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

12 Pages·2000·0.14 MB·English

by  Vilcek J.

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: [phone], fax: [phone], e-mail: [email protected]
DOI: 10.1006/rwcy.2000.02008.
INTRODUCTION
(including TGF(cid:12)s, activins, inhibins, and bone mor-
phogenetic proteins), macrophage migration inhibi-
This chapter will introduce a group of cytokines and tory factor (MIF), osteopontin (OPN), and vascular
cytokine-likemoleculesthat–asissocharacteristicof endothelial growth factor (VEGF), as well as the
most cytokines – engage in very diverse biological angiogenesis inhibitors angiostatin and endostatin.
actions. When Oppenheim (1998) recently asked the It is relatively easy to apply the Oppenheim prin-
rhetoric question ‘Is there any order to the cytokine ciple to the intensely studied and well-defined IFN
chaos?’, his own response was that cytokines can be andTGF(cid:12) ligandandreceptorsuperfamilies,butour
organized into groups that exhibit functional simila- taskbecomesmoreonerouswhenwetrytofitinsome
rities based on a shared utilization of receptors and of the less thoroughly understood factors, such as
shared signal transduction pathways. We shall strive MIF, angiostatin, and endostatin. Although all of
to apply this ‘Oppenheim principle’ in an attempt to these proteins have been molecularly defined, the
introduce some order and logic to the discussion of receptors and signal transduction pathways activated
the somewhat disparate group of mediators covered in their target cells have not yet been identified.
in this section, namely the interferons (IFN(cid:11)/(cid:12) OPN poses a similar problem. Even though it has
superfamily and IFN(cid:13)), the TGF(cid:12) superfamilies been shown that OPN binds to CD44 and integrins,
616 Jan Vilcek
these are not considered to be specific cytokine conventional interferon, the mitogen-induced factor
receptors. was inactivated by exposure to pH2. As pointed out
Inintroducingthisgroupofcytokinesandcytokine- by Billiau and Vandenbroeck in their chapter on
like proteins, we shall try to avoid duplicating infor- IFN(cid:13), IFN(cid:13) was independently described as ‘macro-
mation contained in the chapters describing each of phage activating factor’ (MAF), later to be proved
the specific ligands and receptors in the main section identical with IFN(cid:13) (Le et al., 1983; Nathan et al.,
of the Cytokine Reference. Instead, readers of this 1983). I used to believe that naming the virus inhib-
chapter are provided with links to the appropriate itory protein described by Wheelock an interferon
parts in this main section. was accidental, and that in view of its many immu-
noregulatory activities and the fact that IFN(cid:13) is
structurally unrelated to the IFN(cid:11)/(cid:12) family, it would
have been more appropriate not to call this cytokine
THE INTERFERON (cid:11)/(cid:12)
aninterferon.Ichangedmyviewwhenworkonintra-
SUPERFAMILY AND
cellular signaling revealed that, even though IFN(cid:11)/(cid:12)
INTERFERON (cid:13) and IFN(cid:13) recognize distinct receptors, they utilize
closely related, partly overlapping signaling cascades
Discovery of interferons marks the (see below). It turns out that Wheelock’s original
characterizationoftheproteinas‘IFN-like’wasright
beginning of cytokine research
on target.
Interferons are arguably the oldest known cytokines,
first described over 40 years ago by Isaacs and Overview of the properties of
Lindenmann(1957).True,thereweresomewhatearlier
IFN(cid:11)/(cid:12) and IFN(cid:13)
publications in which phenomena now known to be
mediated by cytokines or growth factors had been
described (Bennett and Beeson, 1953; Levi- Some of the major features of the two IFN families
Montalcini and Hamburger, 1953), but, as pointed are summarized in Table 1. The IFN(cid:11)/(cid:12) or type I
outbythesciencewriterStephenS.Hall(Hall,1997), IFN superfamily is further subdivided into subfami-
itwasthefirstinterferonpublicationsthat‘litthefuse lies termed IFN(cid:11), IFN(cid:12), and IFN!. A distinct
for an explosion of discoveries’ leading to the vast trophoblast IFN (IFN(cid:28)) subfamily has been identi-
accumulation of knowledge about cytokines and the fied in cattle and sheep (Imakawa et al., 1987). IFN(cid:28)
appreciation of their important biological function. is produced in the epithelium of the early preimplan-
In their original publication Isaacs and tation embryo and has been implicated as a factor
Lindenmann (1957) showed that chorioallantoic responsibleforthepreservationofthecorpusluteum,
membranes from developing chick embryos exposed essential for successful completion of the pregnancy.
to heat-inactivated influenza virus released a factor Type I IFN genes form a cluster (in the human
that interfered with the multiplication of influenza species located on the short arm of chromosome 9),
virus in fresh pieces of chorioallantoic membrane. and it is generally believed that they evolved from a
Already in the early days of interferon research, single ancestral gene (Degrave et al., 1981;
Isaacs and his colleagues had hoped that their work Weissmann and Weber, 1986). IFN(cid:12) (earlier known
would lead to the development of a clinically useful as ‘fibroblast IFN’), the most divergent member of
antiviraldrug,ahopethatbecamerealizedonlysome this family, shows about 25–30% homology with the
30 years after the original publication. For a long manymembersoftheIFN(cid:11)subfamily(earlierknown
time,investigatorsinthisfieldhadnotsuspectedthat as‘leukocyteIFN’)attheaminoacidlevelandabout
interferon had biological actions other than the 45% homology in the coding region of the DNA
ability to inhibit virus replication. In fact, more than (Taniguchi et al., 1980). There is also partial homol-
15 years after the first description of interferon, the ogy in the 50-flanking regulatory regions of the IFN(cid:11)
‘probablelackofother[thanantiviral]cellulareffects’ andIFN(cid:12)genes,reflectingthefactthattheyareoften
was still considered to be one of the defining (although not always) coordinately regulated (De
properties of interferons (Lockart, 1973). Maeyerand De Maeyer-Guignard, 1988). It hasbeen
Wheelock (1965) was the first to describe the calculated that the split between the IFN(cid:11) and IFN(cid:12)
protein now termed IFN(cid:13), characterizing it as an genes occurred about 200–400 million years ago,
IFN-like virus-inhibitory protein produced by mito- that is, probably before the emergence of vertebrates
gen-activated human T lymphocytes. Wheelock (Weissmann and Weber, 1986). The split between
termed the protein ‘interferon-like’ because, unlike the more closely related IFN(cid:11) and IFN! subfamilies
Cytokines in Diverse Biological Actions 617
Table 1 Classification and major features of the interferons
IFN(cid:11)/(cid:12) superfamily (type I IFN) IFN(cid:13) (type II IFN)
Subfamilies IFN(cid:11) (at least 12 functional None
genes in humans)
IFN(cid:12)
IFN!
IFN(cid:28) (only in ruminants)
Structural genes Chromosome 9 (human) Chromosome 12 (human)
Chromosome 4 (mouse) Chromosome 10 (mouse)
Introns None 3
Proteinsa,b IFN(cid:11): 165–166aa 143aa (forms dimer)
IFN(cid:12): 166aa
IFN!: 172aa
IFN(cid:28): 172aa
Cysteines in mature proteina 2 None
N-glycosylation sitesa None 2
Receptors Heterodimer of IFNAR1 and IFNAR2 Heterodimer of IFNGR1 and IFNGR2
aReferstohumanproteinsonly.
bPolypeptide lengths refer to mature proteins, as predicted from cDNA sequences, after removal of cleavable signal peptide sequences.
NaturalIFNproteinsmayundergoC-terminalprocessingsothatshorterformsmaybegenerated.
and the numerous IFN(cid:11) subspecies probably oc- type of heterodimeric receptor has not yet been
curred at least 85 million years ago (Degrave et al., fully explained. Recent evidence (reviewed in more
1981). detail in the chapter on the IFN(cid:11)/(cid:12) receptor by
All animal species examined have large IFN(cid:11) HaqueandWilliams)indicatesthatIFN(cid:12) canengage
subfamilies, but most have only one IFN(cid:12) gene. the receptor in a distinct fashion (Lewerenz et al.,
Exceptions are the horse, cow, and pig, in which at 1998).
leastfiveinterrelatedIFN(cid:12)geneshavebeenidentified In the human species, IFN(cid:12) is the predominant
(De Maeyer and De Maeyer-Guignard, 1988). Why species produced by various nonhematopoietic
evolution has favored the emergence of so many cells following virus infection or stimulation with
relatedtypeIIFNgenesandproteinsisnotclear.All double-stranded RNA (De Maeyer and De Maeyer-
typeIIFNscompeteforbindingtothesamereceptor, Guignard, 1988). In contrast, human cells of hema-
and they generally exert similar biological activities topoietic origin tend to produce more readily IFN(cid:11)
(De Maeyer and De Maeyer-Guignard, 1988; Vilcek and IFN! after different forms of stimulation.
and Sen, 1996). However, some clear differences in Relative amounts of induction of individual IFN(cid:11)
biological action have been seen with different type I subspecies also vary depending on both the cell type
IFNs. Some cells from lower animals respond to and inducing stimulus. It seems likely that, in the
human IFN(cid:12) butnot to IFN(cid:13), while the reversemay intact organism, the relative biological significance of
be true for other cell species (Vilcek and Sen, 1996). the individual members of the IFN(cid:11)/(cid:12) superfamily is
Differences have also been noted in the ability of determined primarily by the site and abundance of
individual recombinant IFN(cid:11) subspecies to inhibit their production rather than by their unique func-
the growth of tumor cell lines (Fish et al., 1983) or to tional properties.
produce the activation of natural killer cells (Ortaldo In contrast to the multiple type I IFN genes and
et al., 1984). proteins, only one IFN(cid:13) gene has been found in all
How such divergent biological actions are gener- animalspeciesexamined(seeTable1).WhereastypeI
ated when all IFN(cid:11)/(cid:12) species interact with a single IFNs are produced by many different types of cell,
618 Jan Vilcek
IFN(cid:13) is produced predominantly by lymphoid cells, Receptors and signal transduction
especially TH1 helper T cells and NK cells (De
MaeyerandDeMaeyer-Guignard,1988).Thereisno
Essential aspects of the interaction of the IFN(cid:11)/(cid:12)
structuralhomologybetweentypeIandtypeIIIFNs,
and IFN(cid:13) ligands with their receptors are explained
and these interferons bind to two distinct cell surface
in the individual chapters, and these have also been
receptors. The IFN(cid:13) gene encodes a 143 and 134
reviewed elsewhere (Stark et al., 1998; Mogensen
amino acid basic mature protein in the human and
et al., 1999). The heterodimeric IFN(cid:11)/(cid:12) and IFN(cid:13)
murine species respectively, with only about 40%
receptors (see Table 1) both belong to the class II
homology between these two species (Gray and
cytokine receptor family (Figure 1), containing two
Goeddel, 1983). As a result of this relative lack of
characteristicconservedcysteinepairsandlackingthe
conservation, IFN(cid:13) tends to be more highly species
WSXWS motif present in class I cytokine receptors
specific in its actions than type I IFNs.
(Bazan, 1990).
Both human and murine IFN(cid:13) proteins are N-
IFN(cid:11)/(cid:12) andIFN(cid:13) receptorsutilizemembersofthe
glycosylatedattwosites(DeMaeyerandDeMaeyer-
Janus kinases (JAKs) to activate signal transducers
Guignard, 1988). Mature IFN(cid:13) protein contains no
and activators of transcription (STATs), which then
cysteines, although two cysteine residues are present
translocate to the nucleus and produce activation of
in the 23 amino acid-long cleavable signal peptide
gene expression. The general scenario of IFN signal-
sequence. In agreement with the prediction made on
ing involves the following major steps: (a) crosslink-
the basis of studies with natural IFN(cid:13) (Yip et al.,
ing of the two receptor subunits by the IFN ligand,
1982), X-ray crystallographic analysis indicates that
(b) activation of two members of the JAK tyrosine
the active IFN(cid:13) molecule is a homodimer (Samudzi
kinases (JAK1 and TYK2 by IFN(cid:11)/(cid:12); JAK1 and
et al., 1991; Trotta and Nagabhushan, 1992). As
JAK2 by IFN(cid:13)), leading to the tyrosine phosphor-
already mentioned, the signal transduction pathways
ylation of STATs (STAT1 and STAT2 by IFN(cid:11)/(cid:12);
activated by the IFN(cid:11)/(cid:12) and IFN(cid:13) receptors partly
STAT1byIFN(cid:13)),(c)homo-orheterodimerizationof
overlap, providing an explanation of why these
the phosphorylated STATs, activating them for
structurally unrelated cytokines share so many
(d) transport to the nucleus, and (e) STAT binding
biological functions (Table 2).
Table 2 Major biological actions of IFN(cid:11)/(cid:12) and IFN(cid:13)
Activity Observed with
IFN(cid:11)/(cid:12) IFN(cid:13)
Induction of antiviral state + +
Activation of monocytes/macrophages (cid:6) +
Inhibition of cell growth + +
Induction of class I MHC antigens + +
Induction of class II MHC antigens (cid:6) +
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 (cid:6) +
Inhibition of tumor growth in vivo + +
Pyrogenic action + +
+,positiveeffect; (cid:6),weakorvariableeffect;(cid:255),negative;?,notknown.
Cytokines in Diverse Biological Actions 619
Figure 1 Class II cytokine receptor family. The et al., 1988; Harada et al., 1989). By now, over 10
location of the transmembrane domain is indicated membersoftheIRFfamilyhavebeenidentified,their
by the arrow. Members of this family include recep- roles ranging from cytokine signaling to cell growth
tors for IFN(cid:11)/(cid:12) (IFNAR), IFN(cid:13) (IFNGR), IL-10,
regulation and hematopoietic development (Nguyen
andtissuefactor.Characteristicfeaturesincludecon-
et al., 1997).
served cysteines in the extracellular domain. The
functional IFN(cid:11)/(cid:12), IFN(cid:13), and IL-10 receptors are
heterodimers. Alternative splicing results in the gen- Interferon is potentially fatal
eration of three variants of the second chain of the
IFN(cid:11)/(cid:12)receptor:IFNAR2a(notshowninthefigure),
IFNAR2b, and IFNAR2c, of which only the full- The many important beneficial roles of IFN(cid:11)/(cid:12) and
lengthIFNAR2cvariantcansignal.(Diagramkindly IFN(cid:13) in the intact organism are outlined in the indi-
provided by Dr Michel Aguet.) vidual chapters. Investigations in mice with targeted
deletions of the structural gene for IFN(cid:13) or of
essential IFN(cid:11)/(cid:12) and IFN(cid:13) 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
productioncaninsomesituationsbeharmful,leading
to acute toxicity or chronic disease. Thus, it may not
betoosurprisingthatnaturedevisedmechanismsthat
suppressintracellularsignalingpathwaysactivatedby
interferonsandothercytokines.Onesuchmechanism
relies on members of the SOCS (suppressor of cyto-
kinesignaling)familyofproteins,alsotermedtheCIS
family (Yoshimura, 1998). These are small proteins
withaconservedSH2domainandC-terminalSOCS/
CIS homology domain that can inhibit JAK/STAT
to DNA recognition sequences and stimulation of signaling. Many of the eight known members of the
transcription (Stark et al., 1998). For details of this SOCS family (SOCS-1 to SOCS-7 and CIS) are
process, see also Figure 2 in the IFN(cid:13) receptor inducible by cytokines, suggesting that they represent
chapter. anegativefeedbackloopthatservestopreventharm-
NotonlyweretheIFNsthefirstcytokineligandsto ful cytokine actions.
bedescribedandcharacterized,buttheelucidationof Years ago, Gresser et al. (1981) showed that daily
how IFN receptors work paved the way for progress administration of IFN(cid:11)/(cid:12) to newborn mice for two
in the understanding of cytokine signaling in general. weeks resulted in a fatal disease, characterized by
Specifically, elements of the JAK/STAT signaling severe inhibition of growth and marked fatty degen-
pathway, now known to be important in the erationandnecrosisoftheliver.Incontrast,whenthe
generation of signals from many different cytokine administration of IFN(cid:11)/(cid:12) was delayed until the mice
family receptors, were first identified in the course of were 1 week or more in age, the animals failed to
studies of IFN receptor signaling (Darnell et al., developacomparablesyndrome.TheworkofGresser
1994). Thus, the IFN signaling model represents a and colleagues had, however, been almost forgotten
paradigm for the functioning of many other cytokine untiltworecentpublicationsdemonstratedthepoten-
receptors that utilize the JAK/STAT pathway for tially fatal action of endogenous IFN(cid:13) in newborn
intracellular signal transduction (Ihle, 1996). The mice (Alexander et al., 1999; Marine et al., 1999).
importance of individual members of the JAK kinase These authors sought to explain why mice with a
family and individual STAT proteins varies depend- targeted disruption of the gene encoding SOCS-1 die
ing on the specific cytokine involved, but the basic between 2 and 3 weeks of age of a disease that
principles remain the same. involves fatty degeneration and necrosis of the liver
Research in the interferon field also led to the and multiple hematologic abnormalities. Quite
identification of another important family of tran- unexpectedly, they found that the administration of
scriptionfactorstermedinterferonregulatoryfactors, antibodiestoIFN(cid:13) protectedSOCS-1-nullmicefrom
or IRFs. The first identified members, IRF-1 and the fatal illness. These and other findings indicated
IRF-2, are strongly inducible by the IFNs (Fujita that the pathology is caused by IFN(cid:13) endogenously
620 Jan Vilcek
produced by these mice during the neonatal period. of the anterior pituitary and that it strongly counter-
Macrophages from SOCS-1-null mice showed a acts glucocorticoid-mediated immunosuppression.
greatly increased sensitivity to activation by IFN(cid:13), More recently, it has been demonstrated that MIF
and newborn SOCS-1-null mice were resistant to protein exhibits some enzymatic activities; for exam-
infection with Semliki Forest virus (Alexander et al., ple, it can catalyze keto-enol isomerization reactions.
1999). In addition, Marine et al. (1999) found an Whether this and some other enzymatic activities
increased level of IFN(cid:13) in the serum of SOCS-1-null associated with MIF are physiologically relevant is
mice, suggesting an altered T cell differentiation and apparentlynotknown.MetzandBucalalistanumber
function in these animals. of other significant biological actions associated with
These studies of SOCS-1-null mice raise a number MIF (see also Table 1 in the MIF chapter). The
of intriguing questions. Why do mice show an pathophysiological relevance of MIF is being
increased susceptibility to IFN(cid:13) during the neonatal confirmed by studies in MIF-null mice (Bozza et al.,
period,similartotheuniquesusceptibilityofnewborn 1999).OnemajormissinglinkintheMIFstoryisthe
mice to the toxicity of IFN(cid:11)/(cid:12) earlier demonstrated identification of its receptor. Until such time, the
byGresseretal.(1981)?SOCS-1wasshowntoinhibit exactplaceforMIFinthecytokinehierarchyremains
the actionofalarge numberof cytokinesand growth uncertain.
factors (e.g. IFN(cid:11), IL-6, and growth hormone)
(Yoshimura, 1998), and it is unclear why, under
physiologicalconditions,SOCS-1apparentlyplaysno
OSTEOPONTIN
essential role in the regulation of signals produced by
cytokines other than IFN(cid:13). What is clear is that
SOCS-1functioniscentraltothebalancebetweenthe Firstdescribedin1979as‘secretedphosphoprotein1’
beneficial and the harmful actions of IFN(cid:13). It would (Senger et al., 1979), OPN has a shorter history but
not be surprising if other members of the SOCS shares with MIF some of its mystique. The name
family and/or some related factors were found simi- ‘osteopontin’ derives from the fact that the protein
larly to regulate the balance between the beneficial was found to be present in the bone, and as it is
and harmful effects of other cytokines. 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
MACROPHAGE MIGRATION
and OPN receptors (both authored by Nau), OPN
INHIBITORY FACTOR (MIF)
does not share a structural homology with other
cytokines.Onepotentiallinktoothercytokinesisthe
The fascinating story of MIF is recounted in the very presence of a number characteristic regulatory motifs
informative chapter by Metz and Bucala. Following (including one AP-1, one IRF-1, and numerous NF-
earlier indications that activated leukocytes produced IL6sites)inthepromoterregionoftheOPNgene(see
diffusible factors that could arrest the migration of Table 1 in the Osteopontin chapter). As an apparent
macrophages, two groups of investigators reported a consequence,theOPNgeneisinducibleinavarietyof
partialcharacterizationofasolublefactorresponsible cells by several cytokines, including TNF, IL-1,
for this activity, produced by activated T cells, which IFN(cid:13),andIL-2.OPNisproducedbyactivatedTcells
became known as MIF (Bloom and Bennett, 1966; and macrophages. However, osteopontin is also
David, 1966). MIF became almost forgotten when produced constitutively in a variety of tissues, such
IFN(cid:13) and other cytokines were found to produce as the kidney, ovary, lung, and skin.
some of the actions earlier ascribed to MIF. Finally, The most unusual property of OPN is imparted by
the cloning of the bona fide MIF gene and the thepresenceoftheRGDsequence,whichexplainsthe
expression of the recombinant protein revealed not affinity of OPN for integrin and the fact that, like
only that MIF indeed played a role in the actions other integrin-binding proteins (e.g. fibronectin and
originally ascribed to it (the activation of macro- vitronectin), OPN is often found in association with
phages in vitro and the development of delayed-type the extracellular matrix. Consequently, the best-
hypersensitivityintheintactorganism),butalsosome documented biological activities of OPN are its abi-
newandunexpectedbiologicalproperties(reviewedin litytopromoteadhesionandmigrationinmanytypes
Bucala, 1996; Metz and Bucala, 1997; Swope and of cells, which appears to depend on the presence of
Lolis, 1999). the integrin-binding RGD sequence.
One major surprise was the demonstration that OPNhasbeenfoundtobindwithahighaffinitynot
MIF is efficiently produced by the corticotropic cells only to integrins, but also to CD44, the hyaluronate
Cytokines in Diverse Biological Actions 621
receptorpresentonmosttypesofcell(seethechapter especially to the group of cytokines discussed in this
on OPN receptors). These interactions probably introductory chapter.
account for the observed effects of OPN on cell
adhesion and migration. What links OPN more Gene regulation and
closely to cytokines in its activities is that OPN is
posttranslational modification
oftenclosely associatedwith tissueinjury andinflam-
mation. As explained in the chapter on the OPN
ligand, this protein is associated with both acute and The manner in which biologically active forms of the
chronic inflammatory conditions. Moreover, OPN TGF(cid:12) ligand superfamily are generated is quite
promotestheadhesionandmigrationofmacrophages distinct from that seen with other cytokine families.
in culture, and in the intact organism, OPN has been WeshallusetheexamplesofthemammalianTGF(cid:12)1,
shown to promote the formation of an inflammatory TGF(cid:12)2, and TGF(cid:12)3 proteins to highlight the unique
cell infiltrate. OPN-null mice have been generated, aspects of this process. As described in some detail
and available evidence shows that these mice have an in the chapter on TGF(cid:12), these genes (especially
increased susceptibility to mycobacterial infection. TGF(cid:12)1, the major isoform) can be transcriptionally
Whether the cytokine-like actions of OPN are upregulated in response to stress and injury or
mediated by its interaction with integrin and CD44, by some oncogenes and viral transactivators. Post-
orwhether bindingtosomeotherunknownreceptors transcriptional mechanisms, including the stabiliza-
may be responsible, is not known. tion of mRNA transcripts, also contribute to the
upregulation of TGF(cid:12) expression in some situations.
However, the major form of regulation of TGF(cid:12)
TRANSFORMING GROWTH
activity is by posttranslational modifications, which
FACTOR (cid:12) SUPERFAMILY occur both intracellularly and after the release of
TGF(cid:12) into the extracellular environment. The pro-
Defining features of the TGF(cid:12) teolyticprocessingofalargeTGF(cid:12) precursorprotein
by the endoprotease furin results in the release of the
superfamily
112 amino acid mature TGF(cid:12) protein into the extra-
cellularenvironment.TGF(cid:12) is,however,releasedina
The TGF(cid:12) superfamily of growth factors/cytokines biologically inactive form termed small latent com-
includes proteins with a very broad range of actions. plex, in which the mature TGF(cid:12) molecule is
Ligands included in this superfamily share the noncovalently bound to the latency-associated pep-
following essential features: (a) they form S–S-linked tide (LAP). Another protein, termed latent TGF(cid:12)
dimers,(b)theybindtosimilarreceptorsandactivate bindingprotein(LTBP)maybecovalentlycoupledto
similar signal transduction pathways, and (c) their thesmalllatentcomplex,theresultingstructurebeing
actions are important in development and cell dif- called the ‘large latent complex’. These associations
ferentiation. The TGF(cid:12) family has been conserved prevent TGF(cid:12) binding to receptors.
during evolution, with members identified in ForTGF(cid:12)tobecomebiologicallyactive,itsmature
Drosophila, Caenorhabditis elegans, Xenopus, and sea form needs to be released from the latent complexes,
urchins as well as in vertebrates (fish, birds, and which can be accomplished by proteolytic processing
mammals). The superfamily includes five TGF(cid:12) and by other means (see Figure 2 in the TGF(cid:12)
isoforms, activins and inhibins, bone morphogenetic chapter for a diagrammatic representation of the
proteins (BMPs), and several other secreted factors. structure of latent complexes).
In total, over 50 ligands belonging to the TGF(cid:12) This process of extracellular posttranslational
superfamily are known, and their number continues modification, required for the generation of biologi-
torise.In viewofthe verylargenumber ofgenesand cally active TGF(cid:12), is not known to occur with other
proteins included in this family, it would be a daunt- cytokines and growth factors. It represents a level of
ing task even to attempt to present a comprehensive regulation that can influence the amount of active
overview of the field at this stage. See the individual TGF(cid:12) available at specific sites and under specific
chapters on TGF(cid:12) and its receptors, the BMP family conditions (Munger et al., 1997). The presence of
of ligands and their receptors for further details. LTBP, for example, serves to direct TGF(cid:12) to the
Otherrecentreviews (focusingonTGF(cid:12)s ratherthan extracellular matrix. Plasmin is thought to be the
other members of the superfamily) are by Derynck major physiological activator of TGF(cid:12), and the in-
and Choy (1998) and Massague (1998). We shall creased conversion of plasminogen to plasmin during
attempt to point out some of the unique features of angiogenesis or tumor invasion could lead to an
this family as they relate to cytokines in general and increased activation of latent TGF(cid:12) at these sites.
622 Jan Vilcek
‘Going mad with Smads’ contributes to the unusual pleiotropy of actions of
TGF(cid:12) family members, which is remarkable even
when compared to the pleiotropic actions of other
This eye-catching phrase is the title of a brief recent
multifunctional cytokines. (See chapters on TGF(cid:12)s,
reviewintheNewEnglandJournalofMedicine(Zhou
BMP, and activin for a survey of the many different
etal.,1999).Thetitlereferstoafamilyofintracellular
biological activities of these ligands.)
signaltransducersresponsibleformostofthecomplex
actions of TGF(cid:12) superfamily members. Originally
identified in Drosophila and termed Mad, the family VASCULAR ENDOTHELIAL
of what is now called Smad genes and proteins has
GROWTH FACTOR
grown to at least nine members, of which seven have
been identified in vertebrates (Massague, 1998). Like
VEGF ligand family
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 Although known fora relatively short time, VEGF is
worms to mammals. a well-characterized and important growth factor/
The signaling cascades have been most thoroughly cytokine. Unlike the pleiotropic proteins discussed in
investigated with the prototypical member of this this chapter so far, VEGF is a mitogen for vascular
familyofligands,TGF(cid:12)1.TGF(cid:12) bindinginducesthe endothelialcellsinculture,anditdisplaysarelatively
formation of a heteromeric complex of T(cid:12)R-I and narrow spectrum of other actions in the intact orga-
T(cid:12)R-II (thought to be a tetramer consisting of two nism, virtually all of which concern angiogenesis and
chainseachofT(cid:12)R-IandT(cid:12)R-II).Thephosphoryla- blood vessel function. Placing VEGF in context is
tion of T(cid:12)R-I on the so-called GS domain by the alsogreatlyfacilitatedbythefactthatVEGFdisplays
intrinsic kinase of T(cid:12)R-II leads to the recruitment of significant homology to the A and B chains of
Smad2 and Smad3 and their phosphorylation by the platelet-derived growth factor (PDGF). In addition,
activated T(cid:12)R-I. Phosphorylated Smad2 and Smad3 like PDGF, VEGF signals through tyrosine kinase
then heterodimerize with Smad4, the resulting com- receptors that are structurally and functionally quite
plexesmovingtothenucleus,wheretheycombinewith different from the class I and II cytokine receptors.
other transcription factors and coactivators before (For references, see the VEGF chapter; for recent
they activate the transcription of a variety of target reviews, see Ferrara, 1999; Neufeld et al., 1999;
genes.OtherSmadsarespecificfortheBMPreceptor Ortega et al., 1999.) Another noteworthy property of
signaling pathway. Smad6 and Smad7 exemplify so- VEGF is its ability to bind heparin, in which it
called inhibitory Smads because, rather than activat- resembles some forms of fibroblast growth factors
ing, they inhibit signaling by TGF(cid:12) family members. (FGF) that can also affect angiogenesis.
In some systems, however, Smad6 and Smad7 act as ThealternativeexonsplicingofasingleVEGFgene
transcriptional activators. accounts for the existence of four or five different
In some of its general features, the above signaling molecular species that differ in receptor affinity, in
scenario resembles the events occurring during their ability to bind to cell surface heparan sulfate
activation of the JAK/STAT pathway by interferons proteoglycans, and in their biological activities. In
and many other cytokines. One difference is that additiontothemajorVEGFgene,severalothermem-
T(cid:12)R-I and T(cid:12)R-II contain serine/threonine kinase bers of the VEGF gene family have been identified,
domainswhereasclassIandIIcytokinereceptorsuse including placenta growth factors (PlGF-1 and -2)
extraneous JAK tyrosine kinases. More importantly, and VEGF-B, -C, and -D. The products of these
the Smads, unlike the STATs, do not function as related genes show differences in their receptor
transcriptional activators in their own right but must binding specificity and biological actions.
combinewithcoactivators(e.g.CBP/P300)and,more An important feature in the regulation of VEGF
remarkably, with members of other transcription gene expression is that hypoxia serves as a key
factor families before they can regulate the transcrip- positive regulator. In this respect, VEGF resembles
tion of specific target genes (Raftery and Sutherland, the erythropoietin (EPO) gene. Regulatory regions in
1999; Zhang and Derynck, 1999). Best known is the both the VEGF and EPO genes contain homologous
propensity of the Smad3/4 heterodimer to form sequences that are known to bind hypoxia-inducible
complexes with members of the AP-1 family of factor (HIF-1), a transcription factor whose targeted
transcription factors, such as c-jun, c-fos, and SP-1. deletion is embryonically lethal owing at least partly
The fact that Smads are promiscuous in the choice to defects in embryonic vascularization (Ryan et al.,
of transcription factors they partner undoubtedly 1998;Ferrara,1999;Neufeldetal.,1999;Ortegaetal.,
Cytokines in Diverse Biological Actions 623
1999). Hypoxia developing in necrotic tumors may a very short intracellular domain and are thought to
account for the high level VEGF expression in tumor act as co-receptors rather than signaling receptors.
tissues. In addition, the oncogene v-src can induce A somewhat similar co-receptor function is ascribed
HIF-1 expression in the absence of hypoxia, thereby to cell surface heparan sulfate proteoglycan (Neufeld
leading to increased VEGF synthesis (Jiang et al., etal.,1999).Forinformationonwhatisknownabout
1997). VEGF mRNA expression is also upregulated VEGF receptor signal transduction, the reader is
by many growth factors/cytokines, as detailed in the referred to a recent review by Ortega et al. (1999),
VEGF chapter. available online at http://www.bioscience.org/1999/
The major actions of VEGF in the intact organism v4/d/ortega/list.htm.
include the induction of angiogenesis, the permeabi-
lization of blood vessels, and vasodilation. The key
roleofVEGFinembryonicvasculogenesisandangio-
ANGIOSTATIN AND
genesis has been confirmed by gene targeting studies
in mice: disruption of the VEGF gene resulted in ENDOSTATIN
embryonic lethality between days 11 and 12.
Deregulated VEGF expression promotes the devel-
Ayin/yangrelationshipexistsbetweenVEGFandthe
opment of malignant tumors by increasing tumor
angiogenesis inhibitors angiostatin and endostatin.
angiogenesis. The involvement of VEGF in the
In fact, the discovery and isolation of angiostatin
pathogenesis of a variety of other disorders with
(O’Reilly et al., 1994) and endostatin (O’Reilly et al.,
deregulated angiogenesis (e.g. retinopathies and
1997) were based on the hypothesis that whereas
rheumatoid arthritis) has been documented (Neufeld
angiogenesisinthevascularbedofaprimarytumoris
et al., 1999).
stimulated by angiogenic factors such as VEGF,
angiogenesisinthevascularbedofmetastasesisinhib-
ited by an excess of putative tumor-derived circu-
VEGF receptors
lating angiogenesis inhibitor(s). It had been assumed
that both the angiogenic and antiangiogenic factors
VEGF receptors have been identified, and the signal are produced by the tumor so it came as a surprise
transduction pathways responsible for VEGF actions when the first angiogenesis inhibitor (angiostatin)
are being intensely investigated. Three closely related isolated from the serum and urine of tumor-bearing
signaling receptors, termed VEGFR-1 (flt-1), mice as a result of the application of this bold hypo-
VEGFR-2 (flk-1/KDR), and VEGF-3 (flt-4), have thesis was identified as a 38kDa internal fragment of
been identified. They all contain seven immunoglo- plasminogen. The second tumor angiogenesis inhibi-
bulin-like domains in their extracellular portions and tor (endostatin), isolated with the help of a similar
a split intracellular tyrosine kinase domain (Neufeld strategy, turned out to be the product of the proteo-
et al., 1999). These receptors thus represent a novel lytic cleavage of collagen XVIII.
subfamily of tyrosine kinase receptors. As explained by in the chapters on angiostatin and
The crosslinking of two receptors by the ligand endostatin, neither is actually produced by the tumor
results in an autophosphorylation of the receptors on cells. Instead, plasminogen transcripts are found
specific tyrosine residues. It is unclear whether the primarily in the liver, and collagen XVIII transcripts
tyrosinephosphorylationofVEGFR-1isrequiredfor are made by many different tissues. It is believed that
its function, because deletion of the kinase domain tumorcellsexpressproteasesthatcleaveplasminogen
from this receptor did not impair its ability to pro- toangiostatinandthatothertumor-derivedproteases
mote blood vessel formation (Hiratsuka et al., 1998). are responsible for the proteolytic digestion of colla-
Both VEGFR-1 and VEGFR-2 bind the major form genXVIIItoendostatin.Severalproteasescapableof
of VEGF (VEGF ) but differ with respect to their clearing plasminogen to angiostatin have been identi-
165
affinity for other VEGF family ligands. Gene knock- fied, but the proteases responsible for converting
out studies have shown that mouse embryos with collagen XVIII to endostatin have not.
homozygous disruptions of either VEGFR-1/flt-1 or Itmaynolongermatterwhathypothesishasledto
VEGFR-2/flk-1 genes died in utero, but the patho- the identification of angiostatin and endostatin. Of
logical findings indicated that the two receptors are more concern for us are the questions of how angio-
needed at different stages of vascular development statin and endostatin relate to cytokines and growth
(Fong et al., 1995; Shalaby et al., 1995). factors, and how their actions can be explained in
Two structurally unrelated receptors, termed neu- molecular terms. It appears that, of all the proteins
rophilin-1 and -2, also bind VEGF, but these have reviewed in this chapter, angiostatin and endostatin
624 Jan Vilcek
diverge most from what one might consider a typical Claesson-Welsh,L.,Welsh,M.,Ito,N.,Anand-Apte,B.,Soker,S.,
cytokine. Rather than being encoded by a cytokine- Zetter, B., O’Reilly, M., and Folkman, J. (1998). Angiostatin
inducesendothelialcellapoptosisandactivationoffocaladhe-
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