IL-6 Ligand and Receptor Family

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IL-6 Ligand and Receptor Family Toshio Hirano* and Toshiyuki Fukada Division of Molecular Oncology, Department of Oncology, Biomedical Research Center, Osaka University Graduate School of Medicine (C7), 2-2, Yamada-oka, Suita, Osaka, 565, Japan * corresponding author tel: 81 6 879 3880, fax: 81 6 879 3889, e-mail: [email protected] DOI: 10.1006/rwcy.2000.02007.

SUMMARY

INTRODUCTION

The interleukin-6 (IL-6) family of cytokines is composed of IL-6, leukemia inhibitory factor (LIF), ciliary neurotropic factor (CNTF), oncostatin M (OSM), IL-11, cardiotropin 1 (CT-1), and possibly the novel neurotrophin 1/B cell-stimulating factor 3 (NNT-1/ BSF-3). They play pivotal roles in the immune, hematopoietic, nervous, cardiovascular, and endocrine systems, as well as in bone metabolism, inflammation, and acute-phase response. Gp130, originally identified as a signal-transducing subunit for IL-6 receptor (IL-6R), is shared among the receptors for the IL-6 cytokine family. Janus kinases (JAKs) and signal transducer and activator of transcription (STATs) play essential roles in signal transduction through cytokine rceptors. Other pathways involving src family tyrosine kinases, RAS, mitogen-activated protein kinases (MAPK), and phosphatidylinositol 3-kinase (PI-3 kinase), and the interplay among them, are critically involved in the biological activities of cytokines. Cytokines can simultaneously generate contradictory signals in the same target cells and the balance of each contradictory signal may determine the final output of the cytokine signals to express unified biological activity (signal orchestration). The complex of cytokine and its soluble form of receptor acts like a cytokine with novel target specificity (receptor conversion). These mechanisms may be involved in the generation of functional pleiotrophy of cytokine.

The IL-6 family of cytokines is composed of IL-6, LIF, CNTF, OSM, IL-11, and CT-1. These cytokines are characterized by a four  helix bundle structure (Bazan, 1990) and they play pivotal roles in the immune, hematopoietic, nervous, cardiovascular, and endocrine systems, as well as in bone metabolism, inflammation, and acute-phase response by regulating cell growth, differentiation, cell survival, and expression of a variety of functions (Hirano et al., 1997; Hirano, 1998; Heinrich et al., 1998). Much evidence has been accumulated, leading to the establishment of a variety of concepts about cytokines in general: the establishment of pleiotropy and redundancy as properties of cytokine function, the cytokine receptor superfamily, the sharing of a signal-transducing receptor subunit among several cytokine receptors, and the agonistic activity of the complex of cytokine and its receptors in certain cytokines. The molecular mechanism of functional redundancy is explained at least in part by the sharing of gp130, a signal-transducing receptor subunit, among the receptors for the IL-6 cytokine family and lowaffinity LIFR subunit (LIFR , also called ) among LIFR, CNTFR, and human OSMR (Hirano et al., 1997; Hirano, 1998; Heinrich et al., 1998) (Figure 1). Studies on the signal transduction mechanisms of interferons and erythropoietin have led to insight into the molecular mechanisms of signal transduction through cytokine receptors. It is now known that JAKs and STATs (see chapter on IL-6 Receptor) play

524 Toshio Hirano and Toshiyuki Fukada

Figure 1 Sharing of receptor subunits in cytokine receptors. Gp130 is shared by the receptors for IL-6, IL-11, LIF, CNTF, OSM, and CT-1. m, mouse; h, human. IL-6

IL-11

mOSM

hOSM

LIF

CNTF

CT-1

Conserved cytokines WSXWS motif

CNTFRα IL-6Rα

CT-1Rα?

IL-11Rα Box1 Box2

LIFRβ

OSMRβ gp130

gp130

gp130

essential roles in cytokine function (Darnell et al., 1994; Ihle et al., 1994; Schindler and Darnell, 1995; Ihle, 1996; Darnell, 1997; Horvath and Darnell, 1997). Furthermore, other pathways involving src family tyrosine kinases, Ras, MAP kinase, PI-3 kinase, and as yet unidentified components participate, and the interplay among them is critically involved in the biological activities of cytokines. Moreover, cytokines can simultaneously generate contradictory signals in the same target cells and the balance of each contradictory signal may determine the final output of the cytokine signals to express unified biological activity. Such a mechanism, called signal orchestration, may be involved at least in part in the expression of functional pleiotropy of cytokines.

FUNCTIONAL REDUNDANCY AMONG THE IL-6 FAMILY CYTOKINES IL-6, LIF, CNTF, OSM, IL-11, and CT-1 constitute the IL-6-related cytokine subfamily because of their functional redundancy, structural similarity, and sharing of the receptor subunit (Table 1 and Table 2). They regulate cell growth, cell survival, and cell differentiation in a wide variety of biological systems, including immune response, hematopoiesis, inflammation, neurogenesis, and osteogenesis. Furthermore, they often show functional redundancy; IL-6, LIF,

LIFRβ gp130

LIFRβ gp130

gp130

OSM, and CT-1 induce macrophage differentiation in a myeloid leukemic cell line, M1 (Abe et al., 1991; Hilton and Gough, 1991; Begley, 1994; Pennica et al., 1995). IL-6, IL-11, LIF, and OSM all induce growth of myeloma cells. IL-6, LIF, and IL-11 enhance IL-3dependent colony formation of primitive blast colonyforming cells (Ikebuchi et al., 1987; Musashi et al., 1991). IL-6, LIF, IL-11, and OSM stimulate the biosynthesis of acute-phase proteins in hepatocytes (Leng and Elias, 1997). The functional redundancy observed among these IL-6-related cytokine subfamily is mostly explained by the sharing of receptor subunit gp130 among the IL-6 cytokine family and LIFR among LIFR, CNTFR, and human OSMR. The sharing of a receptor subunit among different cytokine receptors is not a unique feature for the IL-6 cytokine family receptors, but rather a general feature of the cytokine receptor system. GM-CSF, IL-3, and IL-5 receptors share a common subunit (Miyajima et al., 1992). The chain of the IL-2R is shared by IL15R, and the common chain of IL-2R ( c) is shared by IL-4R, IL-7R, IL-9R, and IL-15R (Sugamura et al., 1995; Taniguchi, 1995). Thus, the molecular mechanisms of redundancy in cytokine activity could be explained at least in part by the sharing of receptor subunits among several cytokine receptors. Since OSM functions through the OSM-specific and LIF/ OSM shared receptors in humans, while it functions through the OSM-specific receptor in mice, the reports on biological roles of OSM in mice using human OSM should be interpreted with care (see the chapters on OSM and OSMR).

IL-6 Ligand and Receptor Family

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Table 1 Biochemical and physiological properties of human IL-6-type cytokines

IL-6 (184 aa)

Potential glycosylation sites

No. cysteine residues

No. S±S bonds

Tissues of expression

Stimulus molecules

Functions

2

4

2

Many tissues, including blood, cartilage, bone marrow, skin, lung, and CNS

IL-1, TNF, TGF , OSM, IL-4, IL-11

Hematopoiesis Differentiation and proliferation of B and T cells Stimulation of proliferation of mesangial cells and keratinocytes Regulation of acute phase protein (APP) synthesis Upregulation of TIMP-1 Stimulation of ACTH production Osteoclast development

IL-11 (178 aa)

0

0

0

Hematopoietic tissues, lung, gastrointestinal tract, bone, CNS, thymus, connective tissues, skin, uterus and testis

IL-1, TGF

Hematopoiesis Growth control of epithelial cells Osteoclast development Neurogenesis Stimulator of APP synthesis Upregulation of TIMP-1 Inhibition of adipogenesis

LIF (180 aa)

6

6

3

Many tissues, including heart, liver, endometrium, pituitary, CNS, gut, kidney, lung, and thymus

IL-1, TNF, TGF , IL-8, EGF, IL-3, OSM, LPS, PDGF, IL-4, IL-11

Hematopoiesis Differentiation factor for pituitary corticotropic cells Regulation of APP synthesis Upregulation of TIMP-1 Inhibition of differentiation of ES cells Switch to cholinergic function of sympathetic neurons Proliferation of myoblasts

CNTF (200 aa)

0

1

0

Nervous system

Increased synthesis in astrocytes after injury. Released after injury of peripheral nerve cells

Anti-apoptotic effect after nerve injury Inhibition of developmentally determined apoptosis Promotes the cholinergic phenotype in sympathetic nerves Activation of choline acetyltransferase in motor neurons Activation of outgrowth of neurites in vivo

526 Toshio Hirano and Toshiyuki Fukada Table 1 (Continued ) Potential glycosylation sites

No. cysteine residues

No. S±S bonds

Stimulus molecules

Tissues of expression

Functions

Downregulation of proinflammatory cytokines (IL-1, IL-18) and PGE2 Regulation of APP synthesis Upregulation of CNTFR and NGFR CT-1 (201 aa)

0

2

?

Heart, skeletal muscle, ovary, colon, prostate, testis, fetal kidney, and lung

?

Induction of hypertrophy of neonatal cardiac monocytes Inhibition of cardiac myocyte apoptosis Survival factor for spinal motor neurons Stimulation of cholinergic differentiation of sympathetic neurons Red blood cell counts Inhibition of LPSstimulated TNF production Stimulation of APP synthesis

OSM (196 aa)

2

5

2

Testis, blood

T cell activators, PMA, IL-2, IL-3, EPO

Survival of Sertoli cells and gonocytes Upregulation of LDLR Regulation of APP synthesis Induction of cytokines (IL-6, G-CSF, GM-CSF, LIF, bEGF) Effect on extracellular matrix Upregulation of adhesion molecules in endothelial cells

RECEPTOR CONVERSION

A novel mechanism generating functional diversity of cytokine A complex of IL-6 and a soluble form of IL-6R can activate signal transduction in cells expressing only the gp130 receptor subunit. This type of arrangement is not unique to the IL-6R system. IL-12 consists of a disulfide heterodimer of 40 kDa (p40) and 35 kDa (p35) subunits (Kobayashi et al., 1989). The peptide sequences of p35 and p40 resemble IL-6 and the

soluble form of its receptor, respectively (Gearing and Cosman, 1991), suggesting that IL-12 acts on target cells in a manner similar to the complex of IL-6 and soluble IL-6R. Another example is a CNTFR that is anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) linkage. The complex of soluble CNTFR and CNTF acts on cells expressing LIFR and gp130 (Davis et al., 1993). The complex of IL-11 and the soluble form of IL-11R also functions through gp130 (Baumann et al., 1996; Neddermann et al., 1996). Based on these facts, we originally proposed a novel mechanism by which the cytokine system generates

IL-6 Ligand and Receptor Family

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Table 2 Biochemical and physiological properties of IL-6-type receptors Extracellular domain (aa)

Transmembrane domain (aa)

Intracellular domain (aa)

Potential glycosylation sites

Phenotypes of knockout mice

IL-6 (449 aa)

339

28

82

5

Not examined

IL-11R (400 aa)

343

26

31

2

Female infertility Normal hematopoisis

CNTFR (352 aa)

352

4

Mice die between 12 and 24 hours after birth Defect in motor neuron development

LIFR (1053 aa)

789

26

238

19

Perinatal lethality Defects in placental architecture Decrease in bone volume Reduction of astrocyte number in spinal cord and brainstem Loss in motor neurons of the facial nucleus and lumber spinal cord Reduction in neurons of the nucleus ambiguous Elevated stores of glycogen in late gestation fetal liver

OSMR (952 aa)

712

22

218

15

Not examined

gp130 (896 aa)

597

22

277

10

Embryonic lethality between day 12.5 and term Heart abnormality Hematopoietic abnormality

functional diversity (Figure 2) (Hirano, 1994; Hirano et al., 1994). We wish to call this mechanism receptor conversion. A complex consisting of a soluble cytokine receptor and its corresponding cytokine ligand acquires a different target specificity from the original cytokine, leading to the expression of distinct functions from those of the original cytokine. Actually, double transgenic mice expressing human IL-6 and IL-6R showed myocardial hypertrophy (Hirota et al., 1995), extraordinary expansion of hematopoietic progenitor cells (Peters et al., 1997), and nodular regenerative hyperplasia and adenomas of the liver (Maione et al., 1998), indicating that the complex of IL-6 and the soluble form of IL-6R acts on heart muscle cells and hematopoietic stem cells that express gp130, on which IL-6 alone cannot act. Thus, by forming a complex, IL-6 apparently acquires novel biological activities. Thus, the mechanism of

receptor conversion may be applied to a wide range of receptor systems, for example, the receptors for glial cell line-derived neurotropic factor (GDNF) and neurturin (NTN). Both the GDNF and NTN receptors consist of a ligand-specific GPI-anchored  chain and a common signal-transducing receptor subunit, Ret, which is a receptor tyrosine kinase (Jing et al., 1996; Treanor et al., 1996; Buj-Bello et al., 1997; Klein et al., 1997). Receptor conversion contributes to generating the functional diversity of cytokines and may also play pathological roles in various diseases, since an increase in the serum-soluble form of various cytokine receptors has been reported to occur in a variety of diseases. Furthermore, novel drugs could be designed based on this model. A bioactive designer cytokine is being developed, which is composed of soluble IL-6R and IL-6 linked by a flexible peptide chain (Fischer et al., 1997).

528 Toshio Hirano and Toshiyuki Fukada Figure 2 Receptor conversion, a novel mechanism generating cytokine diversity. A cytokine acts on the cells (target cell 1) that express a specific receptor. With certain cytokines, such as IL-6 and CNTF, a complex composed of the cytokine and a soluble form of its receptor subunit can activate the signal transduction pathway in cells (target cell 2) that express only a receptor subunit and do not respond to the cytokine alone. Cytokine

Complex of cytokine and soluble receptor

Target 1

Target 2

ACTIVATION OF MULTIPLE SIGNAL TRANSDUCTION PATHWAYS BY THE IL-6 FAMILY CYTOKINES

Involvement in regulation of cell growth, differentiation and survival JAK family tyrosine kinases (JAK1, JAK2, JAK3, TYK2) are involved in the signal transduction of cytokines and hormones (Ihle et al., 1994; Ihle, 1996). Cytokines induce receptor aggregation, resulting in the activation of JAK family tyrosine kinases. These events eventually induce the tyrosine phosphorylation of STAT, which was originally identified as an interferon-activated transcription factor by Darnell and his colleagues (Darnell et al., 1994; Schindler and Darnell, 1995). JAK1, JAK2, and TYK2 associate constitutively with gp130 and are tyrosine-phosphorylated in response to IL-6, CNTF, LIF, OSM, or IL-11 (Berger et al., 1994; Lutticken et al., 1994; Matsuda et al., 1994; Stahl et al., 1994). JAK1 is considered to be a major kinase among this family, activating STAT3 through gp130 (Guschin et al., 1995). Cells from JAK1 knockout mice could not respond to the IL-6 family cytokines (Rodig et al., 1998). The IL-6 family cytokines activate STAT3, STAT1, and STAT5 (Akira et al., 1994; Fujitani et al., 1994, 1997;

Zhong et al., 1994; Lai et al., 1995; Nakajima et al., 1995). In response to cytokine stimulation, phosphorylated STATs are dimerized and translocated into the nucleus, leading to the expression of genes with STAT recognition sites. Phosphorylated STAT1 was shown to be associated with subunit (a 97 kDa component) of the nuclear pore-targeting complex via the NPI-1 family of  subunit (a 58 kDa component). STAT1-binding domain of NPI-1 is located in the C-terminal region, which is distinct from the SV40 large T antigen nuclear localization signal-binding region (Sekimoto et al., 1997; Sekimoto and Yoneda, 1998). Furthermore, a nuclear small GTP-binding protein Ran, which is an essential factor for active nuclear protein transport, is involved in, and its GTP hydrolysis activity is required for, the IFN -dependent nuclear transport of STAT1 (Sekimoto et al., 1996). In addition to the JAK/STAT pathway, multiple signaling molecules are tyrosine-phosphorylated in response to the IL-6 family of cytokines (Figure 3). CNTF, LIF, OSM, and IL-6 induce tyrosine phosphorylation of phospholipase C , SHP-2 (a phosphotyrosine phosphatase, also called PTP1-D, SHPTP-2, PTP2C, and Syp), which is a mammalian homolog of Drosophila corkscrew (CWS), pp120, Shc, Grb2, Raf-1, and ERK1 and ERK2 (Boulton et al., 1994). IL-11 induces tyrosine phosphorylation of SHP-2 in mouse 3T3-L1 cells. Furthermore, SHP-2 is inducibly associated with gp130 (Fuhrer et al., 1995; Stahl et al., 1995; Fukada et al., 1996) and JAK2 (Fuhrer et al., 1995). The Ras/MAPK pathway is activated by the IL-6 cytokine family (Nakafuku et al., 1992; Daeipour et al., 1993; Boulton et al., 1994; Kumar et al., 1994; Fukada et al., 1996; Berger and Hawley, 1997). The activation of the Ras/MAPK pathway is possibly mediated by SHP-2 (Fukada et al., 1996; Berger and Hawley, 1997) and/or Shc (Ernst et al., 1994; Kumar et al., 1994), which bind a Grb2/SOS complex. Gp130 stimulation induces tyrosine phosphorylation of both Gab1 and Gab2, which have structural similarities to Drosophila DOS, or daughter of sevenless (Takahashi-Tezuka et al., 1998; Nishida et al., 1999), being complexed with SHP-2 and PI-3 kinase and involved in MAP kinase activation. Gab1 and Gab2 are also tyrosine phosphorylated in response to EGF, insulin, and c-Met stimulation, T and B cell antigen receptors (Gu et al., 1998; Nishida et al., 1999). Both Gab1 and Gab2 have binding sites for PLC , PI-3 kinase, SHP-2, and Grb2 (Holgado-Madruga et al., 1996; Weidner et al., 1996) and show structural similarities with IRS-1, IRS-2, and Drosophila DOS. These DOS-related family molecules may act as universal docking molecules linking a variety of receptors to downstream signaling molecules. In fact, IRS-1 is tyrosine phosphorylated in response to IL-2,

IL-6 Ligand and Receptor Family

529

Figure 3 Distinct cytoplasmic regions of gp130 are involved in different signal transduction pathways.

gp130

JAK PI-3K

JAK

Gab1/2 SHP-2

Y759

Grb2 Sos

Y767 STAT3

STAT3

Y814 Y905 Y915

Ras

?

774 (133)

c-myc Bcl-2

Cyclin D

c-myb Erk1/2

STAT3 p19 ink4D

p27

CDC25A

c-myc

Neurite outgrowth in PC12 cell

p21

Cyclin A

918 (277)

Raf

Antiapoptosis

G1 S G2/M Cell cycle transition

Cell proliferation in BAFB03 cell

Growth arrest and differentiation in M1 cell

IL-4, IL-7, IL-9, IL-15, OSM, and interferons, in addition to insulin (Keegan et al., 1994; Johnston et al., 1995; Yin et al., 1995; Burfoot et al., 1997). IRS-2 acts as an adapter molecule linking growth hormone receptor to PI-3 kinase. Src family tyrosine kinases, such as Btk, Tec, Fes, and Hck (Ernst et al., 1994, 1996; Matsuda et al., 1995a,b) are activated by the IL-6 cytokine family, as well as by a variety of other cytokines (Taniguchi, 1995). Among them, Tec and Btk associate with, and are possibly activated by JAKs, and Tec may be one of the adapter molecules linking the cytokine receptor to PI-3 kinase (Takahashi-Tezuka et al., 1997). These multiple signal transduction pathways are variably involved in the regulation of cell growth, survival, and differentiation by interacting with each other, as described in Figure 3 and in the chapter on the IL-6 receptor.

SIMULTANEOUS GENERATION OF CONTRADICTORY SIGNALS THROUGH A CYTOKINE RECEPTOR

Orchestrating model Cytokines exert a variety of biological activities through specific receptors. Since the expression pattern of each cytokine receptor and that of a

cytokine are different, and since each cytokine receptor has different binding affinities for a variety of signaling molecules, each cytokine is capable of expressing a unique biological activity. Another question is how a single cytokine can exert distinct biological activities on different target cells. There are several points to be considered. First, different sets of signal transduction pathways (despite the existence of partial redundancy among them) could simply be activated in different targets through a given cytokine, due to differences in the expression pattern of each signaling molecule (Figure 4a). Second, even if a cytokine receptor can induce the same set of signal transduction pathways in different targets, each target cell could respond to the cytokine stimulation differently because the expression and/or activation state of other molecules affecting each signal transduction pathway negatively or synergistically is different or because the final transcriptional activation of target genes of the signal transduction pathway is different among different targets (Figure 4b). Third, the balance or interplay (inhibitory or synergistic interaction) among the signaling pathways could determine the eventual outcome of the signal transduction through the receptor in a given target cell (Figure 4c). Relevant findings obtained through studies on gp130-mediated signals are: 1. gp130 stimulation can simultaneously induce opposite signals, e.g. growth-enhancing and

530 Toshio Hirano and Toshiyuki Fukada

al gn si E

gn

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A al gn

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sig l

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G

C

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na

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sig

sig

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gn

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These findings are quite surprising, since one might reasonably expect that a cytokine would only induce well-coordinated signals to express a unified biological activity in a given target cell. A similar observation is reported in tumor necrosis factor stimulation, which elicits simultaneously both apoptotic signals through the caspase cascade and anti-apoptotic signals through NFB activation (Liu et al., 1996). Furthermore, both p21 and G1 cyclins are shown to be activated by growth factors (Schreiber et al., 1999). Thus, cytokine and growth factor receptors have a potential simultaneously to induce contradictory intracellular signaling pathways, and the balance and/or interplay of each pathway could determine the final outcome of the stimulation. Such a situation could be orchestrated by a conductor to exert an unified output. We wish to call this model signal orchestration (Figure 4c). It is likely that the variable combinations of these mechanisms are involved in the determination of the final outcome of the cytokine signaling.

Figure 4 Models for signal transduction pathways involved in the expression of a target-specific biological activities by a single cytokine. (a) Different signal pathways are generated in different target cells through the same cytokine receptor. (b) The same set of signal transduction pathways is generated, but other molecules affecting the signal transduction pathways are differently expressed in each target cell. (c) Orchestrating model: contradictory signal pathways are simultaneously generated in a single target cell and the balance or interplay among them eventually determines the outcome. Such a situation could be orchestrated by a conductor to effect a directed biological action. The variable combination of mechanisms determines the final outcome of the signaling.

B

growth-suppressing signals are induced in M1 cells (Nakajima et al., 1996) 2. gp130 can deliver at the same time both positive and negative signals affecting neurite outgrowth in PC12 cells (Ihara et al., 1997) 3. CNTF promotes differentiation of cortical precurser cells into astrocytes through activation of STAT3, while simultaneous activation of MAPK is rather suppressive for CNTF activation (Bonni et al., 1997) 3. gp130 can drive G1 to S phase cell cycle transition signal and, at the same time, induce p21 cyclindependent kinase inhibitor from the distinct cytoplasmic regions in the same target cells (Fukada et al., 1998).

(c)

A B

C

D

A

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D

POSITIVE AND NEGATIVE FEEDBACK MECHANISMS Conductor

The balance of each signal transduction pathway could be influenced by a variety of factors that determine the duration and intensity of each signaling. In this sense, positive or negative feedback mechanisms may be crucial to determine the balance. The activation state of STAT3 in IL-6-stimulated M1 cells persists for as long as 24 hours after stimulation (Nakajima et al., 1996; Yamanaka et al., 1996). Such prolonged activation of a particular signal transduction pathway should affect the outcome of the signal transduction. The sustained activation of STAT3 in M1 cells may be induced by either the absence of a

Conductor

negative regulator of STAT3, such as a postulated STAT phosphotyrosine phosphatase (Haspel et al., 1996), or the upregulation of STAT3. In fact, the STAT3 gene is autoregulated by STAT3 in M1 cells (Ichiba et al., 1998) and this may partly contribute to the sustained activation of STAT3 in M1 cells. Concerning a negative regulator for STAT, the natural existence of potentially dominantly suppressive variants of STAT3 and STAT5 has been reported (Caldenhoven et al., 1996; Wang et al., 1996).

IL-6 Ligand and Receptor Family MAP kinase is also implicated as a negative regulator for STAT3 activation (Jain et al., 1998; Sengupta et al., 1998). Phosphotyrosine phosphatases are critical negative or positive regulators for cytokine and growth factor-mediated signal transduction pathway. SHP-1, an SH2 domain containing a phosphotyrosine phosphatase, is thought to act as a negative regulator for erythropoietin receptor-mediated signal transduction by inactivating JAK2 (Klingmuller et al., 1995). SHP-2 is also suggested negatively to regulate the expression of acute-phase genes (Kim et al., 1998), although SHP-2 is considered to act as a positive regulator in MAPK activation (Tonks and Neel, 1996; Neel and Tonks, 1996). In addition to these, the family molecules that can bind to SHP-2, SHP-1, and Grb2 have been cloned: these are the signal-regulatory protein (SIRP) family (Kharitonenkov et al., 1997) and SHP substrate 1 (SHPS-1) (Fujioka et al., 1996), which is a member of the SIRP family. SIRP1 is a substrate for activated receptor tyrosine kinases and its tyrosine-phosphorylated form binds SHP-2 through SH2 interactions and acts as its substrate. It has negative regulatory effects on insulin, epidermal growth factor (EGF), and platelet derived growth factor (PDGF)-induced growth, most likely through the inhibition of MAPK activity (Kharitonenkov et al., 1997). Furthermore, STAT3 induces a SH2 domain-containing molecule designated as SOCS-1/JAB/SSI-1 (Endo et al., 1997; Naka et al., 1997; Starr et al., 1997), which is structurally related to CIS, a cytokine-inducible SH2 protein (Yoshimura et al., 1995). SOCS-1/JAB/SSI-1 can bind JAK and inhibit its kinase activity and thereby suppress the tyrosine phosphorylation of gp130 and subsequent activation of STAT. A family of protein inhibitor of activated STAT (PIAS) proteins are identified as another group of STAT inhibitors which bind to STATs and inhibit DNA-binding activity of STATs in a stimulationdependent manner. For instance, PIAS1 blocks the DNA-binding activity of STAT1, but not other STATs, and inhibits STAT1-mediated gene activation in response to interferon. The in vivo PIAS1-STAT1 interaction requires phosphorylation of STAT1 on Tyr701 (Liu et al., 1997). PIAS3 is another member which specifically associates with and inhibits STAT3 (Chung et al., 1997).

CROSSTALK BETWEEN GP130 SIGNALING AND OTHERS Signalings through a cytokine receptor crosstalk with those through other receptors. Such a crosstalk or

531

interplay between a given cytokine and others would modify the final output of a cytokine action. IL-6 stimulation induces association between gp130 and receptor tyrosine kinase erbB2, following tyrosine phosphorylation and kinase activation of erbB2 in prostate carcinoma cells. This activation of erbB2 contributes to the IL-6-induced activation of MAPK ERK2 in prostate carcinoma cells (Qiu et al., 1998). Gp130 can induce neurite outgrowth in PC12 cells when pretreated with NGF. NGF stimulation inhibits IL-6-induced activation of STAT3, which is inhibitory for neurite outgrowth, providing an example of the modification of cytokine signaling by growth factor signaling (Ihara et al., 1997). Such a crosstalk is observed in other cytokines. Growth hormone stimulation induces tyrosine phosphorylation of the intracellular domain of EGF receptor (EGFR), indicating that growth hormone utilizes EGFR to activate the Ras/MAPK pathway (Yamauchi et al., 1997). IFN and TGF have opposite effects on diverse cellular functions, even in the same target cells. IFN inhibits TGF -induced signaling events, such as SMAD3 phosphorylation and activation of TGF -responsive genes. IFN induces an antagonistic SMAD (SMAD7) through activation of JAK1 and STAT1, indicating a mechanism of transmodulation of TGF signaling by IFN (Ulloa et al., 1999). It is possible that such a crosstalk among cytokine receptors is one of the important factors determining the cell's fate, and in line with the notion that different sets of signal transduction could be generated in different targets, through a given cytokine receptor (Figure 4a). Further identification of crosstalk pathways among cytokines and/or growth factors would contribute to a molecular explanation of the features of cytokine functions: functional pleiotropy, and redundancy.

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