hochschule Aachen, D-52074 Aachen,Germany.

Feedback Inhibitors: Suppressors of Cytokine Signaling 573Protein Inhibitors of Activated STATs 573Modulation of the Jak/STAT Pathway Through the

Availability of the Signaling Components 574Endocytosis of the Interleukin-6/Interleukin-6 Receptor

Complex 574Half-Lives of Signaling Components 574

MODULATION OF INTERLEUKIN-6 SIGNALINGTHROUGH THE JAK/STAT PATHWAY BY CROSS-TALKS WITH OTHER SIGNALING CASCADES 575Preactivation of Erk-Type Mitogen-Activated Protein Kinases

Inhibits Interleuken-6–Induced STAT Activation 575Interleukin-6 Signaling Is Down-Modulated by Pretreatment

with Proinflammatory Mediators 576

INTEGRATIVE VIEW ON THE CROSS-TALK BETWEENTHE SIGNALING PATHWAYS OF INTERLEUKIN-6AND PROINFLAMMATORY MEDIATORS 577

and tumor necrosis factor-α (TNF-α) (Fig. 40.1) (reviewedin refs. 1–6). These again induce the synthesis of a range ofsecondary cytokines and chemokines such as IL-6 and IL-8from macrophages, monocytes, endothelial cells, and fibro-blasts. The chemotactic activities of some of these moleculesin turn lead to the attraction of neutrophils and otherimmune effector cells, e.g., lymphocytes, to the site ofinflammation, where the immigrated cells release furtherinflammatory cytokines. This process rapidly enhances thelocal inflammatory response to counteract the inflamma-tory stimulus and to tidy up the cellular debris generated byany associated tissue damage.

This local response may escalate into a systemic reactionof the organism characterized by the induction of neuroen-docrine changes such as, for example, pain, fever, somno-lence, and increased release of such systemically actingmediators as arginine, vasopressin, insulin-like growth fac-tor, corticotropin-releasing hormone, corticotropin, andothers. Furthermore, hematopoietic alterations such asleukocytosis and thrombocytosis, metabolic disturbances

such as cachexia, and modifications of the lipid metabolismand decreased gluconeogenesis belong to the characteristic,systemic phenomena observed during the acute-phase reac-tion.

Apart from these systemic alterations, changes of plasmalevels of several different proteins, known as the acute-phaseproteins have been recognized as a characteristic feature ofthe acute-phase response (Table 40.1). Increases ordecreases in concentrations of these proteins are mainlyattributed to modifications of their synthesis by hepato-cytes. The extent of these changes varies largely anddepends on the species investigated. Thus, α2-macroglobu-lin and α1-acid glycoprotein are major APPs in rats withincreases of about 100-fold during inflammation, whereasthe plasma concentrations of these proteins do not changein humans (7,8). The main acute-phase reactants inhumans are C-reactive protein and serum amyloid A; theirin vivo concentrations rise as much as 1,000-fold during aninflammatory response (9,10).

ACUTE-PHASE RESPONSE OF THE LIVERAND INVOLVEMENT OF THE NEUROENDOCRINE AXIS

The liver plays a pivotal role in the acute-phase response ofthe organism. Its importance for the systemic reactiontoward pathogens is emphasized by the fact that it containsthe largest pool of macrophages (Kupffer cells) of the bodyat a strategically important anatomic and physiologic posi-tion (11,12).

As already mentioned, hepatocytes are the major sites ofAPP synthesis. The list of cytokines capable of inducingAPP production in the liver is extensive and still increasing.It includes the members of the IL-6–type cytokine family:IL-6, leukemia inhibitory factor (LIF), IL-11, oncostatin M(OSM), ciliary neurotrophic factor (CNTF), cardio-

566 Chapter 40

FIGURE 40.1. The acute inflammation process.

TABLE 40.1. MAJOR HUMAN AND RAT ACUTE-PHASE PLASMA PROTEINS

HumanC-reactive proteinSerum amyloid ALPS-binding proteinFibrinogenHaptoglobinα1-Antichymotrypsin

Ratα2-MacroglobulinLPS-binding proteinα1-Acid glycoproteinCysteine proteinase inhibitorSerine proteinase inhibitor 2.3Tissue inhibitor of metalloproteinases-1

LPS, lipopolysaccharide.

trophin-1 (CT-1), and other mediators of growth regula-tion, differentiation, or inflammation such as glucocorti-coids, epidermal growth factor (EGF), hepatocyte growthfactor (HGF), IL-1, and TNF-α. Among these cytokines,IL-6 has been identified as the major stimulator of APPsynthesis in parenchymal cells of the liver. These in vitroobservations have been confirmed by the phenotype of IL-6 knockout mice, where IL-6 has been shown to be crucialfor the acute-phase response during sterile experimentalinflammation (13).

As schematically shown in Fig. 40.2, the inflammatorycascade leading to induction of APP synthesis is primed by

inflammatory stimuli such as viruses, bacteria/lipopolysac-charide (LPS), or tissue injury acting on blood monocytesand resident tissue macrophages such as Kupffer cells. Inturn, these cells release the proinflammatory mediators IL-1 and TNF-α into the circulation, and subsequently induceIL-6 through an autocrine loop. The IL-6 serum levels arefurther increased by IL-6 produced by IL-1– and TNF-α–stimulated endothelial cells, fibroblasts, and other stro-mal cells. Moreover, IL-6 is also produced by cells from theanterior pituitary gland (14). As depicted in Fig. 40.2, IL-6and IL-1 stimulate the secretion of adrenocorticotropic hor-mone (ACTH) from the anterior pituitary gland via the

Interleukin-6 Signaling 567

FIGURE 40.2. Involvement of the neu-roendocrine axis in the acute-phaseresponse of the liver. ACTH, adrenocor-ticotropic hormone; APP, acute-phaseprotein; EC, endothelial cells; F, fibro-blasts; GC, glucocorticoids; IL, inter-leukin; KC, Kupffer cells; MO, mono-cytes; PBMC, peripheral bloodmononuclear cells; parenchymal cells;TNF, tumor necrosis factor.

induction of corticotropin-releasing hormone (CRH),released from the hypothalamus. Furthermore, despite theinduction of ACTH via CRH, IL-6 can directly induce therelease of ACTH, prolactin, growth hormone, and luteiniz-ing hormone from the anterior pituitary gland (4,15).ACTH subsequently leads to the release of glucocorticoidsfrom the adrenal glands. Glucocorticoids are important reg-ulators modulating the inflammatory response, since theyhave been shown to upregulate the production of cytokinereceptors in hepatocytes, as for example IL-6 or interferon-γ (IFN-γ) receptors sensitizing these cells to the respectivecytokines (4,16,17). Moreover, in certain species, glucocor-ticoids directly upregulate the production of a number ofAPPs by hepatocytes (18,19). On the other hand, glucocor-ticoids display an important inhibitory activity againstinflammatory cytokine production by monocytes,macrophages, and other immune effector cells (not shownin Fig. 40.2). In summary, these facts reflect a complex reg-ulatory feedback mechanism between the neuroendocrineand immune system involved in the control of inflamma-tory responses of the organism.

RELEVANCE OF THE ACUTE-PHASERESPONSE IN RELATION TO METABOLICFUNCTIONS

It is interesting that not only the well-known secreted APPschange during the hepatic acute-phase response, but alsokey enzymes of liver-specific metabolic functions. Thus, ithas been shown in rat hepatocyte primary cultures that theglucagon-mediated expression of the gluconeogenic keyenzyme phosphoenolpyruvate-carboxykinase is inhibited byIL-6, IL-1β, and TNF-α (20,21). Surprisingly, the expres-sion of the key enzyme of the glycolytic pathway, glucoki-nase—induced by insulin—is also impaired by IL-6, IL-1β,and TNF-α (20,21). Based on these observations, theauthors conclude that the liver—disturbed in its homeosta-sis—gives priority to the synthesis of APPs instead ofimportant metabolic enzymes in order to cope with the lim-ited amounts of amino acids for protein biosynthesis.

INTERLEUKIN-6–INDUCED ACUTE-PHASEPROTEIN SYNTHESIS IN HEPATOCYTESTHROUGH THE JAK/STAT PATHWAY

Interleukin-6–Type Cytokines and TheirReceptors

IL-6 belongs to a family of cytokines characterized by afour-alpha-helix bundle topology. Besides IL-6, thecytokines IL-11, LIF, CNTF, CT-1, OSM, and the recentlydiscovered B-cell stimulatory factor-3/novel neurotrophin-1 are members of this family. With the exception of CNTFand CT-1, IL-6–type cytokines are classic secretory proteins

synthesized with N-terminal signal peptides (reviewed inref. 3).

The tertiary structure of IL-6 has been solved by nuclearmagnetic resonance (NMR) spectroscopy (22) and x-raycrystallography (23). As shown in Fig. 40.3, helix A (red) isconnected by a long loop with helix B (green) in such a waythat helix B lies parallel to helix A. Helix B is separated fromhelix C (yellow) by a very short loop allowing only anantiparallel packaging. Helix C is again joined by a longloop with helix D (blue), resulting in parallel packaging ofthe C-terminal helices. As a consequence the overall foldshows an up-up-down-down topology of the four longalpha-helices. Some biochemical properties of human IL-6are listed in Table 40.2.

IL-6 exerts its action via a specific surface receptor complexon hepatocytes consisting of an α-receptor subunit, gp80, anda signal transducing subunit, gp130. Both receptor chains aretype I membrane proteins characterized by an extracellular N-terminus and one transmembrane domain. Gp80 and gp130both belong to the cytokine receptor class I family defined bythe presence of at least one cytokine-binding module consist-ing of two fibronectin type III–like domains of which the N-terminal domain contains a set of four conserved cysteineresidues and the C-terminal domain, a WSXWS motif (24).

568 Chapter 40

AD

B

C

FIGURE 40.3. The three-dimensional structure of interleukin-6(IL-6) (ribbon representation). The four long α-helices (A, B, C,and D) and the connecting loops (gray), as far as they have beendefined, are shown. The Brookhaven Databank accession num-ber is 1IL6.

Figure 40.4 shows the domain structures of the two IL-6receptor chains and Table 40.2 their biochemical properties.Both IL-6 receptor subunits contain an Ig-like domain locatedat the N-terminus. Gp130 has three additional membrane-proximal fibronectin type III–like domains.

The binding of IL-6 to its α-receptor gp80 has beenstudied in great detail. Whereas the Ig-like domain of gp80is dispensable for biologic activity, the residues crucial forligand binding are located in the cytokine-binding module.Mutagenesis studies have shown that residues in the loopsnear the hinge region between the domains of the cytokine-binding module are involved in ligand recognition (25,26).The IL-6/gp80 complex forms a ternary complex with twosignal transducing receptor subunits gp130. Based on thegrowth hormone–growth hormone receptor complex struc-ture (27), a model for the IL-6/gp80/gp130 ternary com-plex has been proposed and used for mutagenesis studies. Inthis model (Fig. 40.5) domains D2 and D3 of gp130 (Fig.40.4) are in contact with IL-6. Point mutations of tyrosine190 and phenylalanine 191 in D2 and valine 252 in D3 ledto the loss of binding of gp130 to IL-6/IL-6R complexes aswell as to impaired signal transduction (28,29). Most inter-estingly, also the N-terminal domain D1 of gp130 turnedout to be crucial for ligand binding and signaling (29,30).

The role of the membrane-proximal domains D4, D5,and D6 of gp130 in receptor activation has been investi-gated by construction of deletion mutants lacking D4, D5,and D6. Deletion of D5 did not alter the affinity of thereceptor to its ligand, but this mutant did not transduce anysignal in response to IL-6. Thus, it has been concluded that

high-affinity ligand binding is not sufficient for receptoractivation, but an adjustment of a well-defined gp130dimer conformation is required for gp130 activation andsignal transduction (31).

Due to the lack of a three-dimensional (3D) structure ofa comparable cytokine-receptor complex, no molecularmodel that shows the binding of the second gp130 mole-cule to the IL-6/IL-6R complex is presently available.

Interleukin-6 Signaling

The major steps in IL-6 signal transduction have beenworked out independently in two laboratories (32,33). Thefirst event in IL-6 signaling is the binding of the ligand toits α-receptor, followed by the homodimerization of the sig-nal transducer gp130 by formation of a ternary complex.The IL-6–induced dimerization of gp130 initiates a phos-phorylation cascade (Fig. 40.6). The first step in this cas-cade is the autophosphorylation of tyrosine kinases of theJanus (Jak) family. The Janus kinases Jak1, Jak2, and

Interleukin-6 Signaling 569

TABLE 40.2. BIOCHEMICAL PROPERTIES OFHUMAN INTERLEUKIN-6 (IL-6) AND ITS RECEPTOR SUBUNITS

Property IL-6 IL-6R gp130

Number of amino acidsPrecursor 212 468 918Mature protein 184 449 896Extracellular domain 339 597Transmembrane domain 28 22Intracellular domain 82 277

Molecular mass (kd)Predicted 20.8 49.9 101Observed 21–28 80 130–150

GlycosylationPotential N-glycosylation sites 2 5 10N-glycosylation demonstrated Yes 1–2 Yes Yes

Number of cysteine residues 4Number of S-S bridges 2mRNA size (kb) 1.3 5 7Number of exons 5 n.d. 17Chromosomal localization 7p21-p14 n.d. 5,17Soluble forms Yesa,b Yesa

aGenerated by alternative splicing.bGenerated by shedding.mRNA, messenger RNA.

FIGURE 40.4. Domain composition of IL-6 receptor subunits.Predicted immunoglobulin (Ig)-like domains are shown in lightgray, fibronectin type III–like domains in medium gray, andcytokine-binding modules (CBMs) in dark gray. The horizontalbars in the CBMs define the conserved cysteine residues (thinwhite lines) or the WSXWS motif (broad white bars). The lengthsof the cytoplasmic parts correspond to the respective numbers ofamino acids. Tyrosine residues in the cytoplasmic domain ofgp130 are represented as dark lines, box 1 and box 2 as graybars, and the di-leucine motif as a dark gray bar.

Tyk2—all constitutively bound to the membrane-proximalpart of the cytoplasmic tail of gp130—become tyrosine-phosphorylated and thus enzymatically active. Subse-quently, tyrosine residues in the cytoplasmic part of gp130are phosphorylated. These phosphotyrosines function asdocking sites for transcription factors of the STAT (signaltransducers and activators of transcription) family recruit-ing unphosphorylated predimerized STAT factors (34,35)via their Src homology 2 (SH2) domains. In turn, theSTAT factors are also phosphorylated at tyrosine residuesnear their C-termini and released from the receptor com-plex. Phosphorylated homo- or heterodimeric STATs aretranslocated to the nucleus where they bind to IL-6–responsive elements in the 5�-flanking regions of targetgenes, e.g., acute-phase protein genes.

The importance of these signaling components not onlyfor IL-6 but also for the signal transduction of othercytokines is emphasized by the fact that gene knockouts ofSTAT3 (36), gp130 (37), and Jak1 (38) showed lethal phe-notypes, whereas the phenotypes of mice deficient for onlyone IL-6–type cytokine displayed relatively mild defects.

In the following subsections the different steps and the keyplayers involved in IL-6 signaling are discussed in more detail.

Janus Kinases

Janus kinases (Jaks) are intracellular tyrosine kinases withmolecular masses of 120 to 140 kd. Four members areknown in mammalian cells: Jak1, Jak2, and Tyk2 are widelyexpressed, and Jak3 is mainly found in cells of hematopoi-etic origin. The structural organization of Jaks is shown inFig. 40.7. A typical kinase domain, also called JH1 (Jakhomology-1) domain, is located at the C-terminus. It ispreceded by a kinase-like domain (JH2). The N-terminalhalf of the Jaks contains five additional regions with highsequence similarity between the different Jaks (JH3 to JH7)(reviewed in refs. 39–41).

Within the kinase domain, Jaks show considerable simi-larity to other kinases with respect to an activation loopimplicated in regulation of kinase activity (reviewed in ref.42). Ligand-induced receptor dimerization is thought tobring the associated Jaks into close proximity, leading totheir activation via inter- or intramolecular phosphoryla-tion at sites necessary for catalytic activity (40,41). The sig-nificance of the kinase-like domain is not clear. Thisdomain has been described to have an influence on thekinase activity, although no clear picture emerges from the

570 Chapter 40

gp130

IL-6

IL-6R

Tyr 190

Phe 191

Val 252

FIGURE 40.5. Model of the Interleukin(IL)-6–IL-6R-gp130 ternary complex.Amino acid side chains analyzed bymutagenesis are depicted in the insertand labeled in orange: tyrosine 190,phenylalanine 191, and valine 252.

Interleukin-6 Signaling 571

FIGURE 40.6. Interleukin-6 (IL-6) signal transduction through the gp130/Jak/STAT pathway. APRE,acute phase response element; encircled Y, tyrosine; black P in gray circle, phosphate; Jak, Januskinases; STAT, signal transducer and activator of transcription; SH2, Src homology domain 2.

FIGURE 40.7. Structural organization of Janus kinases (JAK), STAT (signal transducer and activa-tor of transcription) factors, SH2-domain–containing tyrosine phosphatase (SHP2), and suppres-sors of cytokine signaling (SOCS) proteins.

literature as to whether this is a positive or a negative one.The N-terminal half of the Jak regions JH7 to JH3 (Fig.40.7) is involved in receptor association (40,41).

It should be noted that Jaks, apart from being receptor-associated enzymes, may fulfill further functions. For Tyk2a structural role has been demonstrated: it is necessary forsurface expression of the IFNARI receptor as well as forhigh-affinity binding of IFN-α (43,44).

IL-6 leads to the activation of Jak1, Jak2, and Tyk2(32,33,45). This holds true also for the other IL-6–typecytokines IL-11, LIF, OSM, CT-1, and CNTF. Whichkinases and to what extent a certain kinase is activatedvaries between cells (33,46) and possibly reflects differentexpression levels of Jaks. Among the Jaks, Jak1 plays a cru-cial role for signal transduction of IL-6–type cytokines asdemonstrated by studies with Jak1-deficient fibrosarcomacells and with cells derived from Jak1 knockout animals(38,47).

STAT Family of Transcription Factors

Seven mammalian STAT genes have been cloned so far andlocalized in three chromosomal clusters, suggesting that thisfamily of proteins has evolved by gene duplication. Themammalian STAT factors are designated as STAT1, 2, 3, 4,5a, 5b, and 6 (reviewed in ref. 40). Except for STAT2, alter-natively spliced forms have been described. In the case ofSTAT4 and STAT6, the corresponding isoforms could notbe identified.

With the exception of STAT4, STAT factors are ubiqui-tously expressed. STAT 4 expression is more restricted tomyeloid cells and testis (48). The regulation of synthesis ofSTATs does not seem to play a major role in cytokine sig-naling. STAT activity is predominantly regulated by post-translational modifications, i.e., tyrosine and serine phos-phorylation. STATs are mainly activated after stimulationof cytokine receptors. However, there are a growing numberof reports demonstrating STAT activation also via receptortyrosine kinases: EGR receptor (EGF-R), fibroblast growthfactor receptor (FGF-R), c-met, platelet-derived growth fac-tor receptor (PDGF-R), colony-stimulating factor-1 recep-tor (CSF-1-R), c-kit, and insulin-R (49–58), and G-pro-tein–coupled receptors (angiotensin-R) (59). Ligandssignaling through the same class of receptor complexes acti-vate usually the same set of STAT factors (49); e.g., all IL-6–type cytokines activate STAT3 and STAT1.

STATs are proteins with a conserved structural organiza-tion (Fig. 40.7). They consist of 750 to 850 amino acids(e.g., STAT1, 750 aa; STAT3, 770 aa). Various domainswithin the STAT molecules have been defined: a tetramer-ization domain at the N-terminus, a DNA-binding domainin the middle, an SH2-domain, and a transactivationdomain at the C-terminal end. In all STATs a tyrosineresidue near the C-terminus is phosphorylated upon activa-

tion (tyrosine 701 for STAT1 and tyrosine 705 for STAT3)(reviewed in refs. 3 and 40).

The function of the highly conserved SH2-domain iswell established. This domain is responsible for the bindingof the STATs to tyrosine-phosphorylated receptor motifs(60–62) and also for homo- and heterodimerization. A pre-association of unphosphorylated STAT factors has beendescribed (34,35). The mechanism responsible for thisinteraction, however, needs to be elucidated.

The activity of the C-terminal transactivation domain ofSTATs is at least partially regulated by a serine phosphory-lation (S727 in STAT1 and STAT3) (63–66). Recent exper-iments of Jain et al. (67) have shown that STAT3 serinephosphorylation after IL-6 stimulation is due to the actionof protein kinase Cδ (PKCδ).

The tyrosine phosphorylated STAT dimers translocatefrom the cytoplasm to the nucleus (Fig. 40.6); upon IL-6treatment of liver cells, nuclear translocation of STAT3occurs within minutes. The translocation is transient. Themechanism by which STAT factors enter the nucleus isunknown. A nuclear localization sequence (NLS) responsi-ble for the transport of proteins to the cell nucleus has notbeen identified in any of the STAT molecules cloned so far.Therefore, nuclear translocation of STATs might beachieved either via an untypical NLS or via an NLS-con-taining shuttle protein that associates with activated STATs.In this respect it should be noted that activated STAT5 (68)as well as STAT3 (69) form complexes with the glucocorti-coid receptor known to contain two NLS (70).

After nuclear translocation, STATs bind to specificenhancer sequences and stimulate—and in certain casespossibly also repress—transcription of respective targetgenes. During the past few years new target genes for IL-6have been identified and functional STAT binding siteshave been found in the promoter regions of these genes.STAT3 involvement in the transcriptional regulation ofmany of the well-known APPs such as C-reactive protein,α1-antichymotrypsin, α2-macroglobulin, lipopolysaccha-ride-binding protein, and tissue inhibitor of metallopro-teinases-1 has been shown in hepatocytes in vitro and invivo (71–75).

Besides APPs, a variety of STAT-activated genes has beendescribed: Jun B, c-Fos, interferon regulatory factor-1,CCAAT enhancer binding protein-β (C/EBPβ), intestinalcollagenase, vasoactive intestinal peptide, proopiome-lanocortin, heat shock protein (hsp)90, and IL-6 signaltransducer gp130 (reviewed in ref. 3). The promoter analy-sis of STAT-regulated genes revealed that STATs often asso-ciate with other transcription factors, resulting in a cooper-ative action. It has been shown, for example, thatSTAT3—the major transcription factor activated in livercells upon IL-6 treatment—interacts with C/EBPβ/nuclearfactor (NF)-IL-6 (74,76), NFκB (77), activating protein(AP)-1 (74,75,78,79), and glucocorticoid receptor (69).

572 Chapter 40

Moreover, tandem arrangement of STAT binding siteshas been reported for rat α2-macroglobulin and human α1-antichymotrypsin promoters, suggesting that STAT dimersmight form multimers on clustered binding sites. Such amultimerization has been demonstrated for STAT1. Forthis process the N-terminus has been shown to be crucial(80,81). These two modes of cooperative action support thenotion that gene regulation of IL-6–responsive genes is anintegrative process in which several transcription factorstogether modulate the rate of transcription of a target gene.

Although much detailed information on STAT enhancerinteraction became available during the last years, it is stillnot known how STAT factors and their cooperating tran-scription factors communicate to the basal transcriptionmachinery. In the case of STAT1, an interaction with cyclicAMP response-element–binding protein (CBP) and p300has been described (82,83). Also STAT3 has been found tointeract with CBP, resulting in an enhanced transcription ofthe human α1-antichymotrypsin gene (Schniertshauer, the-sis Aachen 1998).

NEGATIVE REGULATION OF INTERLEUKIN-6SIGNALING

In most systems STAT activation is transient, suggesting theexistence of efficient mechanisms for STAT inactivation.Various mechanisms of STAT inactivation have been pro-posed:

� gp130-, Jak-kinase-, and STAT-dephosphorylation bytyrosine phosphatases;

� induction of feedback inhibitors inactivating Januskinases;

� complex formation of activated STAT-dimers with spe-cific protein inhibitors.

Tyrosine Phosphatases

Among various tyrosine phosphatases known, SH2-domain–containing tyrosine phosphatase (SHP-2) seems toplay a pivotal role in IL-6 signaling. After IL-6–typecytokine stimulation, not only Jaks and STATs but also thetyrosine phosphatase SHP-2 is recruited to gp130 and sub-sequently phosphorylated. Whereas the role of the activatedSTATs in signaling is well established, that of SHP-2 is lessclear.

SHP-2 is a ubiquitously expressed tyrosine phosphataseof 585 amino acids and a molecular mass of about 65 kd.SHP-2, like its homologue SHP-1, contains two SH2domains at its N-terminus (Fig. 40.7). Both SH2-domainsare required for the recruitment of SHP-2 to the phospho-tyrosine motif of the activated gp130.

SHP-2 binds to phosphotyrosine 759 of gp130 (60,84).SHP-2 also interacts with Grb2 and very likely links the

gp130/Jak/STAT pathway to the Ras/Raf/MAP kinasepathway (85). Exchange of Y759 by phenylalanine ingp130 abrogates SHP-2 tyrosine phosphorylation (60,86)and in turn leads to elevated and prolonged STAT1- andSTAT3-activation, resulting in an enhanced APP geneinduction (87–89).

SHP-2 can be phosphorylated by many tyrosine kinasessuch as Src (90), bcr-abl (91), and Jaks. A Jak/SHP-2 inter-action and the phosphorylation of SHP-2 by Jaks have beendemonstrated (92). Although several reports on the role ofSHP-2 in IL-6 signaling have been published, further clar-ification is needed.

Feedback Inhibitors: Suppressors ofCytokine Signaling

A new family of feedback inhibitors of cytokine signalinghas been discovered in three different laboratories. Theseproteins are referred to as suppressors of cytokine signaling(SOCS) (93), Jak-binding proteins (JAB) (94), and STAT-induced STAT inhibitors (SSIs) (95). The proteins of thisfamily are relatively small molecules, about 200 amino acidsin length, containing a central SH2-domain, a kinaseinhibitory region (KIR), and a carboxy-terminal domaincalled the SOCS box (Fig. 40.7). The SOCS box plays animportant role in the regulation of degradation of theseproteins (96). The SH2-domain was shown to directlyinteract with the kinase domain of Jak1, Jak2, and Tyk2,thereby preventing receptor phosphorylation and activationof the STAT-factors (97–99). SOCS proteins are rapidlyinduced by a variety of cytokines, particularly through theJak/STAT pathway. Due to their potent action on Jakkinase activity, SOCS proteins represent powerful feedbackinhibitors of the Jak/STAT pathway (Fig. 40.8). Recently, ithas been found that inhibition of tyrosine phosphorylationof the phosphatase SHP-2 correlates with an enhancedinduction of SOCS3 messenger RNA (mRNA). On theother hand, overexpression of SOCS3 protein decreased thelevel of tyrosine phosphorylated SHP-2 after IL-6 stimula-tion. Interestingly, SOCS3, but not SOCS1, requires andbinds to the SHP-2 recruitment site of the cytoplasmicregion (Y759) of gp130 to exert its negative function on IL-6 signaling (100).

Protein Inhibitors of Activated STATs

In various human tissues protein inhibitors of activatedSTATs (designated as PIASs) have been discovered(101,102). It is speculated that there may exist a specificPIAS for each phosphorylated STAT factor. For example,STAT3, but not STAT1, activity is regulated by PIAS3 (Fig.40.8). However, it is still not understood how PIAS pro-teins are regulated.

Interleukin-6 Signaling 573

Modulation of the Jak/STAT PathwayThrough the Availability of the SignalingComponents

Modulation of the Jak/STAT pathway occurs in the follow-ing ways:

� escape from overstimulation by ligand/receptor internal-ization;

� regulation of the availability of the signaling moleculesby different half-lives.

Endocytosis of the Interleukin-6/Interleukin-6 Receptor Complex

Most cells escape from being overstimulated by surface recep-tor internalization. After binding to its receptor, IL-6 is effi-ciently internalized and the α-receptors/gp80 are downregu-lated (Fig. 40.8), resulting in a complete depletion of IL-6surface binding sites within 30 to 60 minutes (103,104). Toreplenish IL-6 binding sites, de novo protein synthesis isrequired, suggesting that ligand and gp80 have beendegraded after internalization, most likely in the lysosomalcompartment. It has been previously demonstrated that theIL-6 signal transducer gp130 contains a di-leucine internal-ization motif within its cytoplasmic tail necessary for theendocytosis of the IL-6 receptor complex (105). Since gp80per se is internalized very inefficiently, the observed downreg-ulation of the IL-6 α-receptor can be explained by the for-mation of a ternary receptor complex consisting of IL-6/gp80and gp130 in which gp130 not only mediates signal trans-duction, but also promotes efficient endocytosis of the IL-

6/IL-6 receptor complex (Fig. 40.8). Recently, the activationof the Jak/STAT pathway via the IL-6 receptor complex oragonistic antibodies against gp130 have been shown not tobe required for efficient endocytosis to occur (106), suggest-ing that signaling and endocytosis are independent processes.Interestingly, the signal transducer gp130 undergoes consti-tutive endocytosis independent of the presence of IL-6. Inter-nalization of gp130 occurs most likely via clathrin-coatedpits, since a constitutive interaction between gp130 and theplasma membrane adaptor protein complex AP-2 has beenobserved (107).

Half-Lives of Signaling Components

Whereas considerable information has been accumulated con-cerning the time course of activation for the individual signal-ing molecules, data on the availability of the proteins involvedin IL-6–type cytokine signal transduction are scarce. Never-theless, the availability of these molecules, determined by thebalance of protein synthesis and degradation, also influencesIL-6 signal transduction. The turnover rates for the variousproteins differ substantially (108). Three groups of signalingproteins can be discriminated; whereas the feedback inhibitorsSOCS1, SOCS2, and SOCS3 are very short-lived (1 to 1.5hours), STAT1, STAT3, and SHP2 have an extremely lowturnover (8.5 to 20 hours). The Janus kinases Jak1, Jak2,Tyk2, and gp130 show intermediate half-lives (2 to 3 hours).Based on these observations it is concluded that signalingcomponents activated by posttranslational modifications arelong-lived, whereas the activities of short-lived proteins ismainly regulated at the transcriptional level.

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FIGURE 40.8. Negative regulation of theinterleukin-6 (IL-6)–type cytokine signaltransduction pathway. APP, acute-phase pro-tein; PIAS, protein inhibitors of activatedSTATs; SH2, Src homology 2; SOCS, suppres-sors of cytokine signaling.

MODULATION OF INTERLEUKIN-6SIGNALING THROUGH THE JAK/STATPATHWAY BY CROSS-TALKS WITH OTHERSIGNALING CASCADES

Preactivation of Erk-Type Mitogen-Activated Protein Kinases InhibitsInterleukin-6–Induced STAT Activation

A number of mediators has been reported to downregulateJak/STAT activation, e.g., transforming growth factor-β(TGF-β), granulocyte/macrophage colony-stimulating fac-tor (GM-CSF), and angiotensin II (109–111). The protein

kinase C activator phorbol 12-myristate 13-acetate (PMA)was recently shown to inhibit IL-6–induced STAT3 activa-tion via Erk/MAP kinases (112,113). These studies havebeen extended by the use of the Erk activators basic FGF(bFGF) and constitutively active raf (113a). Moreover,phosphotyrosine-759 of gp130—the docking site for SHP2(see Tyrosine Phosphatases, above) and SOCS3—is crucialfor the inhibitory effect of PMA-induced mitogen-activatedprotein (MAP) kinases on IL-6 signaling. Both PMA andbFGF rapidly stimulate SOCS3 mRNA expression. Thesefindings are schematically summarized in Fig. 40.9. Asmentioned above (Fig. 40.8) SOCS3 in turn inhibits IL-6

Interleukin-6 Signaling 575

FIGURE 40.9. Modulation of interleukin-6 (IL-6) signaling through the Jak/STAT pathway bycross-talks with other signaling cascades. bFGF, basic fibroblast growth factor; Jak, Janus kinases;LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; STAT, signal transducer and acti-vator of transcription; SOCS, suppressors of cytokine signaling; TNF, tumor necrosis factor.

signaling via inactivation of Jak kinases and thereby APPinduction. Accordingly, the IL-6–induced SOCS3 expres-sion is impaired.

In conclusion, it is intriguing that preactivation of theMAP kinase cascade by PMA or bFGF impairs the IL-6–dependent stimulation of the gp130/Jak/STAT pathwayvia induction of the negative feedback inhibitor SOCS3 (Fig.40.9). This cross-talk between the Jak/STAT- and the MAP-kinase pathways is even more complicated, since Erk-typeMAP-kinases are activated through gp130-associated SHP-2.

Interleukin-6 Signaling Is Down-Modulated by Pretreatment withProinflammatory Mediators

Pretreatment of rat Kupffer cells as well as humanmacrophages with the proinflammatory mediators TNF-α

or LPS largely decreases or even completely abolishesSTAT3 activation after IL-6 stimulation (114). This inhibi-tion closely correlates with the induction of SOCS3 mRNAby LPS or TNF-α. Since neither LPS nor TNF-α influ-ences IL-6–induced STAT3-activation in HepG2 cells orrat hepatocytes, this effect might be macrophage-specific.Both LPS and TNF-α are well-known activators of the p38MAP kinase (115,116). Interestingly, inhibition of the p38activity not only neutralizes particularly the TNF-α actionon IL-6–induced STAT3-activation, but also suppressesTNF-α–mediated induction of SOCS3 mRNA. Therefore,it can be concluded that inhibition of STAT3-activationand induction of SOCS3 mRNA are functionally linked(Fig. 40.9).

Another remarkable mechanism for the modulation ofIL-6 signaling has recently been observed. IL-1—but notTNF-α—has been shown to inhibit dose-dependently IL-

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FIGURE 40.10. Inhibition of interleukin-6 (IL-6)–induced luciferase activity by IL-1β in HepG2cells. A: HepG2 cells were transiently cotransfected with complementary DNAs (cDNAs) codingfor the α2-macroglobulin promoter luciferase and IκBα and subsequently stimulated with IL-6, IL-1β, or IL-6 and IL-1β. After cell lysis, luciferase activity was determined and normalized to cotrans-fected β-galactosidase activity. B: Promoter region of the rat α2-macroglobulin gene. The twoSTAT3 binding sites (distal and proximal APREs) are underlined, the two putative overlappingNFκB binding sites are represented as hatched bars.

A

B

6–induced APP synthesis in and secretion by primary cul-tures of hepatocytes (117). As shown in Fig. 40.10, overex-pression of IκBα and consequently prevention of NFκBactivation blocks the inhibitory effect of IL-1β on IL-6–induced α2-macroglobulin promoter activation (Bode etal., unpublished data). This observation might be explainedby a competition of NFκB and STAT3 for overlappingbinding elements in the α2-M promoter (118) (Figs. 40.9,and 40.10 lower panel).

INTEGRATIVE VIEW ON THE CROSS-TALKBETWEEN THE SIGNALING PATHWAYS OFINTERLEUKIN-6 AND PROINFLAMMATORYMEDIATORS

As mentioned above, LPS as well as TNF-α inhibit IL-6–stimulated STAT3 activation in macrophages. This inhi-bition correlates with the induction of SOCS-3. Further-more, IL-1 interferes with the α2-macroglobulin promoteractivation through STAT3 induced by IL-6 (Fig. 40.9).

With respect to these observations, it is important tonote that IL-6—besides its proinflammatory properties—also exerts antiinflammatory actions. For example, IL-6does not upregulate other inflammatory mediators, it doesnot induce cyclooxygenase activity leading to the produc-tion of prostaglandins, and it does not induce metallopro-teinases responsible for tissue degradation (119). In contrastto IL-1 and TNF-α, which are poorly tolerated by mam-mals since they cause shock, IL-6 has no such effect (120).

An important antiinflammatory property of IL-6 is itspotency to inhibit the synthesis of TNF-α and IL-1, both invitro and in vivo (121). It is also of interest to note that IL-6acts on human monocytes leading to the release of IL-1receptor antagonist (122). Moreover, it was shown that IL-6suppresses macrophage colony-stimulating factor–inducedproliferation and differentiation of both tissue and bone mar-row macrophages (123). Considering these antiinflammatoryproperties of IL-6, it is attractive to speculate that theSTAT3-dependent IL-6 signaling cascade needs to be down-regulated by the proinflammatory mediators LPS, IL-1, orTNF-α in vivo in order to enforce the inflammatoryresponse. Subsequently, the proinflammatory phase is termi-nated through the IL-6–dependent inhibition of IL-1 andTNF-α production. This inhibition of proinflammatorycytokine synthesis is reinforced by the induction of glucocor-ticoids resulting in a most likely STAT3-dependent dominat-ing antiinflammatory response (Fig. 40.2).

ACKNOWLEDGMENTS

We thank Gerhard Müller-Newen, Iris Behrmann, andFred Schaper for their critical reading of this manuscript,Peter Freyer for his help with the artwork, and Silvia Cottin

for secretarial assistance. The experimental work performedin the Department of Biochemistry in Aachen and men-tioned in this chapter has been supported by grants fromthe Deutsche Forschungsgemeinschaft (Bonn), and theFonds der Chemischen Industrie (Frankfurt).

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The Liver: Biology and Pathobiology, Fourth Edition, edited by I. M. Arias, J. L. Boyer, F. V. Chisari, N. Fausto, D. Schachter, and D. A. Shafritz. Lippincott Williams & Wilkins, Philadelphia © 2001.