Cytokines Engaged in Antiviral Action, Macrophage Activation, Angiogenesis, and Regulation of Cell Growth and Differentiation

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Cytokines Engaged in Antiviral Action, Macrophage Activation, Angiogenesis, and Regulation of Cell Growth and Differentiation Jan Vilcek* Department of Microbiology and Kaplan Cancer Center, New York University School of Medicine, 550 First Avenue, New York, NY 10016-6402, USA * corresponding author tel: 212-263-6756, fax: 212-263-7933, e-mail: [email protected] DOI: 10.1006/rwcy.2000.02008.

INTRODUCTION This chapter will introduce a group of cytokines and cytokine-like molecules that ± as is so characteristic of most cytokines ± engage in very diverse biological actions. When Oppenheim (1998) recently asked the rhetoric question `Is there any order to the cytokine chaos?', his own response was that cytokines can be organized into groups that exhibit functional similarities based on a shared utilization of receptors and shared signal transduction pathways. We shall strive to apply this `Oppenheim principle' in an attempt to introduce some order and logic to the discussion of the somewhat disparate group of mediators covered in this section, namely the interferons (IFN/ superfamily and IFN ), the TGF superfamilies

(including TGF s, activins, inhibins, and bone morphogenetic proteins), macrophage migration inhibitory factor (MIF), osteopontin (OPN), and vascular endothelial growth factor (VEGF), as well as the angiogenesis inhibitors angiostatin and endostatin. It is relatively easy to apply the Oppenheim principle to the intensely studied and well-defined IFN and TGF ligand and receptor superfamilies, but our task becomes more onerous when we try to fit in some of the less thoroughly understood factors, such as MIF, angiostatin, and endostatin. Although all of these proteins have been molecularly defined, the receptors and signal transduction pathways activated in their target cells have not yet been identified. OPN poses a similar problem. Even though it has been shown that OPN binds to CD44 and integrins,

616 Jan Vilcek these are not considered to be specific cytokine receptors. In introducing this group of cytokines and cytokinelike proteins, we shall try to avoid duplicating information contained in the chapters describing each of the specific ligands and receptors in the main section of the Cytokine Reference. Instead, readers of this chapter are provided with links to the appropriate parts in this main section.

THE INTERFERON / SUPERFAMILY AND INTERFERON

Discovery of interferons marks the beginning of cytokine research Interferons are arguably the oldest known cytokines, first described over 40 years ago by Isaacs and Lindenmann (1957). True, there were somewhat earlier publications in which phenomena now known to be mediated by cytokines or growth factors had been described (Bennett and Beeson, 1953; LeviMontalcini and Hamburger, 1953), but, as pointed out by the science writer Stephen S. Hall (Hall, 1997), it was the first interferon publications that `lit the fuse for an explosion of discoveries' leading to the vast accumulation of knowledge about cytokines and the appreciation of their important biological function. In their original publication Isaacs and Lindenmann (1957) showed that chorioallantoic membranes from developing chick embryos exposed to heat-inactivated influenza virus released a factor that interfered with the multiplication of influenza virus in fresh pieces of chorioallantoic membrane. Already in the early days of interferon research, Isaacs and his colleagues had hoped that their work would lead to the development of a clinically useful antiviral drug, a hope that became realized only some 30 years after the original publication. For a long time, investigators in this field had not suspected that interferon had biological actions other than the ability to inhibit virus replication. In fact, more than 15 years after the first description of interferon, the `probable lack of other [than antiviral] cellular effects' was still considered to be one of the defining properties of interferons (Lockart, 1973). Wheelock (1965) was the first to describe the protein now termed IFN , characterizing it as an IFN-like virus-inhibitory protein produced by mitogen-activated human T lymphocytes. Wheelock termed the protein `interferon-like' because, unlike

conventional interferon, the mitogen-induced factor was inactivated by exposure to pH2. As pointed out by Billiau and Vandenbroeck in their chapter on IFN , IFN was independently described as `macrophage activating factor' (MAF), later to be proved identical with IFN (Le et al., 1983; Nathan et al., 1983). I used to believe that naming the virus inhibitory protein described by Wheelock an interferon was accidental, and that in view of its many immunoregulatory activities and the fact that IFN is structurally unrelated to the IFN/ family, it would have been more appropriate not to call this cytokine an interferon. I changed my view when work on intracellular signaling revealed that, even though IFN/ and IFN recognize distinct receptors, they utilize closely related, partly overlapping signaling cascades (see below). It turns out that Wheelock's original characterization of the protein as `IFN-like' was right on target.

Overview of the properties of IFN/ and IFN Some of the major features of the two IFN families are summarized in Table 1. The IFN/ or type I IFN superfamily is further subdivided into subfamilies termed IFN, IFN , and IFN!. A distinct trophoblast IFN (IFN) subfamily has been identified in cattle and sheep (Imakawa et al., 1987). IFN is produced in the epithelium of the early preimplantation embryo and has been implicated as a factor responsible for the preservation of the corpus luteum, essential for successful completion of the pregnancy. Type I IFN genes form a cluster (in the human species located on the short arm of chromosome 9), and it is generally believed that they evolved from a single ancestral gene (Degrave et al., 1981; Weissmann and Weber, 1986). IFN (earlier known as `fibroblast IFN'), the most divergent member of this family, shows about 25±30% homology with the many members of the IFN subfamily (earlier known as `leukocyte IFN') at the amino acid level and about 45% homology in the coding region of the DNA (Taniguchi et al., 1980). There is also partial homology in the 50 -flanking regulatory regions of the IFN and IFN genes, reflecting the fact that they are often (although not always) coordinately regulated (De Maeyer and De Maeyer-Guignard, 1988). It has been calculated that the split between the IFN and IFN genes occurred about 200±400 million years ago, that is, probably before the emergence of vertebrates (Weissmann and Weber, 1986). The split between the more closely related IFN and IFN! subfamilies

Cytokines in Diverse Biological Actions 617

Table 1 Classification and major features of the interferons

Subfamilies

IFN/ superfamily (type I IFN)

IFN (type II IFN)

IFN (at least 12 functional genes in humans)

None

IFN IFN! IFN (only in ruminants) Structural genes

Chromosome 9 (human)

Chromosome 12 (human)

Chromosome 4 (mouse)

Chromosome 10 (mouse)

Introns

None

3

Proteinsa,b

IFN: 165±166 aa

143 aa (forms dimer)

IFN : 166 aa IFN!: 172 aa IFN: 172 aa a

Cysteines in mature protein N-glycosylation sites Receptors

a

2

None

None

2

Heterodimer of IFNAR1 and IFNAR2

Heterodimer of IFNGR1 and IFNGR2

a

Refers to human proteins only. Polypeptide lengths refer to mature proteins, as predicted from cDNA sequences, after removal of cleavable signal peptide sequences. Natural IFN proteins may undergo C-terminal processing so that shorter forms may be generated. b

and the numerous IFN subspecies probably occurred at least 85 million years ago (Degrave et al., 1981). All animal species examined have large IFN subfamilies, but most have only one IFN gene. Exceptions are the horse, cow, and pig, in which at least five interrelated IFN genes have been identified (De Maeyer and De Maeyer-Guignard, 1988). Why evolution has favored the emergence of so many related type I IFN genes and proteins is not clear. All type I IFNs compete for binding to the same receptor, and they generally exert similar biological activities (De Maeyer and De Maeyer-Guignard, 1988; Vilcek and Sen, 1996). However, some clear differences in biological action have been seen with different type I IFNs. Some cells from lower animals respond to human IFN but not to IFN , while the reverse may be true for other cell species (Vilcek and Sen, 1996). Differences have also been noted in the ability of individual recombinant IFN subspecies to inhibit the growth of tumor cell lines (Fish et al., 1983) or to produce the activation of natural killer cells (Ortaldo et al., 1984). How such divergent biological actions are generated when all IFN/ species interact with a single

type of heterodimeric receptor has not yet been fully explained. Recent evidence (reviewed in more detail in the chapter on the IFN/ receptor by Haque and Williams) indicates that IFN can engage the receptor in a distinct fashion (Lewerenz et al., 1998). In the human species, IFN is the predominant species produced by various nonhematopoietic cells following virus infection or stimulation with double-stranded RNA (De Maeyer and De MaeyerGuignard, 1988). In contrast, human cells of hematopoietic origin tend to produce more readily IFN and IFN! after different forms of stimulation. Relative amounts of induction of individual IFN subspecies also vary depending on both the cell type and inducing stimulus. It seems likely that, in the intact organism, the relative biological significance of the individual members of the IFN/ superfamily is determined primarily by the site and abundance of their production rather than by their unique functional properties. In contrast to the multiple type I IFN genes and proteins, only one IFN gene has been found in all animal species examined (see Table 1). Whereas type I IFNs are produced by many different types of cell,

618 Jan Vilcek IFN is produced predominantly by lymphoid cells, especially TH1 helper T cells and NK cells (De Maeyer and De Maeyer-Guignard, 1988). There is no structural homology between type I and type II IFNs, and these interferons bind to two distinct cell surface receptors. The IFN gene encodes a 143 and 134 amino acid basic mature protein in the human and murine species respectively, with only about 40% homology between these two species (Gray and Goeddel, 1983). As a result of this relative lack of conservation, IFN tends to be more highly species specific in its actions than type I IFNs. Both human and murine IFN proteins are Nglycosylated at two sites (De Maeyer and De MaeyerGuignard, 1988). Mature IFN protein contains no cysteines, although two cysteine residues are present in the 23 amino acid-long cleavable signal peptide sequence. In agreement with the prediction made on the basis of studies with natural IFN (Yip et al., 1982), X-ray crystallographic analysis indicates that the active IFN molecule is a homodimer (Samudzi et al., 1991; Trotta and Nagabhushan, 1992). As already mentioned, the signal transduction pathways activated by the IFN/ and IFN receptors partly overlap, providing an explanation of why these structurally unrelated cytokines share so many biological functions (Table 2).

Table 2

Receptors and signal transduction Essential aspects of the interaction of the IFN/ and IFN ligands with their receptors are explained in the individual chapters, and these have also been reviewed elsewhere (Stark et al., 1998; Mogensen et al., 1999). The heterodimeric IFN/ and IFN receptors (see Table 1) both belong to the class II cytokine receptor family (Figure 1), containing two characteristic conserved cysteine pairs and lacking the WSXWS motif present in class I cytokine receptors (Bazan, 1990). IFN/ and IFN receptors utilize members of the Janus kinases (JAKs) to activate signal transducers and activators of transcription (STATs), which then translocate to the nucleus and produce activation of gene expression. The general scenario of IFN signaling involves the following major steps: (a) crosslinking of the two receptor subunits by the IFN ligand, (b) activation of two members of the JAK tyrosine kinases (JAK1 and TYK2 by IFN/ ; JAK1 and JAK2 by IFN ), leading to the tyrosine phosphorylation of STATs (STAT1 and STAT2 by IFN/ ; STAT1 by IFN ), (c) homo- or heterodimerization of the phosphorylated STATs, activating them for (d) transport to the nucleus, and (e) STAT binding

Major biological actions of IFN/ and IFN

Activity

Induction of antiviral state

Observed with IFN/

IFN

+

+

Activation of monocytes/macrophages



+

Inhibition of cell growth

+

+

Induction of class I MHC antigens

+

+

Induction of class II MHC antigens



+

Promotion of TH1 response, inhibition of TH2 response

?

+

Promotion of antigen processing and presentation

?

+

Activation of natural killer cells

+

+

Activation of cytotoxic T cells

+

+

Modulation of Ig synthesis in B cells

+

+

Induction of apoptosis

+

?

Inhibition of the growth of nonviral intracellular pathogens



+

Inhibition of tumor growth in vivo

+

+

Pyrogenic action

+

+

+, positive effect; , weak or variable effect; ÿ, negative; ?, not known.

Cytokines in Diverse Biological Actions 619 Figure 1 Class II cytokine receptor family. The location of the transmembrane domain is indicated by the arrow. Members of this family include receptors for IFN/ (IFNAR), IFN (IFNGR), IL-10, and tissue factor. Characteristic features include conserved cysteines in the extracellular domain. The functional IFN/ , IFN , and IL-10 receptors are heterodimers. Alternative splicing results in the generation of three variants of the second chain of the IFN/ receptor: IFNAR2a (not shown in the figure), IFNAR2b, and IFNAR2c, of which only the fulllength IFNAR2c variant can signal. (Diagram kindly provided by Dr Michel Aguet.)

to DNA recognition sequences and stimulation of transcription (Stark et al., 1998). For details of this process, see also Figure 2 in the IFN receptor chapter. Not only were the IFNs the first cytokine ligands to be described and characterized, but the elucidation of how IFN receptors work paved the way for progress in the understanding of cytokine signaling in general. Specifically, elements of the JAK/STAT signaling pathway, now known to be important in the generation of signals from many different cytokine family receptors, were first identified in the course of studies of IFN receptor signaling (Darnell et al., 1994). Thus, the IFN signaling model represents a paradigm for the functioning of many other cytokine receptors that utilize the JAK/STAT pathway for intracellular signal transduction (Ihle, 1996). The importance of individual members of the JAK kinase family and individual STAT proteins varies depending on the specific cytokine involved, but the basic principles remain the same. Research in the interferon field also led to the identification of another important family of transcription factors termed interferon regulatory factors, or IRFs. The first identified members, IRF-1 and IRF-2, are strongly inducible by the IFNs (Fujita

et al., 1988; Harada et al., 1989). By now, over 10 members of the IRF family have been identified, their roles ranging from cytokine signaling to cell growth regulation and hematopoietic development (Nguyen et al., 1997).

Interferon is potentially fatal The many important beneficial roles of IFN/ and IFN in the intact organism are outlined in the individual chapters. Investigations in mice with targeted deletions of the structural gene for IFN or of essential IFN/ and IFN receptor genes have generated a wealth of information about the specific functions of the two interferon families in host defenses (van den Broek et al., 1995; Vilcek et al., 1998). As is true for many other cytokines, interferon production can in some situations be harmful, leading to acute toxicity or chronic disease. Thus, it may not be too surprising that nature devised mechanisms that suppress intracellular signaling pathways activated by interferons and other cytokines. One such mechanism relies on members of the SOCS (suppressor of cytokine signaling) family of proteins, also termed the CIS family (Yoshimura, 1998). These are small proteins with a conserved SH2 domain and C-terminal SOCS/ CIS homology domain that can inhibit JAK/STAT signaling. Many of the eight known members of the SOCS family (SOCS-1 to SOCS-7 and CIS) are inducible by cytokines, suggesting that they represent a negative feedback loop that serves to prevent harmful cytokine actions. Years ago, Gresser et al. (1981) showed that daily administration of IFN/ to newborn mice for two weeks resulted in a fatal disease, characterized by severe inhibition of growth and marked fatty degeneration and necrosis of the liver. In contrast, when the administration of IFN/ was delayed until the mice were 1 week or more in age, the animals failed to develop a comparable syndrome. The work of Gresser and colleagues had, however, been almost forgotten until two recent publications demonstrated the potentially fatal action of endogenous IFN in newborn mice (Alexander et al., 1999; Marine et al., 1999). These authors sought to explain why mice with a targeted disruption of the gene encoding SOCS-1 die between 2 and 3 weeks of age of a disease that involves fatty degeneration and necrosis of the liver and multiple hematologic abnormalities. Quite unexpectedly, they found that the administration of antibodies to IFN protected SOCS-1-null mice from the fatal illness. These and other findings indicated that the pathology is caused by IFN endogenously

620 Jan Vilcek produced by these mice during the neonatal period. Macrophages from SOCS-1-null mice showed a greatly increased sensitivity to activation by IFN , and newborn SOCS-1-null mice were resistant to infection with Semliki Forest virus (Alexander et al., 1999). In addition, Marine et al. (1999) found an increased level of IFN in the serum of SOCS-1-null mice, suggesting an altered T cell differentiation and function in these animals. These studies of SOCS-1-null mice raise a number of intriguing questions. Why do mice show an increased susceptibility to IFN during the neonatal period, similar to the unique susceptibility of newborn mice to the toxicity of IFN/ earlier demonstrated by Gresser et al. (1981)? SOCS-1 was shown to inhibit the action of a large number of cytokines and growth factors (e.g. IFN, IL-6, and growth hormone) (Yoshimura, 1998), and it is unclear why, under physiological conditions, SOCS-1 apparently plays no essential role in the regulation of signals produced by cytokines other than IFN . What is clear is that SOCS-1 function is central to the balance between the beneficial and the harmful actions of IFN . It would not be surprising if other members of the SOCS family and/or some related factors were found similarly to regulate the balance between the beneficial and harmful effects of other cytokines.

MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) The fascinating story of MIF is recounted in the very informative chapter by Metz and Bucala. Following earlier indications that activated leukocytes produced diffusible factors that could arrest the migration of macrophages, two groups of investigators reported a partial characterization of a soluble factor responsible for this activity, produced by activated T cells, which became known as MIF (Bloom and Bennett, 1966; David, 1966). MIF became almost forgotten when IFN and other cytokines were found to produce some of the actions earlier ascribed to MIF. Finally, the cloning of the bona fide MIF gene and the expression of the recombinant protein revealed not only that MIF indeed played a role in the actions originally ascribed to it (the activation of macrophages in vitro and the development of delayed-type hypersensitivity in the intact organism), but also some new and unexpected biological properties (reviewed in Bucala, 1996; Metz and Bucala, 1997; Swope and Lolis, 1999). One major surprise was the demonstration that MIF is efficiently produced by the corticotropic cells

of the anterior pituitary and that it strongly counteracts glucocorticoid-mediated immunosuppression. More recently, it has been demonstrated that MIF protein exhibits some enzymatic activities; for example, it can catalyze keto-enol isomerization reactions. Whether this and some other enzymatic activities associated with MIF are physiologically relevant is apparently not known. Metz and Bucala list a number of other significant biological actions associated with MIF (see also Table 1 in the MIF chapter). The pathophysiological relevance of MIF is being confirmed by studies in MIF-null mice (Bozza et al., 1999). One major missing link in the MIF story is the identification of its receptor. Until such time, the exact place for MIF in the cytokine hierarchy remains uncertain.

OSTEOPONTIN First described in 1979 as `secreted phosphoprotein 1' (Senger et al., 1979), OPN has a shorter history but shares with MIF some of its mystique. The name `osteopontin' derives from the fact that the protein was found to be present in the bone, and as it is localized in the osteoid matrix, it is believed that it can form a bridge between bone and the adjacent cellular tissues (Oldberg et al., 1986). As outlined in the informative chapters on OPN and OPN receptors (both authored by Nau), OPN does not share a structural homology with other cytokines. One potential link to other cytokines is the presence of a number characteristic regulatory motifs (including one AP-1, one IRF-1, and numerous NFIL6 sites) in the promoter region of the OPN gene (see Table 1 in the Osteopontin chapter). As an apparent consequence, the OPN gene is inducible in a variety of cells by several cytokines, including TNF, IL-1, IFN , and IL-2. OPN is produced by activated T cells and macrophages. However, osteopontin is also produced constitutively in a variety of tissues, such as the kidney, ovary, lung, and skin. The most unusual property of OPN is imparted by the presence of the RGD sequence, which explains the affinity of OPN for integrin and the fact that, like other integrin-binding proteins (e.g. fibronectin and vitronectin), OPN is often found in association with the extracellular matrix. Consequently, the bestdocumented biological activities of OPN are its ability to promote adhesion and migration in many types of cells, which appears to depend on the presence of the integrin-binding RGD sequence. OPN has been found to bind with a high affinity not only to integrins, but also to CD44, the hyaluronate

Cytokines in Diverse Biological Actions 621 receptor present on most types of cell (see the chapter on OPN receptors). These interactions probably account for the observed effects of OPN on cell adhesion and migration. What links OPN more closely to cytokines in its activities is that OPN is often closely associated with tissue injury and inflammation. As explained in the chapter on the OPN ligand, this protein is associated with both acute and chronic inflammatory conditions. Moreover, OPN promotes the adhesion and migration of macrophages in culture, and in the intact organism, OPN has been shown to promote the formation of an inflammatory cell infiltrate. OPN-null mice have been generated, and available evidence shows that these mice have an increased susceptibility to mycobacterial infection. Whether the cytokine-like actions of OPN are mediated by its interaction with integrin and CD44, or whether binding to some other unknown receptors may be responsible, is not known.

TRANSFORMING GROWTH FACTOR SUPERFAMILY

Defining features of the TGF superfamily The TGF superfamily of growth factors/cytokines includes proteins with a very broad range of actions. Ligands included in this superfamily share the following essential features: (a) they form S±S-linked dimers, (b) they bind to similar receptors and activate similar signal transduction pathways, and (c) their actions are important in development and cell differentiation. The TGF family has been conserved during evolution, with members identified in Drosophila, Caenorhabditis elegans, Xenopus, and sea urchins as well as in vertebrates (fish, birds, and mammals). The superfamily includes five TGF isoforms, activins and inhibins, bone morphogenetic proteins (BMPs), and several other secreted factors. In total, over 50 ligands belonging to the TGF superfamily are known, and their number continues to rise. In view of the very large number of genes and proteins included in this family, it would be a daunting task even to attempt to present a comprehensive overview of the field at this stage. See the individual chapters on TGF and its receptors, the BMP family of ligands and their receptors for further details. Other recent reviews (focusing on TGF s rather than other members of the superfamily) are by Derynck and Choy (1998) and Massague (1998). We shall attempt to point out some of the unique features of this family as they relate to cytokines in general and

especially to the group of cytokines discussed in this introductory chapter.

Gene regulation and posttranslational modification The manner in which biologically active forms of the TGF ligand superfamily are generated is quite distinct from that seen with other cytokine families. We shall use the examples of the mammalian TGF 1, TGF 2, and TGF 3 proteins to highlight the unique aspects of this process. As described in some detail in the chapter on TGF , these genes (especially TGF 1, the major isoform) can be transcriptionally upregulated in response to stress and injury or by some oncogenes and viral transactivators. Posttranscriptional mechanisms, including the stabilization of mRNA transcripts, also contribute to the upregulation of TGF expression in some situations. However, the major form of regulation of TGF activity is by posttranslational modifications, which occur both intracellularly and after the release of TGF into the extracellular environment. The proteolytic processing of a large TGF precursor protein by the endoprotease furin results in the release of the 112 amino acid mature TGF protein into the extracellular environment. TGF is, however, released in a biologically inactive form termed small latent complex, in which the mature TGF molecule is noncovalently bound to the latency-associated peptide (LAP). Another protein, termed latent TGF binding protein (LTBP) may be covalently coupled to the small latent complex, the resulting structure being called the `large latent complex'. These associations prevent TGF binding to receptors. For TGF to become biologically active, its mature form needs to be released from the latent complexes, which can be accomplished by proteolytic processing and by other means (see Figure 2 in the TGF chapter for a diagrammatic representation of the structure of latent complexes). This process of extracellular posttranslational modification, required for the generation of biologically active TGF , is not known to occur with other cytokines and growth factors. It represents a level of regulation that can influence the amount of active TGF available at specific sites and under specific conditions (Munger et al., 1997). The presence of LTBP, for example, serves to direct TGF to the extracellular matrix. Plasmin is thought to be the major physiological activator of TGF , and the increased conversion of plasminogen to plasmin during angiogenesis or tumor invasion could lead to an increased activation of latent TGF at these sites.

622 Jan Vilcek

`Going mad with Smads' This eye-catching phrase is the title of a brief recent review in the New England Journal of Medicine (Zhou et al., 1999). The title refers to a family of intracellular signal transducers responsible for most of the complex actions of TGF superfamily members. Originally identified in Drosophila and termed Mad, the family of what is now called Smad genes and proteins has grown to at least nine members, of which seven have been identified in vertebrates (Massague, 1998). Like the ligands and the receptors of this superfamily, Smads have been remarkably preserved in evolution: they are found in species ranging from fruit flies and worms to mammals. The signaling cascades have been most thoroughly investigated with the prototypical member of this family of ligands, TGF 1. TGF binding induces the formation of a heteromeric complex of T R-I and T R-II (thought to be a tetramer consisting of two chains each of T R-I and T R-II). The phosphorylation of T R-I on the so-called GS domain by the intrinsic kinase of T R-II leads to the recruitment of Smad2 and Smad3 and their phosphorylation by the activated T R-I. Phosphorylated Smad2 and Smad3 then heterodimerize with Smad4, the resulting complexes moving to the nucleus, where they combine with other transcription factors and coactivators before they activate the transcription of a variety of target genes. Other Smads are specific for the BMP receptor signaling pathway. Smad6 and Smad7 exemplify socalled inhibitory Smads because, rather than activating, they inhibit signaling by TGF family members. In some systems, however, Smad6 and Smad7 act as transcriptional activators. In some of its general features, the above signaling scenario resembles the events occurring during activation of the JAK/STAT pathway by interferons and many other cytokines. One difference is that T R-I and T R-II contain serine/threonine kinase domains whereas class I and II cytokine receptors use extraneous JAK tyrosine kinases. More importantly, the Smads, unlike the STATs, do not function as transcriptional activators in their own right but must combine with coactivators (e.g. CBP/P300) and, more remarkably, with members of other transcription factor families before they can regulate the transcription of specific target genes (Raftery and Sutherland, 1999; Zhang and Derynck, 1999). Best known is the propensity of the Smad3/4 heterodimer to form complexes with members of the AP-1 family of transcription factors, such as c-jun, c-fos, and SP-1. The fact that Smads are promiscuous in the choice of transcription factors they partner undoubtedly

contributes to the unusual pleiotropy of actions of TGF family members, which is remarkable even when compared to the pleiotropic actions of other multifunctional cytokines. (See chapters on TGF s, BMP, and activin for a survey of the many different biological activities of these ligands.)

VASCULAR ENDOTHELIAL GROWTH FACTOR

VEGF ligand family Although known for a relatively short time, VEGF is a well-characterized and important growth factor/ cytokine. Unlike the pleiotropic proteins discussed in this chapter so far, VEGF is a mitogen for vascular endothelial cells in culture, and it displays a relatively narrow spectrum of other actions in the intact organism, virtually all of which concern angiogenesis and blood vessel function. Placing VEGF in context is also greatly facilitated by the fact that VEGF displays significant homology to the A and B chains of platelet-derived growth factor (PDGF). In addition, like PDGF, VEGF signals through tyrosine kinase receptors that are structurally and functionally quite different from the class I and II cytokine receptors. (For references, see the VEGF chapter; for recent reviews, see Ferrara, 1999; Neufeld et al., 1999; Ortega et al., 1999.) Another noteworthy property of VEGF is its ability to bind heparin, in which it resembles some forms of fibroblast growth factors (FGF) that can also affect angiogenesis. The alternative exon splicing of a single VEGF gene accounts for the existence of four or five different molecular species that differ in receptor affinity, in their ability to bind to cell surface heparan sulfate proteoglycans, and in their biological activities. In addition to the major VEGF gene, several other members of the VEGF gene family have been identified, including placenta growth factors (PlGF-1 and -2) and VEGF-B, -C, and -D. The products of these related genes show differences in their receptor binding specificity and biological actions. An important feature in the regulation of VEGF gene expression is that hypoxia serves as a key positive regulator. In this respect, VEGF resembles the erythropoietin (EPO) gene. Regulatory regions in both the VEGF and EPO genes contain homologous sequences that are known to bind hypoxia-inducible factor (HIF-1), a transcription factor whose targeted deletion is embryonically lethal owing at least partly to defects in embryonic vascularization (Ryan et al., 1998; Ferrara, 1999; Neufeld et al., 1999; Ortega et al.,

Cytokines in Diverse Biological Actions 623 1999). Hypoxia developing in necrotic tumors may account for the high level VEGF expression in tumor tissues. In addition, the oncogene v-src can induce HIF-1 expression in the absence of hypoxia, thereby leading to increased VEGF synthesis (Jiang et al., 1997). VEGF mRNA expression is also upregulated by many growth factors/cytokines, as detailed in the VEGF chapter. The major actions of VEGF in the intact organism include the induction of angiogenesis, the permeabilization of blood vessels, and vasodilation. The key role of VEGF in embryonic vasculogenesis and angiogenesis has been confirmed by gene targeting studies in mice: disruption of the VEGF gene resulted in embryonic lethality between days 11 and 12. Deregulated VEGF expression promotes the development of malignant tumors by increasing tumor angiogenesis. The involvement of VEGF in the pathogenesis of a variety of other disorders with deregulated angiogenesis (e.g. retinopathies and rheumatoid arthritis) has been documented (Neufeld et al., 1999).

VEGF receptors VEGF receptors have been identified, and the signal transduction pathways responsible for VEGF actions are being intensely investigated. Three closely related signaling receptors, termed VEGFR-1 (flt-1), VEGFR-2 (flk-1/KDR), and VEGF-3 (flt-4), have been identified. They all contain seven immunoglobulin-like domains in their extracellular portions and a split intracellular tyrosine kinase domain (Neufeld et al., 1999). These receptors thus represent a novel subfamily of tyrosine kinase receptors. The crosslinking of two receptors by the ligand results in an autophosphorylation of the receptors on specific tyrosine residues. It is unclear whether the tyrosine phosphorylation of VEGFR-1 is required for its function, because deletion of the kinase domain from this receptor did not impair its ability to promote blood vessel formation (Hiratsuka et al., 1998). Both VEGFR-1 and VEGFR-2 bind the major form of VEGF (VEGF165) but differ with respect to their affinity for other VEGF family ligands. Gene knockout studies have shown that mouse embryos with homozygous disruptions of either VEGFR-1/flt-1 or VEGFR-2/flk-1 genes died in utero, but the pathological findings indicated that the two receptors are needed at different stages of vascular development (Fong et al., 1995; Shalaby et al., 1995). Two structurally unrelated receptors, termed neurophilin-1 and -2, also bind VEGF, but these have

a very short intracellular domain and are thought to act as co-receptors rather than signaling receptors. A somewhat similar co-receptor function is ascribed to cell surface heparan sulfate proteoglycan (Neufeld et al., 1999). For information on what is known about VEGF receptor signal transduction, the reader is referred to a recent review by Ortega et al. (1999), available online at http://www.bioscience.org/1999/ v4/d/ortega/list.htm.

ANGIOSTATIN AND ENDOSTATIN A yin/yang relationship exists between VEGF and the angiogenesis inhibitors angiostatin and endostatin. In fact, the discovery and isolation of angiostatin (O'Reilly et al., 1994) and endostatin (O'Reilly et al., 1997) were based on the hypothesis that whereas angiogenesis in the vascular bed of a primary tumor is stimulated by angiogenic factors such as VEGF, angiogenesis in the vascular bed of metastases is inhibited by an excess of putative tumor-derived circulating angiogenesis inhibitor(s). It had been assumed that both the angiogenic and antiangiogenic factors are produced by the tumor so it came as a surprise when the first angiogenesis inhibitor (angiostatin) isolated from the serum and urine of tumor-bearing mice as a result of the application of this bold hypothesis was identified as a 38 kDa internal fragment of plasminogen. The second tumor angiogenesis inhibitor (endostatin), isolated with the help of a similar strategy, turned out to be the product of the proteolytic cleavage of collagen XVIII. As explained by in the chapters on angiostatin and endostatin, neither is actually produced by the tumor cells. Instead, plasminogen transcripts are found primarily in the liver, and collagen XVIII transcripts are made by many different tissues. It is believed that tumor cells express proteases that cleave plasminogen to angiostatin and that other tumor-derived proteases are responsible for the proteolytic digestion of collagen XVIII to endostatin. Several proteases capable of clearing plasminogen to angiostatin have been identified, but the proteases responsible for converting collagen XVIII to endostatin have not. It may no longer matter what hypothesis has led to the identification of angiostatin and endostatin. Of more concern for us are the questions of how angiostatin and endostatin relate to cytokines and growth factors, and how their actions can be explained in molecular terms. It appears that, of all the proteins reviewed in this chapter, angiostatin and endostatin

624 Jan Vilcek diverge most from what one might consider a typical cytokine. Rather than being encoded by a cytokinelike gene, these two mediators are derived by proteolytic cleavage from relatively mundane proteins that serve completely different functions. On the other hand, the described biological activities of angiostatin and endostatin show characteristics that are very much cytokine-like. Not unlike VEGF, angiostatin acts on vascular endothelial cells derived from many sources, but it fails to affect a large variety of other normal or transformed cells in culture. (VEGF, of course, acts as a potent mitogen whereas angiostatin inhibits the proliferation of the same target cells.) Angiostatin was also shown to induce apoptosis and to activate a kinase in endothelial cells (Claesson-Welsh et al., 1998). The less extensively studied endostatin protein similarly inhibits the proliferation and migration of vascular endothelial cells but fails to affect several other types of cells examined. The understanding of how angiostatin and endostatin work is hampered by the absence of definitive evidence for their cell surface receptors (Moser et al., 1999). It seems likely that the very restricted target cell specificity of angiostatin and endostatin is determined by the presence or absence of cellular receptors. The identification of receptors and the characterization of signal transduction pathways activated by these intriguing mediators should help us to better appreciate their relationship to cytokines and growth factors.

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