Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration.

Myogenesis in the embryo and the adult mammal consists of a highly organized and regulated sequence of cellular processes to form or repair muscle tissue that include cell proliferation, migration, and differentiation. Data from cell culture and in vivo experiments implicate both FGFs and HGF as critical regulators of these processes. Both factors require heparan sulfate glycosaminoglycans for signaling from their respective receptors. Since syndecans, a family of cell-surface transmembrane heparan sulfate proteoglycans (HSPGs) are implicated in FGF signaling and skeletal muscle differentiation, we examined the expression of syndecans 1-4 in embryonic, fetal, postnatal, and adult muscle tissue, as well as on primary adult muscle fiber cultures. We show that syndecan-1, -3, and -4 are expressed in developing skeletal muscle tissue and that syndecan-3 and -4 expression is highly restricted in adult skeletal muscle to cells retaining myogenic capacity. These two HSPGs appear to be expressed exclusively and universally on quiescent adult satellite cells in adult skeletal muscle tissue, suggesting a role for HSPGs in satellite cell maintenance or activation. Once activated, all satellite cells maintain expression of syndecan-3 and syndecan-4 for at least 96 h, also implicating these HSPGs in muscle regeneration. Inhibition of HSPG sulfation by treatment of intact myofibers with chlorate results in delayed proliferation and altered MyoD expression, demonstrating that heparan sulfate is required for proper progression of the early satellite cell myogenic program. These data suggest that, in addition to providing potentially useful new markers for satellite cells, syndecan-3 and syndecan-4 may play important regulatory roles in satellite cell maintenance, activation, proliferation, and differentiation during skeletal muscle regeneration.     

Syndecans-2 and -4; close cousins, but not identical twins

The vertebrate syndecans, which make up a four-member family of small type I transmembrane heparan sulfate proteoglycans, constitute evolutionarily conserved family proteins. In particular, sequences in the transmembrane and cytoplasmic domains are a unifying feature within the family. However, the extracellular domain sequences are molecule-specific, implying that different syndecans have evolved to carry out similar, but non-identical, functions. While all four syndecans have been implicated in regulation of the cytoskeleton, their roles are clearly complex. Recent developments indicate that the closely related syndecan-2 and -4 have separable functions, though both bind a number of ligands through their heparan sulfate chains. The specification of these activities is probably core protein related, but is it due to a distinct expression pattern or molecule-specific regulatory mechanisms? Although there is not yet enough data to provide unambiguous answers, here we shall review the known functions and regulatory mechanisms of syndecan-2 and -4.     

Syndecans in tumor cell adhesion and signaling

Anchorage of cells to "heparin" – binding domains that are prevalent in extracellular matrix (ECM) components is thought to occur primarily through the syndecans, a four-member family of transmembrane heparan sulfate proteoglycans that communicate environmental cues from the ECM to the cytoskeleton and the signaling apparatus of the cell. Known activities of the syndecans trace to their highly conserved cytoplasmic domains and to their heparan sulfate chains, which can serve to regulate the signaling of growth factors and morphogens. However, several emerging studies point to critical roles for the syndecans' extracellular protein domains in tumor cell behavior to include cell adhesion and invasion. Although the mechanisms of these activities remain largely unknown, one possibility involves "co-receptor" interactions with integrins that may regulate integrin function and the cell adhesion-signaling phenotype. Thus, alterations in syndecan expression, leading to either overexpression or loss of expression, both of which take place in tumor cells, may have dramatic effects on tumor cell invasion.     

Syndecan-2 expression in colorectal cancer-derived HT-29 M6 epithelial cells induces a migratory phenotype

Members of the heparan sulfate proteoglycan family, the syndecans have emerged as integrators of extracellular signals, such as ECM components or growth factors, that activate cytoplasmic signaling cascades and regulate cytoskeletal functions. Specifically, syndecan-2 has been implicated in various cellular processes, from differentiation to migration, including its participation in cell-cell and cell-matrix adhesion. Here, we focused on the involvement of syndecan-2 in epithelial versus mesenchymal differentiation. Colorectal cancer-derived HT-29 M6 epithelial cells were stably transfected with full-length syndecan-2 cDNA, and the effect on cell morphology, adhesion, and mobility was evaluated. Characteristic features of migratory cells such as loss of intercellular contacts, flatter shape and multiple membrane projections were observed in syndecan-2 transfectants. Western blot analysis of the major component of epithelial adherens junctions, E-cadherin, revealed decreased expression levels. Furthermore, syndecan-2 induced stronger adhesion to collagen type I, specifically inhibited by heparin. This was correlated with an increased ability for migration, as demonstrated by wound healing experiments and transwell assays, without affecting their growth rate. These results indicate that syndecan-2 expression in mucus-secreting HT-29 M6 cells induces differentiation toward a migratory mesenchymal-like phenotype.     

Syndecan-2 induces filopodia by active cdc42Hs

The syndecans, a family of transmembrane heparan sulfate proteoglycans, are ubiquitous molecules whose intracellular function is still unknown. To examine the function of syndecan-2, one of the most abundant heparan sulfate proteoglycan in fibroblasts, we performed transfection studies in COS-1 and Swiss 3T3 cells. Endogenous syndecan-2 colocalized with F-actin in cortical structures. Overexpression of full-length syndecan-2 induced the formation of long filopodia-like structures. These changes correlated with a rearrangement of the actin cytoskeleton, which strongly colocalized with syndecan-2. Overexpression of syndecan-2 lacking the extracellular domain increased the number of microspikes on the cell surface but failed to induce filopodia. Addition of heparin blocked the effect of full-length syndecan-2, suggesting that heparan sulfate chains in the extracellular domain are necessary to induce filopodia. Coexpression of cdc42Hs negative-dominant N17 blocked syndecan-2-induced filopodia and cdc42Hs positive-dominant V12 had a synergic effect. This indicates that active cdc42Hs is necessary for syndecan-2 induction of filopodia. These results provide a link between syndecan-2, actin cytoskeleton, and cdc42Hs.     

Chondroitin sulfate chains on syndecan-1 and syndecan-4 from normal murine mammary gland epithelial cells are structurally and functionally distinct and cooperate with heparan sulfate chains to bind growth factors. A novel function to control binding of midkine, pleiotrophin, and basic fibroblast growth factor

A comparative analysis was carried out of heparan sulfate (HS) and chondroitin sulfate (CS) chains of the ectodomains of hybrid type transmembrane proteoglycans, syndecan-1 and -4, synthesized simultaneously by normal murine mammary gland epithelial cells. Although the HS chains were structurally indistinguishable, intriguingly the CS chains were structurally and functionally distinct, probably reflecting the differential regulation of sulfotransferases involved in the synthesis of HS and CS. The CS chains of the two syndecans comprised nonsulfated, 4-O-, 6-O-, and 4,6-O-disulfated N-acetylgalactosamine-containing disaccharide units and were significantly different, with a higher degree of sulfation for syndecan-4. Functional analysis using a BIAcore system showed that basic fibroblast growth factor (bFGF) specifically bound only to the HS chains of both syndecans, whereas midkine (MK) and pleiotrophin (PTN) bound not only to the HS but also to the CS chains. Stronger binding of MK and PTN to the CS chains of syndecan-4 than those of syndecan-1 was revealed, supporting the structural and functional differences. Intriguingly, removal of the CS chains decreased the association and dissociation rate constants of MK, PTN, and bFGF for both syndecans, suggesting the simultaneous binding of these growth factors to both types of chains, producing a ternary complex that transfers the growth factors to the corresponding cell surface receptors more efficiently compared with the HS chains alone. The involvement of the core protein was also shown in the binding of MK and PTN to syndecan-1, suggesting the possibility of cooperation with the HS and/or CS chains in the binding of these growth factors and their delivery to the cell surface receptors.     

A novel function of syndecan-2, suppression of matrix metalloproteinase-2 activation, which causes suppression of metastasis

The syndecans comprise a family of cell surface heparan sulfate proteoglycans exhibiting complex biological functions involving the interaction of heparan sulfate side chains with a variety of soluble and insoluble heparin-binding extracellular ligands. Here we demonstrate an inverse correlation between the expression level of syndecan-2 and the metastatic potential of three clones derived from Lewis lung carcinoma 3LL. This correlation was proved to be a causal relationship, because transfection of syndecan-2 into the higher metastatic clone resulted in the suppression of both spontaneous and experimental metastases to the lung. Although the expression levels of matrix metalloproteinase-2 (MMP-2) and its cell surface activators, such as membrane-type 1 matrix metalloproteinase and tissue inhibitor of metalloproteinase-2, were similar regardless of the metastatic potentials of the clones, elevated activation of MMP-2 was observed in the higher metastatic clone. Removal of heparan sulfate from the cell surface of low metastatic cells by treatment with heparitinase-I promoted MMP-2 activation, and transfection of syndecan-2 into highly metastatic cells suppressed MMP-2 activation. Furthermore, transfection of mutated syndecan-2 lacking glycosaminoglycan attachment sites into highly metastatic cells did not have any suppressive effect on MMP-2 activation, suggesting that this suppression was mediated by the heparan sulfate side chains of syndecan-2. Actually, MMP-2 was found to exhibit a strong binding ability to heparin, the dissociation constant value being 62 nM. These results indicate a novel function of syndecan-2, which acts as a suppressor for MMP-2 activation, causing suppression of metastasis in at least the metastatic system used in the present study.     

Protein kinase C regulates the recruitment of syndecan-4 into focal contacts.

Cell surface heparan sulfate proteoglycans have been implicated as co-receptors facilitating cell adhesion and growth factor binding. Recent studies on the role of a family of transmembrane heparan sulfate proteoglycans, syndecans, in cell adhesion has identified one member, syndecan-4, to be present within focal contacts. The current study investigates the mechanisms regulating the association of syndecan-4 with focal contacts based upon its immunolocalization with vinculin in quiescent, serum-stimulated, and 12-0-tetradecanoylphorbol 13-acetate (TPA)-induced cultures. In quiescent cells, syndecan-4 did not localize to focal contacts. However, activation of protein kinase C by TPA or serum induces the active recruitment of syndecan-4 into focal contacts. This induction preferentially localizes syndecan-4 to focal contacts behind the leading lamella, the subnuclear region, and along the trailing edge of migratory cells. Focal contacts in either freshly adhered cells or in the leading lamellae of migrating cells did not stain for syndecan-4. In addition to the observed subcellular distribution and recruitment, syndecan-4 was observed to co-localize with endogenously synthesized fibronectin fibrils within focal contacts as well as with fibrils present in the matrix. These findings suggest that protein kinase C activation results in syndecan-4 recruitment to focal contacts and its association with sites of matrix deposition.     

Syndecan-1 expression in epithelial cells is induced by transforming growth factor beta through a PKA-dependent pathway

Syndecans comprise a major family of cell surface heparan sulfate proteoglycans (HSPGs). Syndecans bind and modulate a wide variety of biological molecules through their heparan sulfate (HS) moiety. Although all syndecans contain the ligand binding HS chains, they likely perform specific functions in vivo because their temporal and spatial expression patterns are different. However, how syndecan expression is regulated has yet to be clearly defined. In this study, we examined how syndecan-1 expression is regulated in epithelial cells. Our results showed that among several bioactive agents tested, only forskolin and three isoforms of TGFbeta (TGFbeta1-TGFbeta3) significantly induced syndecan-1, but not syndecan-4, expression on various epithelial cells. Steady-state syndecan-1 mRNA was not increased by TGFbeta treatment and cycloheximide did not inhibit syndecan-1 induction by TGFbeta, indicating that TGFbeta induces syndecan-1 in a post-translational manner. However, TGFbeta induction of syndecan-1 was inhibited by transient expression of a dominant-negative construct of protein kinase A (PKA) and by specific inhibitors of PKA. Further (i) syndecan-1 cytoplasmic domains were Ser-phosphorylated when cells were treated with TGFbeta and this was inhibited by a PKA inhibitor, (ii) PKA was co-immunoprecipitated from cell lysates by anti-syndecan-1 antibodies, (iii) PKA phosphorylated recombinant syndecan-1 cytoplasmic domains in vitro, and (iv) expression of a syndecan-1 construct with its invariant Ser(286) replaced with a Gly was not induced by TGFbeta. Together, these findings define a regulatory mechanism where TGFbeta signals through PKA to phosphorylate the syndecan-1 cytoplasmic domain and increases syndecan-1 expression on epithelial cells.     

Transmembrane proteoglycans control stretch-activated channels to set cytosolic calcium levels

Transmembrane heparan sulfate proteoglycans regulate multiple aspects of cell behavior, but the molecular basis of their signaling is unresolved. The major family of transmembrane proteoglycans is the syndecans, present in virtually all nucleated cells, but with mostly unknown functions. Here, we show that syndecans regulate transient receptor potential canonical (TRPCs) channels to control cytosolic calcium equilibria and consequent cell behavior. In fibroblasts, ligand interactions with heparan sulfate of syndecan-4 recruit cytoplasmic protein kinase C to target serine714 of TRPC7 with subsequent control of the cytoskeleton and the myofibroblast phenotype. In epidermal keratinocytes a syndecan–TRPC4 complex controls adhesion, adherens junction composition, and early differentiation in vivo and in vitro. In Caenorhabditis elegans, the TRPC orthologues TRP-1 and -2 genetically complement the loss of syndecan by suppressing neuronal guidance and locomotory defects related to increases in neuronal calcium levels. The widespread and conserved syndecan–TRPC axis therefore fine tunes cytoskeletal organization and cell behavior.     

Removal of syndecan-1 promotes TRAIL-induced apoptosis in myeloma cells

Syndecan is the major transmembrane proteoglycan in cells. Of the four syndecans, syndecan-1 is the dominant form expressed in multiple myeloma and is an indicator of poor prognosis. In the current study, we observed that early TRAIL-induced apoptotic processes were accompanied by cleavage of syndecan-1 intracellular region, and explored the possibility whether removal of syndecan-1 promotes apoptotic processes. We found that syndecan-1 knockdown by specific small interfering RNA in multiple myeloma enhanced TRAIL-induced apoptosis, even though the expression of TRAIL receptors and several apoptosis-associated molecules was unaffected. The enhanced TRAIL-mediated apoptosis in syndecan-1-deficient cells was not due to a decrease in surface heparan sulfate or a reduction in TRAIL receptor endocytosis. The increase in TRAIL-induced cell death was accompanied by an elevated caspase-8 activation and an enhanced formation of death-inducing signaling complexes, which could be attributed to an increased expression of TRAIL receptor O-glycosylation enzyme in syndecan-1-deficient cells. We also found that in H9 lymphoma and Jurkat cells, knockdown of the predominant syndecan member also led to an increase in Fas ligand-induced apoptosis. Our results demonstrate that syndecan plays a negative role in death receptor-mediated cell death, suggesting potential application of syndecan downregulation in the treatment of myeloma in combination with TRAIL.     

Augmented synthesis and differential localization of heparan sulfate proteoglycans in Duchenne muscular dystrophy

Muscular dystrophies are characterized by continuous cycles of degeneration and regeneration that result in extensive fibrosis and a progressive diminution of muscle mass. Cell surface heparan sulfate proteoglycans are found almost ubiquitously on the surface and in the extracellular matrix (ECM) of mammalian cells. These macromolecules interact with a great variety of ligands, including ECM constituents, adhesion molecules, and growth factors. In this study, we evaluated the expression and localization of three heparan sulfate proteoglycans in the biopsies of Duchenne muscular dystrophy (DMD) patients. Through SDS-PAGE analyses followed by specific identification of heparitinase-digested proteins with an anti-Delta-heparan sulfate specific monoclonal antibodies, we observed an increase of three forms of heparan sulfate proteoglycans, corresponding to perlecan, syndecan-3, and glypican-1. Immunohistochemistry analyses indicated a differential localization for these proteoglycans: glypican-1 and perlecan were found mainly associated to ECM structures, while syndecan-3 was associated to muscle fibers. These results suggest that the amount of specific heparan sulfate proteoglycans is augmented in skeletal muscle in DMD patients presenting a differential localization.     

The Conserved Phenylalanine in the Transmembrane Domain Enhances Heteromeric Interactions of Syndecans

The transmembrane domain (TMD) of the syndecans, a family of transmembrane heparin sulfate proteoglycans, is involved in forming homo- and heterodimers and oligomers that transmit signaling events. Recently, we reported that the unique phenylalanine in TMD positively regulates intramolecular interactions of syndecan-2. Besides the unique phenylalanine, syndecan-2 contains a conserved phenylalanine (SDC2-Phe-169) that is present in all syndecan TMDs, but its function has not been determined. We therefore investigated the structural role of SDC2-Phe-169 in syndecan TMDs. Replacement of SDC2-Phe-169 by tyrosine (S2F169Y) did not affect SDS-resistant homodimer formation but significantly reduced SDS-resistant heterodimer formation between syndecan-2 and -4, suggesting that SDC2-Phe-169 is involved in the heterodimerization/oligomerization of syndecans. Similarly, in an in vitro binding assay, a syndecan-2 mutant (S2(F169Y)) showed a significantly reduced interaction with syndecan-4. FRET assays showed that heteromolecular interactions between syndecan-2 and -4 were reduced in HEK293T cells transfected with S2(F169Y) compared with syndecan-2. Moreover, S2(F169Y) reduced downstream reactions mediated by the heterodimerization of syndecan-2 and -4, including Rac activity, cell migration, membrane localization of PKCα, and focal adhesion formation. The conserved phenylalanine in syndecan-1 and -3 also showed heterodimeric interaction with syndecan-2 and -4. Taken together, these findings suggest that the conserved phenylalanine in the TMD of syndecans is crucial in regulating heteromeric interactions of syndecans.     

Heparan sulfate chains of syndecan-1 regulate ectodomain shedding

Matrix metalloproteinases release intact syndecan-1 ectodomains from the cell surface giving rise to a soluble, shed form of the proteoglycan. Although it is known that shed syndecan-1 controls diverse pathophysiological responses in cancer, wound healing, inflammation, infection, and immunity, the mechanisms regulating shedding remain unclear. We have discovered that the heparan sulfate chains present on syndecan core proteins suppress shedding of the proteoglycan. Syndecan shedding is dramatically enhanced when the heparan sulfate chains are enzymatically degraded or absent from the core protein. Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparan sulfate must be attached to the core protein to suppress shedding. Regulation of shedding by heparan sulfate occurs in multiple cell types, for both syndecan-1 and syndecan-4 and in murine and human syndecans. Mechanistically, the loss of heparan sulfate enhances the susceptibility of the core protein to proteolytic cleavage by matrix metalloproteinases. Enhanced shedding of syndecan-1 following loss of heparan sulfate is accompanied by a dramatic increase in core protein synthesis. This suggests that in response to an increase in the rate of shedding, cells attempt to maintain a significant level of syndecan-1 on the cell surface. Together these data indicate that the amount of heparan sulfate present on syndecan core proteins regulates both the rate of syndecan shedding and core protein synthesis. These findings assign new functions to heparan sulfate chains, thereby broadening our understanding of their physiological importance and implying that therapeutic inhibition of heparan sulfate degradation could impact the progression of some diseases.     

Identification of an invasion regulatory domain within the core protein of syndecan-1

Among the four members of the syndecan family there exists a high level of divergence in the ectodomain core protein sequence. This has led to speculation that these core proteins bear important functional domains. However, there is little information regarding these functions, and thus far, the biological activity of syndecans has been attributed largely to their heparan sulfate chains. We have previously demonstrated that cell surface syndecan-1 inhibits invasion of tumor cells into three-dimensional gels composed of type I collagen. Inhibition of invasion is dependent on the syndecan heparan sulfate chains, but a role for the syndecan-1 ectodomain core protein was also indicated. To more closely examine this possibility and to map the regions of the ectodomain essential for syndecan-1-mediated inhibition of invasion, a panel of syndecan-1 mutational constructs was generated, and each construct was transfected individually into myeloma tumor cells. The anti-invasive effect of syndecan-1 is dramatically reduced by deletion of an ectodomain region close to the plasma membrane. Further mutational analysis identified a stretch of 5 hydrophobic amino acids, AVAAV (amino acids 222-226), critical for syndecan-1-mediated inhibition of cell invasion. This invasion regulatory domain is 26 amino acids from the start of the transmembrane domain. Importantly, this domain is functionally specific because its mutation does not affect syndecan-1-mediated cell binding to collagen, syndecan-1-mediated cell spreading, or targeting of syndecan-1 to specific cell surface domains. This invasion regulatory domain may play an important role in inhibiting tumor cell invasion, thus explaining the observed loss of syndecan-1 in some highly invasive cancers.     

Trans-regulation of Syndecan Functions by Hetero-oligomerization

Syndecans, a family of transmembrane heparansulfate proteoglycans, are known to interact through their transmembrane domains to form non-covalently linked homodimers, a process essential for their individual functions. Because all syndecan transmembrane domains are highly conserved and thus might mediate interactions between different members of the syndecan family, we investigated syndecan interactions in detail. All recombinant syndecan-2 and -4 protein variants containing the transmembrane domain formed not only sodium dodecyl sulfate (SDS)-resistant homodimers but also SDS-resistant heterodimers. Biochemical and structural data revealed that recombinant syndecan-2 and -4 formed intermolecular interactions in vitro, and the GXXXG motif in transmembrane domain mediated this interaction. When exogenously expressed in rat embryonic fibroblasts, syndecan-2 interacted with syndecan-4 and vice versa. Furthermore, bimolecular fluorescence complementation-based assay demonstrated specific hetero-molecular interactions between syndecan-2 and -4, supporting hetero-oligomer formation of syndecans in vivo. Interestingly, hetero-oligomerization significantly reduced syndecan-4-mediated cellular processes such as protein kinase Cα activation and protein kinase Cα-mediated cell adhesion as well as syndecan-2-mediated tumorigenic activities in colon cancer cells such as migration and anchorage-independent growth. Taken together, these data provide evidence that hetero-oligomerization produces distinct syndecan functions and offer insights into the underlying signaling mechanisms of syndecans.     

Unlocking the secrets of syndecans: transgenic organisms as a potential key

Heparan sulfate proteoglycans are known to modulate the activity of a large number of extracellular ligands thereby having the potential to regulate a great diversity of biological processes. The long-term studies in our laboratory have focused on the syndecans, one of the major cell surface heparan sulfate proteoglycan families. Most early work on syndecans involved biochemical studies that provided initial information on their structure and putative biological roles. In recent years, the development of transgenic organisms has allowed a more complete understanding of syndecan function. Studies with transgenic syndecan-1 and syndecan-3 mice have demonstrated an unforeseen role for syndecans in the regulation of feeding behavior. Syndecan-1 knockout mice display a reduced susceptibility to both Wnt-induced tumorigenesis and microbial pathogenesis. Experiments with Drosophila show that syndecan is first expressed upon cellularization in the early embryo, and may play a role in the early developmental stages of the fly. This review focuses on these diverse functions of the syndecans that have been elucidated by the use of transgenic mice and Drosophila as model systems. Published in 2003.