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(Circulation Research. 2005;96:488.)
© 2005 American Heart Association, Inc.

Review

Syndecans

New Kids on the Signaling Block

Eugene Tkachenko, John M. Rhodes, Michael Simons

From the Angiogenesis Research Center, Section of Cardiology, and Departments of Medicine and Pharmacology and Toxicology, Dartmouth Medical School, Lebanon, N.H.

Correspondence to Michael Simons, MD, Section of Cardiology, Dartmouth-Hitchcock Medical Center, One Medical Center Dr, Lebanon, NH 03756. E-mail michael.simons{at}dartmouth.edu

This Review is part of a thematic series on Angiogenesis, which includes the following articles:

Endothelial Progenitor Cells: Characterization and Role in Vascular Biology
Bone Marrow–Derived Cells for Enhancing Collateral Development: Mechanisms, Animal Data, and Initial Clinical Experiences
Influence of Mechanical, Cellular, and Molecular Factors on Collateral Artery Growth (Arteriogenesis)
Innate Immunity and Angiogenesis
Syndecans: New Kids on the Signaling Block
Growth Factors and Blood Vessels: Differentiation and Maturation

Ralph Kelly Guest Editor

	Abstract

Top Abstract Introduction Syndecan Structure Regulation of Syndecan... Syndecan Function in Development Modulation of Outside-In... Cell Adhesion and the... Summary References

 
Cell-associated proteoglycans provide highly complex and sophisticated systems to control interactions of extracellular cell matrix components and soluble ligands with the cell surface. Syndecans, a conserved family of heparan- and chondroitin-sulfate carrying transmembrane proteins, are emerging as central players in these interactions. Recent studies have demonstrated the essential role of syndecans in modulating cellular signaling in embryonic development, tumorigenesis, and angiogenesis. In this review, we focus on new advances in our understanding of syndecan-mediated cell signaling.

Key Words: proteoglycan • angiogenesis • signal transduction • cytoskeleton • migration

	Introduction

Top Abstract Introduction Syndecan Structure Regulation of Syndecan... Syndecan Function in Development Modulation of Outside-In... Cell Adhesion and the... Summary References

 
Cell interactions with their environment are critical to a large number of processes including growth, migration, adhesion, and apoptosis, among many others. In addition to a variety of specialized receptor systems that have evolved to transmit signals from specific ligands, other systems have also evolved to inform cells of the broader extracellular context. Thus, adhesion receptors such as integrins can be activated by a number of extracellular matrix proteins, whereas selectins and various cellular adhesion molecules participate in cell–cell interactions.

Over the last few years, it has become increasingly clear that cells possess yet another unique system that integrates signaling from circulating ligands such as growth factors and extracellular matrix proteins with other cellular receptor systems such as integrins. This unique function, performed by the syndecan family of proteins, places them at the center of signal integration in the cell and has earned them the name of "tuners of transmembrane signaling."¹

	Syndecan Structure

Top Abstract Introduction Syndecan Structure Regulation of Syndecan... Syndecan Function in Development Modulation of Outside-In... Cell Adhesion and the... Summary References

 
Syndecans are a family of transmembrane core proteins capable of carrying heparan sulfate (HS) and chondroitin sulfate (CS) chains. Whereas invertebrates have only one syndecan, four syndecan genes (syndecan-1, -2, -3, and -4) are present in vertebrates. Each syndecan has a short cytoplasmic domain, a single-span transmembrane domain (TM), and an extracellular domain with attachment sites for three to five HS or CS chains (Figure 1). The presence of HS chains allows interactions with a large number of proteins, including heparin-binding growth factors such as fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs), transforming growth factor-ß (TGF-ß), and platelet-derived growth factors. Furthermore, HSs facilitate interactions with various extracellular matrix proteins, including fibronectin and plasma proteins such as antithrombin-1. The role of CS chains is less clear. A recent study has suggested that syndecan-1 and syndecan-4 CS chains cooperate with HS chains in binding to the heparin-binding growth factors midkine and pleitrophin² and to the extracellular matrix protein laminin.³

View larger version (37K): [in this window] [in a new window]   Figure 1. Syndecan structure. Top, Representation of the vertebrate syndecan structure. Cytoplasmic domains are shown in red, and glycosaminoglycam chains are shown in green. Bottom, Protein alignment of mouse syndecan cytoplasmic domains. Amino acids in color identify post-translational or binding motifs: red, phosphorylation in situ; brown, phosphorylation in transfectants or a synthetic peptide; green, PDZ2-binding domains; dark blue, c-Src or Fyn kinase activity in a complex with cortactin; light blue, ezrin-binding site. MW indicates molecular weight; C1, conserved domain 1; C2, conserved domain 2.

Extracellular Domain
The structural diversity of HS and CS chains results from a series of post-translational modifications beginning with the attachment of the first hexosamine to the linkage region tetrasaccharide, which is O-linked to serine or threonine in the core transmembrane protein committing the oligosaccharide chain to either HS (N-acetylglucosamine, uronic acid) or CS (N-acetylgalactosamine, uronic acid) disaccharide polymer.⁴ Then HS and CS glycosaminoglycan (GAG) backbones are extended by different sets of polymerizing glycosyltransferases, resulting in HS and CS chains of 20 to 80 disaccharides in average length. Then these newly synthesized chains are modified by epimerization of glucuronic to iduronic acid residues and sulfation of the C2 position of glucuronic and iduronic acid. A myriad of specific CS (4-O and 6-O N-acetylgalactosaminyl) and HS (2-O, 3-O, and 6-O N-acetylglucosaminyl and N-deacetylase/N-) sulfotransferases complete the decoration of these GAG chains.⁵

Various combinations of enzymes involved in post-translational HS chain modifications produce unique binding motifs that selectively recognize different proteins. Thus, a specific combination of 2-O and 6-O sulfation is necessary for synthesis of the FGF2-binding site, whereas 3-O-sulfotransferase 1 activity is needed for generation of the antithrombin-1–binding site. Modifications of sulfotransferase expression and activity can significantly modulate syndecan functions as demonstrated, for example, in the case of 2-O-sulfotransferase (2-OST) deficiency that results in marked abnormalities of FGF signaling.⁶ On the other hand, augmentation of 2-OST expression, as occurs in the setting of hypoxia, leads to enhanced FGF responsiveness.⁷

The recent discovery of specific mammalian sulfatases (Sulf 1 and Sulf2), which can modify GAG chains extracellularly, adds another layer of complexity to the system. Sulf1 and Sulf2 are secreted, HS-specific 6-O-sulfatases,^8,9 and their activity can modify heparin-binding growth factor signaling.¹⁰ In quail, Qsulf1, the avian homolog of Sulf1 and Sulf2, has been shown to remodel HS on the cell surface to promote Wnt-1 signaling;¹¹ whereas in human cancer cell lines, Hsulf1 expression inhibited FGF2 and hepatocyte growth factor (HGF) stimulation of cell growth.¹²

It has been generally assumed that all syndecan HS and CS chains are created equal; that is, there is no preference for specific HS/CS sequences on specific syndecans.¹³ However, a recent study has suggested that CS and HS chains on syndecan-1 and syndecan-4 are structurally different.² If confirmed, this will add yet more complexity to syndecan biology. In addition to interacting with extracellular protein via their HS and CS chains, the syndecan-4 core can directly engage in protein–protein interactions.¹⁴ The extracellular domain of syndecan-4 binds specifically to human foreskin fibroblasts (IC₅₀=10^–8 M) and mouse aortic endothelial cells, as well as other cell types, whereas same-species syndecan family members did not efficiently compete with these interactions.¹⁴

Transmembrane and Intracellular Domains
Although the degree of conservation in the extracellular domain of syndecans is fairly low, the TM is highly conserved. As with classic type I membrane protein architecture, syndecans have a single-pass TM. Uniquely though, the TM and a small sequence adjacent to it have a high affinity for self-association, which, in the case of syndecan-4, has been shown to be required for protein kinase C- (PKC) activation.¹⁵

The cytoplasmic domain, despite being relatively short, has a number of important regions. The domain is divided into three regions: conserved regions 1 and 2 (C1 and C2) and a variable (V) region (Figure 1). The C1 domain, immediately adjacent to the plasma cell membrane, is virtually identical in all four mammalian syndecans. It is thought to be involved in syndecan dimerization (all syndecans probably exist as homodimers and higher-order oligomers) and in binding of several intracellular proteins, including ezrin, tubulin, Src kinase, and cortactin.^16–18 The universally conserved C-terminal C2 domain contains a postsynaptic density-95/disc large protein/zonula occludens-1 (PDZ₂)-binding site at the C-terminal end and two tyrosine residues. There are four identified syndecan-interacting PDZ domain–containing proteins: syntenin, synectin, synbindin, and calcium/calmodulin-dependent serine protein kinase.^19–23

The V domain is highly heterogeneous among the four mammalian syndecans. This region has been studied most extensively in syndecan-4 because of its unique features. The syndecan-4 V-region sequence includes a phosphatidylinositol-4,5-bisphosphate (PIP₂) binding site that is involved in syndecan-4 dimerization, in which two syndecan-4 cytoplasmic domains are linked by two PIP₂ molecules in antiparallel fashion (Figure 2).²⁴

View larger version (21K): [in this window] [in a new window]   Figure 2. Nuclear magnetic resonance (NMR) structure of syndecan-4 cytoplasmic domain; NMR structure of the syndecan-4 dimer in the presence and absence of PIP2. Note that interaction with PIP2 results in reorientation of PDZ-binding domains (blue).

In addition, PIP₂ binding to the V region plays a critical role in binding and activation of PKC, an enzyme that plays a key role in syndecan-4 signaling (see below).^15,25–29 Indeed, this feature places PKC in the class of receptor-activated kinases and allows it to play a role not dissimilar from that of integrin-linked kinases. -Actinin and PKC compete for syndecan-4 binding, possibly providing a mechanism for regulation of syndecan-4–cytoskeletal interaction.³⁰ Another syndecan-4–specific binding protein is syndesmos.³¹ Although the precise nature of its interaction with syndecan-4 has not been defined, it requires C1 and V syndecan-4 domains for binding. Syndesmos binding to focal adhesion adaptor proteins paxillin and Hic-5 may be an alternative to an -actinin mechanism, linking syndecan-4 to focal adhesions.³²

Nuclear magnetic resonance structural analysis demonstrates that the C-terminal orientation of syndecan-4 changes on PIP₂ binding, implying a possible regulation of PDZ interactions either by PIP₂ or the PDZ-binding partners (Figure 2).^24,33

	Regulation of Syndecan Expression

Top Abstract Introduction Syndecan Structure Regulation of Syndecan... Syndecan Function in Development Modulation of Outside-In... Cell Adhesion and the... Summary References

 
Each syndecan family member has a distinct temporal and spatial pattern of expression, with every mammalian cell expressing at least one syndecan that is highly regulated during development.³⁴ Syndecan-1 is expressed predominantly in epithelial and mesenchymal tissues, syndecan-2 in cells of mesenchymal origin and neuronal and epithelial cells, and syndecan-3 almost exclusively in neuronal and musculoskeletal tissue, whereas syndecan-4 is found in virtually every cell type.³⁵

Given their role in tuning numerous signaling events, it is not surprising that syndecan levels are tightly regulated. Growth factors play an important role in regulation of syndecan expression. Thus, tumor necrosis factor- (TNF-) upregulates syndecan-2 and downregulates syndecan-1 in endothelial cells.³⁶ Similarly, TGF-ß2 upregulates syndecan-4 and downregulates syndecan-1 in epithelial cells.³⁷ In aortic smooth muscle cells, FGF2 induces syndecan-4 but not syndecan-1 or syndecan-2 expression.³⁸ Mechanical stress is also a prominent inducer of syndecan-4 expression in smooth muscle during arteriogenesis.³⁹ Syndecan-4 levels are markedly upregulated in several disease states, including arterial injury⁴⁰ and acute myocardial infarction.⁴¹ Another condition associated with a pronounced increase in syndecan-1 and syndecan-4 expression is wound healing.^42,43 Although there are probably numerous factors responsible for this increase, an interesting mediator is an inflammatory cell-derived peptide, PR39, which has a unique ability to markedly upregulate syndecan-1 and syndecan-4 expression in vitro and in vivo.^44,45

Very little is known about regulation of syndecan-2 and syndecan-3 expression. Syndecan-2 levels increase during transformation of fat cells into myofibroblasts, whereas the level of syndecan-1, syndecan-3, and syndecan-4 remains constant.⁴⁶ Syndecan-3 expression is highly regulated during development. In rodents, its level in central nervous system rises at birth, peaks on day 7, and then declines to the level of adult.⁴⁷ This period of high level of syndecan-3 expression corresponds with oligodendrocyte differentiation and myelin formation in the central nervous system. The temporal dermal expression of syndecan-3 suggests the possibility of its involvement in feather development.⁴⁸ During chick embryo limb development, syndecan-3 is transiently expressed during the period of mesenchymal condensation.⁴⁹

Interestingly, changes in the gene expression of one syndecan family member may affect others. For example, increased syndecan-3 expression in satellite cells leads to the downregulation of syndecan-4–transduced FGF2 and HGF signaling,⁵⁰ whereas syndecan-3 expression is reduced in syndecan-4^–/– satellite cells.

	Syndecan Function in Development

Top Abstract Introduction Syndecan Structure Regulation of Syndecan... Syndecan Function in Development Modulation of Outside-In... Cell Adhesion and the... Summary References

 
Recent studies have begun clarifying the roles played by various syndecans during the developmental process (Table). Homozygous disruption of the syndecan-1 gene in mice leads to viable offspring. The mice are grossly normal but demonstrate abnormally slow re-epithelialization after injury⁵¹ and increased leukocyte adhesion.⁵² Syndecan-1 appears to be involved in modulation of Wnt-1 signaling because mammary gland–specific expression of Wnt-1 leads to development of tumors in wild-type but not in syndecan-1 knockout mice.⁵³

View this table: [in this window] [in a new window]   Table 1. Syndecans Function in Animal Models

Studies in zebrafish using anti-syndecan-2 morpholino oligonucleotides demonstrated that this syndecan plays an important role in vascular development involving modulation of VEGF signaling. Syndecan-2 knockdown resulted in suppression of intersegmental vessels, whereas formation of dorsal vessels (aorta, cardinal vein) was not affected.⁵⁴ Overexpression of VEGF₁₆₅ in syndecan-2 morphans was not able to induce ectopic vessel formation, whereas coexpression of syndecan-2 and VEGF₁₆₅ resulted in an increase of vessel formation compared with VEGF alone. Application of moderate doses of syndecan-2 and VEGF morpholinos demonstrated synergistic inhibition of angiogenesis. Expression of cytoplasmic domain-truncated syndecan-2 mimics the phenotype produced by syndecan-2 knockdown. On the basis of these data, syndecan-2 appears to potentiate VEGF-induced capillary sprouting.

Syndecan-2 is also involved in left–right asymmetry regulation.³⁵ In Xenopus, syndecan-2 is phosphorylated by PKC only in right animal cap ectodermal cells.⁵⁵ Overexpression of the cytoplasmic domain-truncated form of syndecan-2 results in perturbed determination of left–right asymmetry of embryo and also randomizes expression of lefty, a left–right symmetry regulator.⁵⁵

Syndecan-3 levels in hypothalamus have been demonstrated to physiologically regulate feeding behavior, and mice with homozygous disruption of both syndecan-3 alleles displayed reduced feeding behavior in response to food deprivation⁵⁶ as well as impaired performance in tasks using hippocampal functioning, indicating learning and memory abnormalities.⁵⁷ They develop muscular dystrophy characterized by fibrosis, deteriorated locomotion, and hyperplasia of myonuclei and satellite cells.⁵⁰

Syndecan-4 deficiency has generated much interest. Null mice are viable and fertile but display a number of subtle defects. However, syndecan-4^–/– embryos demonstrate much more frequent thrombi formation in the vessels of the placental labyrinth than the littermate controls, resulting in much higher embryo loss and implicating syndecan-4 in regulation of blood clotting.⁵⁸ The adult syndecan-4^–/– mice have increased mortality after lipopolysaccharide injection, suggesting their inability to clear pathogens⁵⁹ and to downregulate TNF-ß—induced suppression of interleukin-1ß (IL-1ß) expression in macrophages.⁵⁷ The syndecan-4^–/– mice also have increased susceptibility to -carrageenan–induced renal damage, presumably because of increased deposition in renal collecting ducts.⁶⁰

Other interesting observations include impaired skin wound healing that is thought to be secondary to defective angiogenesis.⁶¹ However, no other angiogenesis deficiency phenotypes have been reported to date. Finally, satellite cells from syndecan-4^–/– mice fail to reconstitute damaged muscles, suggesting that syndecan-4 presence is required for migration of these skeletal muscle progenitor cells.⁵⁰ This may in part be attributable to the defective FGF2- and HGF-induced activation of extracellular signal-regulated kinase (Erk)-1/2.⁵⁰

	Modulation of Outside-In Signaling

Top Abstract Introduction Syndecan Structure Regulation of Syndecan... Syndecan Function in Development Modulation of Outside-In... Cell Adhesion and the... Summary References

 
The preceding section demonstrates that disruption of various syndecan genes leads to phenotypes consistent with the "tuning" role of syndecans in signal transduction. The precise mechanism of this tuning has generated much interest and appears to be different for different syndecans. In the discussion that follows, although we consider signaling events associated with individual syndecans, it should be kept in mind that considerable overlap between various syndecan-mediated events probably exists and that the "specific" signaling events described for an individual syndecan may just as well reflect the limitation of our knowledge as the real biological specificities. Nevertheless, there is also a considerable degree of specificity. In particular, it is useful to think in the signaling context of syndecan-1 and syndecan-3 as one subfamily and syndecan-2 and syndecan-4 as another. This classification reflects homology of V domain between corresponding syndecans as well as functional similarities. For example, syndecan-1 and syndecan-3 expression is mostly associated with inhibition of cell growth, whereas syndecan-2 and syndecan-4 expression leads to its stimulation.

HS and CS Chains
The way in which syndecans engage in signal transduction remains a matter of active research and controversy. The first and the oldest hypothesis postulates that syndecans serve as coreceptors for various heparin-binding growth factors. This is thought to occur because of GAG chains binding growth factors, thereby restricting their presence to the membrane surface and facilitating their subsequent interactions with corresponding high-affinity receptors.⁶² Numerous studies have shown that inhibition of HS chain synthesis grossly affects signaling. For example, heparinase treatment of cultured smooth muscle cells decreases their responsiveness to FGF2,⁶³ whereas heparinase treatment in vivo inhibits angiogenesis.⁶⁴

Genetic data are equally compelling. Inhibition of HS formation attributable to mutations of EXT1 and EXT2 genes, which catalyze polymerization of glucuronic acid and N-acetylglucosamine, the crucial step in HS synthesis, results in hereditary multiple exostoses, an autosomal skeletal disorder characterized by inappropriate chondrocyte proliferation and bone growth.⁶⁵ The defect is thought to be attributable to abnormal diffusion of hedgehog (Hh) proteins.⁶⁶ A homozygous disruption of Ext-1 expression in mice results in gastrulation defects and early embryonic lethality.⁶⁷

More subtle changes in the HS chain composition also have profound effects. Sulfation of 2-O and 6-O HS sites is thought to be required for formation of FGF-binding sites. A homozygous deletion of 2-OST results in mice that survive until birth but die perinatally because of the complete failure of kidney formation.⁶ Similarly, a knockdown of the 6-O-sulfotransferase in Drosophila severely perturbs tracheal development, an FGF-dependent process.⁶⁸ Deletions of other genes involved in HS biosynthesis have equally profound phenotypic effects.⁶⁹

In addition to HS-dependent signaling, syndecans also clearly can engage in protein–protein interactions via their protein core ectodomains, an event that can also initiate intracellular signaling, as is discussed below. Furthermore, syndecan–growth factor interaction, whether accomplished via the chain or the core-based interaction, does not simply serve to present the factor to its high-affinity receptor. Rather, such binding can initiate signaling events via the cytoplasmic syndecan domains that have their own unique aspects, as is further discussed. Finally, each of the syndecans has its own characteristic biology that we briefly review.

Syndecan-1 and Cell–Matrix Interaction
Syndecan-1 is an important regulator of cell–cell and cell–extracellular matrix interactions. Downregulation of its expression in epithelial cells by antisense mRNA results in loss of cell polarity associated with a reduced level of E-cadherin on the cell surface, suggesting involvement in the epithelial–mesenchymal switch during development and in wound healing.⁷⁰ Overexpression of syndecan-1 or shedding of its ectodomain inhibits FGF2-induced cell proliferation.⁷¹ Mice overexpressing syndecan-1 have delayed dermal wound repair because of the inhibitory effect of soluble syndecan-1 ectodomain.⁷² However, under certain circumstances, heparinases can convert syndecan-1 ectodomain from an inhibitor to an activator of FGF2.⁷³ This reaction enables FGF2 interaction with syndecan-4 (see below), although a specific protein sequence in the syndecan-1 ectodomain may also play a role.⁷⁴ The ability of syndecan-1 overexpression to inhibit cell growth and migration could be an explanation of the aggressive invasive behavior of tumor cells lacking syndecan-1.^75,76

Interestingly, downregulation of syndecan-1 expression in carcinoma cells results in impaired cell spreading on vitronectin but not on fibronectin.⁷⁷ This seems to be mediated by the ability of syndecan-1 to modulate vitronectin interaction with its ß3 integrin receptor because glycosylphosphatidylinositol (GPI)–syndecan-1 extracellular domain construct expression in syndecan-1^–/– cells converts their "no spreading phenotype" to normal spreading.

Syndecan-1 plays an important role in regulation of inflammation,⁷⁸ as suggested by increased leukocyte–endothelial interactions in syndecan-1 null mice.⁷⁹ It also plays an important role in chemokine gradient formation for trans-endothelial and trans-epithelial migration of neutrophils.⁷⁸ The former process is facilitated by an IL-8/syndecan-1 complex and regulated by shedding of syndecan-1.⁸⁰ Trans-epithelial neutrophil migration is dependent on matrilysin-mediated shedding of syndecan-1 from the mucosal surface of epithelium.⁸¹ As would be expected, matrix-metalloproteinase matrilysin–deficient mice demonstrate impaired trans-epithelial migration of neutrophils.

Finally, syndecan-1 may also play a role in regulation of ephrin signaling that is involved in venoarterial differentiation. Ephrin-B4 (EphB4) is expressed in venule endothelium. Homozygous disruption of EphB4 or one of its ligands, ephrin-B2, results in fatal vascular defects.^82,83 Activation of EphB4-positive endothelial cells causes upregulation of syndecan-1,⁸⁴ resulting in suppression of FGF2 signaling. However, in certain settings, the role of syndecan-1 could be the opposite, depending on the presence of heparinases. Degradation of heparan by platelet heparanase, for example, produces heparin-like HS fragments, capable of promoting FGF2 mitogenicity.⁷³

Syndecan-2 and TGF-ß Signaling
Syndecan-2 is the predominant syndecan expressed during embryonic development. Although its role in adult cells has not been well established, recent studies suggest its involvement in regulating TGF-ß signaling. Whereas TGF-ß can bind to HS chains, syndecan-2, but not other syndecans, binds TGF-ß directly via a protein–protein interaction. Furthermore, syndecan-2 coimmunoprecipitates with the TGF-ß as well as with the type III TGF-ß receptor (betaglycan),⁸⁵ and expression of a syndecan-2 mutant with a truncated cytoplasmic domain results in impaired response to TGF-ß.⁸⁵ These data correlate with a previously reported inability of cells overexpressing this construct to assemble laminin or fibronectin into a fibrillar matrix.⁸⁶

The mechanism of syndecan-2–TGF-ß interactions is complex and not entirely clear (Figure 3). TGF-ß binding to betaglycan transfers it to the type II receptor, which undergoes autophosphorylation and then trans-phosphorylates betaglycan and the type I receptor. The phosphorylated type I/type II receptor complex then engages in downstream signaling. Betaglycan binds to synectin (GAIP-C terminus protein) that retains betaglycan on the cell surface, thus preventing its degradation.⁸⁷ However, synectin also binds syndecan-2. As already mentioned, expression of a syndecan-2 mutant with a truncated cytoplasmic domain increases plasma membrane betaglycan levels.⁸⁵ Whereas an increase in syndecan-2 protein increases type I and type II TGF-ß receptor expression and decreases betaglycan, probably as the consequence of competition for synectin binding,⁸⁵ this decrease in betaglycan expression possibly leads to disregulated activity of the type-I/type-II TGF-ß receptor complex.

View larger version (43K): [in this window] [in a new window]   Figure 3. Modulation of TGF-ß signaling by syndecan-2. Syndecan-2 effects on TGF-ß signaling are shown for normal cells and cells overexpressing syndecan-2 constructs. Under normal circumstances, TGF-ß binds to betaglycan, which then transfers it to the TGF-ßRI/TGF-ßRII complex (top left). Expression of syndecan-2 PDZ–mutant results in increase of betaglycan on the cell surface and impaired signaling (top right). In contrast, overexpression of full-length syndecan-2 results in plasma membrane accumulation of TGF-ßRI and TGF-ßII and reduced levels of betaglycan (TGF-ßRIII; bottom).

Similar to syndecan-1, syndecan-2 may also play a role in ephrin signaling. Cell surface ephrin–Eph signaling induces clustering of syndecan-2 and recruitment of cytoplasmic molecules, which leads to localized actin polymerization via Rho family GTPases, N-Wiscott-Aldrich syndrome protein, and the Arp2/3 complex.⁸⁸

Syndecan-3 and Growth Factor Signaling
Syndecan-3 plays an important role in regulation of skeletal muscle differentiation and development. Its levels are transiently elevated in the developing limb bud but are absent in adult skeletal muscle. Skeletal muscle myoblasts are held in an undifferentiated state until they receive signals to further differentiate. The maintenance of these cells in their undifferentiated state has been shown to be controlled by specific growth factors, including FGF2, HGF/scatter factor, and TGF-ß.⁸⁹ Syndecan-3 inhibition results in expression of myogenin, a master transcription factor for muscle differentiation, and in accelerated skeletal muscle differentiation and myoblast fusion.⁹⁰

In the model of skeletal muscle regeneration induced by barium chloride injection, syndecan-3 showed the earliest and largest increase in satellite cells.⁹¹ Myoblast grafting of C₂C₁₂ cells transfected with antisense syndecan-3 resulted in cells with a normal proliferation rate but defective in fusion and formation of skeletal muscle fibers. Further, examination of syndecan-3 null mice demonstrates mislocalization of MyoD (a myocyte differentiation transcription factor) and an aberrant differentiation of skeletal muscle.⁵⁰

Syndecan-3 also appears to be involved in regulation of Hh signaling. Hh is a family of secreted signaling proteins that play an important role in embryogenesis and differentiation. One of the mammalian family of Hh members, indian Hh (iHh), is produced by prehypertrophic chondrocytes and regulates chondrocyte proliferation. Proper spatial and temporal regulation of iHh is regulated by syndecan-3.⁹²

Syndecan-4, FGF Signaling, and the Extracellular Matrix
Syndecan-4 has become the most extensively studied member of the syndecan family. The proteoglycan has a number of activities, including modulation of FGF2 signaling, regulation of cell migration via cross-talk with ß1 integrin, and control of adhesion via cytoskeletal modifications. All these various events are achieved via actions of a 33-aa cytoplasmic tail, one of the most overworked small proteins among the signaling giants.

FGF Signaling and Endocytosis
In addition to being able to bind FGF2 to HS chains and to present it to FGF tyrosine kinase receptors, syndecan-4 directly initiates a number of intracellular signaling events. The most direct demonstration of syndecan-4 signaling came from studies of syndecan-4/glypican chimeras in endothelial cells. Expression of a full-length syndecan-4 (S4), syndecan-1 (S1), glypican-1 (G1), or of chimera constructs consisting of the ectoplasmic domain of G1 linked to the transmembrane/cytoplasmic domain of syndecan-4 (G1-S4c) or of the ectoplasmic domain of syndecan-4 linked to the G1 GPI anchor (S4-GPI), significantly increased cell-associated HS mass and the number of low-affinity FGF2-binding sites. However, only cells expressing S4 and G1-S4c constructs but not G1, S1, or S4-GPI cells demonstrated enhanced responsiveness to FGF2. Thus, the presence of syndecan-4 cytoplasmic domain and not simply an increase in the cell surface HS mass with a corresponding increase in the number of the low-affinity FGF2-binding sites is required for signaling. However, removal of the syndecan-4 HS chains also blocked enhanced FGF2 responsiveness, demonstrating that syndecan-4 HS chains and the syndecan-4 cytoplasmic domain are required for FGF2 signaling.⁹³ Syndecan-3 cannot replace syndecan-4 to promote FGF2- and HGF-induced cell migration.⁵⁰

Additional evidence of direct syndecan-4 signaling ability comes from studies in vascular smooth muscle cells expressing dominant-negative FGF receptor. FGF2 was able to transiently stimulate Erk 1/2 activation and induce cell migration, activities that were inhibited by removal of HS chains.⁹⁴ It should be noted that FGF2-induced Erk activation in the absence of functional FGF receptor 1 is transient and does not lead to cell proliferation.

The final piece of evidence demonstrating syndecan-4 modulation of FGF2 signaling comes from studies that explored the effect of dominant-negative syndecan-4 construct expression on endothelial cell responses to FGF2 and other growth factors. Introduction of syndecan-4 constructs with the mutated PDZ- or PIP₂-binding regions that respectively eliminated syndecan-4 ability to bind PDZ proteins or PIP₂ inhibited FGF2- but not serum-induced endothelial cell growth, migration, and the ability to form vascular structures on Matrigel.²⁷

Although it is clear that syndecan-4 can modulate FGF2 signaling, the precise mechanism of this modulation is uncertain. A sustained activation of FGF2 signaling requires internalization and perhaps nuclear transport of the ligand.⁹⁵ Syndecan-4 is directly involved in FGF2 endocytosis that proceeds in a clathrin- and dynamin-independent fashion and requires syndecan-4–dependent activation of Rac1.⁹⁶ The sensitivity of this process to amiloride as well as colocalization of internalized FGF2 with dextran suggests that FGF2 uptake proceeds via macropinocytosis.⁹⁶ An interesting feature of this process is the fact that FGF2 uptake proceeds from lipid rafts, a specialized plasma membrane domain thought to be involved in signaling.

Endocytosis, Lipid Rafts, and Caveolae
Plasma membrane location of syndecan-4 and its participation in FGF2 endocytosis raises the issue of its relation to lipid rafts, plasma membrane domains that provide fluid platforms to segregate membrane components and dynamically compartmentalize membranes.⁹⁷ Those phospholipid- and cholesterol-rich domains contain specific sets of proteins that include GPI-anchored proteins, doubly acetylated proteins (Src family tyrosine kinases), G subunit of heterotrimeric G-proteins, and palmitylated proteins including endothelial NO synthase.^98,99

Whereas in unstimulated cells, syndecan-4 is predominantly present in the nonraft compartments, clustering with FGF2 or anti–syndecan-4 antibody induces its shift to lipid rafts.¹⁰⁰ This shift is necessary for the initiation of FGF2 endocytosis because its blockade by cholesterol depletion from the plasma cell membrane completely blocks FGF2 uptake.⁹⁶ Similarly, low-density lipoprotein (LDL) causes clustering and clathrin-independent uptake of syndecan-1, syndecan-2, and syndecan-4.¹⁰¹ In the model in which syndecan-1 was used, LDL clustering also induced a shift of syndecan into the lipid rafts.¹⁰²

Caveolae are a subset of lipid rafts. They are flask-shaped invaginations of the plasma membrane¹⁰³ that play a role in regulation of certain endocytic pathways and regulation of enzymes such as endothelial NO synthase. A variety of protein- and lipid-signaling molecules are concentrated in caveolae. These include PKC, Rho GTPases, and nonreceptor tyrosine kinases such as c-Src, Yes, Fyn, phosphatidylinositol 3-kinase, and phosphatidylinositol kinases, among others.^103–105 Caveolins are the principal building blocks of caveolae and are usually accepted as its marker. Caveolin-1 oligomeric complex creates a stable membrane structure that transiently interacts with plasma membrane and endosomes.¹⁰⁶

On initiation of cell migration, pools of syndecan-1, syndecan-4, and caveolin-1 are directed into the same retracting region of the moving cell.^107–109 The caveolin property of inhibiting kinase activity of a variety of proteins involved in leading edge formation was speculated to play a key role in cell migration.¹¹⁰ In contrast to the deactivating role of caveolin, syndecan-4 is needed for activation of several kinases, including focal adhesion kinase (FAK), which results in increased turnover of focal adhesions.^111–113 Silencing of caveolin-1 gene expression or introduction of a dominant-negative mutant of syndecan-4 leads to slow migration and impaired tube formation of endothelial cells on Matrigel.^27,107 Syndecan-4 and caveolin-1 knockout mice demonstrate reduced postnatal angiogenesis that may be related to the impairment of endothelial cell migration.^61,110

Whereas syndecan-4 does not colocalize with caveolin-1 at the cell surface, syndecan-4– and caveolin-1–containing vesicles frequently move in tandem, presumably along microtubules.¹⁰⁰ Remarkably, internalization of caveolae depends on Src and PKC activation.¹¹⁴ The ability of syndecan-4 to activate PKC is well established and is discussed in detail below. Similar to syndecan-3, it can also activate Src.¹⁷ Thus, syndecan-4 may be responsible for modulation of caveolae, pinching from the plasma membrane in response to heparin-binding growth factors.^115,116

The association of syndecan-4 with fast-moving caveolin-1–positive vesicles could also be used for a fast delivery of syndecan-4 pinosomal cargo to intracellular destinations. This process might be needed for proper distribution of macropinosome-associated small GTPases, leading to migratory polarization of the cell.^117,118 This interaction could also be involved in extracellular matrix turnover. Downregulation of caveolin-1 results in inhibition of fibronectin internalization and degradation,¹¹⁹ suggesting the participation of fibronectin receptors such as integrins or syndecans in caveolae-dependent uptake of fibronectin.

Activation of PKC
Another feature of syndecan-4 signaling is activation of PKC in the absence of Ca²⁺. This unique ability of syndecan-4 to activate a calcium-dependent PKC was first shown by Oh et al to require core protein multimerization and the presence of PIP₂.^15,120 Syndecan-4 oligomerization in the presence of PIP₂ depends on the phosphorylation status of the Ser¹⁸³ site in its cytoplasmic domain.²⁷ Phosphorylation of this site markedly reduces its affinity for PIP₂²⁶ and prevents PKC activation in vitro and in vivo.^26,121 Interestingly, the Ser¹⁸³ site is phosphorylated by PKC.²⁸ This sets up a system that allows one PKC to regulate activity of another (Figure 4). The Ser dephosphorylation is performed by as yet undefined type I/IIa serine phosphatase.¹²¹

View larger version (53K): [in this window] [in a new window]   Figure 4. Syndecan-4–dependent modulation of PKC activity. Under normal circumstances, Ser183 site is phosphorylated by PKC. Exposure to FGF2 results in dephosphorylation of the site that, in turn, allows syndecan-4 oligomerization, binding of PIP2, and activation of PKC. Any signal that would activate PKC would impair syndecan-4–dependent PKC activation.

The molecular details of syndecan-4–dependent PKC activation have not been established. The results of yeast two hybrid screening demonstrated that although a full-length PKC weakly binds the V domain of syndecan-4, the PKC constructs that lack the pseudosubstrate region or that encode only the whole catalytic domain interacted more strongly.^15,122 A mutation of Tyr¹⁹² in the syndecan-4 cytoplasmic domain abolished this interaction.¹²² Interestingly, the corresponding binding site in the catalytic domain of PKC (amino acid sequence 513 to 672) encompasses the regulatory autophosphorylation sites implicated in control of PKC activation and stability.¹²² On the other hand, in vitro surface plasmon resonance studies suggested that binding affinity of a full-length PKC to the syndecan-4 cytoplasmic domain is very low in the absence of PIP₂.²⁶

Although the precise role of syndecan-4–dependent PKC activation has not been defined, colocalization of the two proteins in focal adhesions¹²⁰ suggests a role in cell adhesion, spreading, and stress fiber formation. Studies in cultured fibroblasts have shown that overexpression of syndecan-4, but not a mutant lacking its cytoplasmic domain, specifically increases the level of endogenous PKC and enhances the translocation of PKC in the plasma cell membrane in general and in the membrane raft fractions in particular, and increases the activity of membrane PKC.¹²³

	Cell Adhesion and the Cytoskeleton

Top Abstract Introduction Syndecan Structure Regulation of Syndecan... Syndecan Function in Development Modulation of Outside-In... Cell Adhesion and the... Summary References

 
The involvement of members of the syndecan family in regulation of cell adhesion suggests that these proteins likely modulate cytoskeletal rearrangements. Indeed, fibronectin binds to syndecan-4 HS chains, and this interaction plays an important role in regulation of cell adhesion and spreading.¹²⁴ Fibroblasts use integrin receptors and syndecan-4 to induce Rho-dependent spreading in fibronectin.^125,126 Overexpression of syndecan-4 results in the flattening of Chinese hamster ovary K1 (CHO-K1) cells, promotion of focal adhesion formation, and a decrease in cell migration.¹²⁷ Syndecan-4 null fibroblasts plated on fibronectin exhibit enhanced lamellipodia formation and increased level of Rac1¹²⁸ and low Rho¹¹¹ activities compared with wild-type cells. Expression of syndecan-4 in these knockout cells downregulates Rac1 activity.¹²⁸ In endothelial cells also plated on fibronectin, clustering of syndecan-4 construct by antibody results in upregulation of Rac1 activity.⁹⁶ Those seemingly contrary results might be attributable to the differences in localization changes followed by binding to matrix versus soluble ligand. In this way, growth factors might modulate cell–matrix interactions by removal of the syndecan component via endocytosis.⁹⁶

In vascular smooth muscle cells, shear stress causes syndecan-4 dissociation from the focal adhesions.³⁹ Overexpression of syndecan-4 blocks this dissociation and also results in reduced mechanical stress-induced cell migration. This observation suggests that syndecan-4 might be involved in regulation of smooth muscle migration during arteriogenesis.

Another role of syndecan-4 involves regulation of fibronectin signaling and matrix contraction together with tenascin-C. Tenascin-C is an extracellular matrix protein involved in regulation of cellular response to fibronectin by preventing cell spreading.¹²⁹ Syndecan-4 knockout fibroblasts, while spreading normally on a 2D matrix but failing to spread in a 3D fibronectin matrix, do not activate RhoA and, consequently, do not form focal adhesions and stress fibers. Anti–syndecan-4 antibody treatment of wild-type fibroblasts similarly prevents spreading on fibronectin, decreases RhoA, and activates FAK. Stimulation of RhoA by lysophosphatidic acid in syndecan-4^–/– cells rescues syndecan functions by activating RhoA and inducting cell spreading and matrix contraction. Addition of tenascin-C blocks spreading of control but not syndecan-4^–/– fibroblasts on a 2D fibronectin matrix, whereas syndecan-4 overexpression overcomes this tenascin-C effect.¹³⁰

Activated B lymphocytes, when seeded on syndecan-4 antibodies, exhibit dramatic morphological changes,¹³¹ the most profound being filopodial extensions, implying the possibility of CDC42 activation. Overexpression of a truncated form of syndecan-4 lacking an intracellular domain demonstrated that the extracellular domain is sufficient to generate a response, indicating the need for a transmembrane partner to transmit signal. One such potential partner is the chemokine receptor 4 (CXCR4). Syndecan-4, but neither syndecan-1 nor syndecan-2, coimmunoprecipitate with CXCR4.¹³² This syndecan-4/CXCR4 complex is likely a functional unit involved in chemokine stromal cell–derived factor-1 (SDF-1) binding that may play a role in SDF-1–dependent stem cell recruitment.

Syndecan-2 and syndecan-3 overexpression also induce filopodia formation in COS-1 and CHO-K1 cells.^133,134 In the case of syndecan-2, this process is CDC42 dependent.^133,134 The deletion of V or C2 domains, but not entire cytoplasmic part of syndecan-3, results in more filopodia formation, whereas extensive membrane blebbing is observed in cells expressing a C2-truncated mutant.¹³⁴ The blebs are distributed uniformly over the cell surface rather than next to the leading edge, suggesting improper targeting or activation of Rac1 resulted to bleb formations away from cell edges as a possible mechanism.

Members of the ezrin-radixin-moesin family have NH₂- and C-terminal domains that associate with the plasma membrane and the actin cytoskeleton, respectively. After overexpression of a constitutively active form of RhoA, association of syndecan-2 with actin cytoskeleton through ezrin binding was observed in COS-1 cells.^16,18 Thus, ezrin provides Rho GTPases a regulated link between syndecan-2 and actin. The ezrin-binding motif is identical between syndecan-2 and syndecan-4, implying the possibility of syndecan-4–ezrin interactions (Figure 1).¹⁸ Such regulation of cytoskeleton by Rho GTPases promises to be one of the most important tuning functions of syndecans.

	Summary

Top Abstract Introduction Syndecan Structure Regulation of Syndecan... Syndecan Function in Development Modulation of Outside-In... Cell Adhesion and the... Summary References

 
As we hope this review demonstrates, syndecans have been shown to function as potent and specific regulators of cell signaling in a variety of settings. This regulatory function is very complex because of the involvement of different family members, differences in level and cellular location of expression, GAG chain modifications, and extracellular domain shedding. Overall, given their ability to participate in signal transduction involving the extracellular matrix, cell surface molecules, and soluble ligands, syndecans are emerging as conductors of the entire signaling orchestra.

	Acknowledgments

 
This work was supported in part by National Institutes of Health grants HL62289 and HL63609 (M.S.). We would like to thank members of the Simons’ laboratory and the Angiogenesis Research Center colleagues for helpful discussions and incisive comments. Certain areas of syndecans signaling dealing with their role in the CNS have been omitted in this review. We also would like to apologize to authors whose primary articles were not included because of space limitations.

	Footnotes

 
Original received December 27, 2004; revision received January 27, 2005; accepted February 8, 2005.

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Top Abstract Introduction Syndecan Structure Regulation of Syndecan... Syndecan Function in Development Modulation of Outside-In... Cell Adhesion and the... Summary References

 

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