Nodes of Ranvier correspond to specialized axonal domains where voltage-gated sodium channels are highly concentrated. In the peripheral nervous system, they are covered by Schwann cells microvilli, where three homologous cytoskeletal-associated proteins, ezrin, radixin and moesin (ERM proteins) are enriched. These glial processes are thought to play a crucial role in organizing axonal nodal domains during development. However, little is known about the molecules present in Schwann cell processes that could mediate axoglial interactions. The aim of this study is to identify by immunocytochemistry transmembrane proteins enriched in Schwann cells processes that could interact, directly or indirectly, with axonal proteins. Syndecan-3 (S3) and syndecan-4 (S4), two proteoglycans expressed in Schwann cells, are enriched in perinodal processes in rat sciatic nerves. S3 labeling is localized in close vicinity of sodium channels as early as post-natal day 2, and highly concentrated at nodes of Ranvier in the adult. S4 immunoreactivity accumulates at nodes later, and is also prominent in internodal regions of myelinated fibers. Both S3 and S4 co-localize with ezrin in perinodal processes. These data identify S3 and S4 as transmembrane proteins specifically enriched in Schwann cell perinodal processes, and suggest that S3 may be involved in early axoglial interactions during development (Goutebroze, 2003).
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 (Deepa, 2004).
The transmembrane heparan sulfate proteoglycan syndecan-1 was identified from a human placenta cDNA library by the expression cloning method as a gene product that interacts with membrane type matrix metalloproteinase-1 (MT1-MMP). Co-expression of MT1-MMP with syndecan-1 in HEK293T cells promotes syndecan-1 shedding, and concentration of cell-associated syndecan-1 is reduced. Treatment of cells with MMP inhibitor BB-94 or tissue inhibitor of MMP (TIMP)-2 but not TIMP-1 interferes with the syndecan-1 shedding promoted by MT1-MMP expression. In contrast, syndecan-1 shedding induced by 12-O-tetradecanoylphorbol-13-acetate treatment is inhibited by BB-94 but not by either TIMP-1 or TIMP-2. Shedding of syndecan-1 is also induced by MT3-MMP but not by other MT-MMPs. Recombinant syndecan-1 core protein is cleaved by recombinant MT1-MMP or MT3-MMP preferentially at the Gly245-Leu246 peptide bond. HT1080 fibrosarcoma cells stably transfected with the syndecan-1 cDNA (HT1080/SDC), that express endogenous MT1-MMP, spontaneously shed syndecan-1. Migration of HT1080/SDC cells on collagen-coated dishes is significantly slower than that of control HT1080 cells. Treatment of HT1080/SDC cells with BB-94 or TIMP-2 induces accumulation of syndecan-1 on the cell surface, concomitant with further retardation of cell migration. Substitution of Gly245 of syndecan-1 with Leu significantly reduces shedding from HT1080/SDC cells and cell migration. These results suggest that the shedding of syndecan-1 promoted by MT1-MMP through the preferential cleavage of Gly245-Leu246 peptide bond stimulates cell migration (Endo, 2003).
The syndecan family of cell surface heparan sulfate proteoglycans interacts via their cytoplasmic C-terminal tail with the PDZ domain of CASK/LIN-2, a membrane-associated guanylate kinase homolog. The syndecan-CASK interaction may be involved in intercellular signaling and/or cell adhesion. Syndecan-1 to syndecan-4 have distinctive mRNA distributions in adult rat brain by in situ hybridization, with syndecan-2 and -3 being the major syndecans expressed in neurons of the forebrain. At the protein level, syndecan-2 and -3 are differentially localized within neurons; syndecan-3 is concentrated in axons, whereas syndecan-2 is localized in synapses. The synaptic accumulation of syndecan-2 occurs late in synapse development. CASK is a cytoplasmic-binding partner for syndecans, and its subcellular distribution changes strikingly during development, shifting from a primarily axonal distribution in the first 2 postnatal weeks to a somatodendritic distribution in adult brain. This change in CASK distribution correlates temporally and spatially with the expression patterns of syndecan-3 and -2, consistent with the association of both of these syndecans with CASK in vivo. In support of this, a complex of CASK and syndecan-3 was coimmunoprecipitated from brain extracts. These results indicate that specific syndecans are differentially expressed in various cell types of the brain and are targeted to distinct subcellular compartments in neurons, where they may serve specialized functions. Moreover, CASK is appropriately expressed and localized to interact with both syndecan-2 and -3 in different compartments of the neuron throughout postnatal development (Hsueh, 1999).
CASK, the rat homolog of a gene (LIN-2) required for vulval differentiation in Caenorhabditis elegans, is expressed in mammalian brain, but its function in neurons is unknown. CASK is distributed in a punctate somatodendritic pattern in neurons. By immunogold EM, CASK protein is concentrated in synapses, but is also present at nonsynaptic membranes and in intracellular compartments. This immunolocalization is consistent with biochemical studies showing the presence of CASK in soluble and synaptosomal membrane fractions and its enrichment in postsynaptic density fractions of rat brain. By yeast two-hybrid screening, a specific interaction was identified between the PDZ domain of CASK and the COOH terminal tail of syndecan-2, a cell surface heparan sulfate proteoglycan (HSPG). The interaction was confirmed by coimmunoprecipitation from heterologous cells. In brain, syndecan-2 localizes specifically at synaptic junctions where it shows overlapping distribution with CASK, consistent with an interaction between these proteins in synapses. Cell surface HSPGs can bind to extracellular matrix proteins, and are required for the action of various heparin-binding polypeptide growth/differentiation factors. The synaptic localization of CASK and syndecan suggests a potential role for these proteins in adhesion and signaling at neuronal synapses (Hsueh, 1998).
The formation of distinctive basic FGF-heparan sulfate complexes is essential for the binding of bFGF to its cognate receptor. In previous experiments, cell-surface heparan sulfate proteoglycans extracted from human lung fibroblasts could not be shown to promote high affinity binding of bFGF when added to heparan sulfate-deficient cells that express FGF receptor-1 (FGFR1). In alternative tests to establish whether cell-surface proteoglycans can support the formation of the required complexes, K562 cells were first transfected with the IIIc splice variant of FGFR1 and then transfected with constructs coding for either syndecan-1, syndecan-2, syndecan-4 or glypican, or with an antisense syndecan-4 construct. Cells cotransfected with receptor and proteoglycan show a two- to three- fold increase in neutral salt-resistant specific 125I-bFGF binding in comparison to cells transfected with only receptor or cells cotransfected with receptor and anti-syndecan-4. Exogenous heparin enhances the specific binding and affinity cross-linking of 125I-bFGF to FGFR1 in receptor transfectants that are not cotransfected with proteoglycan, but has no effect on this binding and decreases the yield of bFGFR cross-links in cells that are cotransfected with proteoglycan. Receptor-transfectant cells show a decrease in glycophorin A expression when exposed to bFGF. This suppression is dose-dependent and obtained at significantly lower concentrations of bFGF in proteoglycan-cotransfected cells. Complementary cell-free binding assays indicate that the affinity of 125I-bFGF for an immobilized FGFR1 ectodomain is increased threefold when the syndecan-4 ectodomain is coimmobilized with receptor. Equimolar amounts of soluble syndecan-4 ectodomain, in contrast, have no effect on this binding. It is concluded that, at least in K562 cells, syndecans and glypican can support bFGF-FGFR1 interactions and signaling, and that cell-surface association may augment their effectiveness (Steinfeld, 1996).
The outgrowth of the mesoderm of the developing limb bud in response to the apical ectodermal ridge (AER) is mediated at least in part by members of the FGF family. Recent studies have indicated that FGFs need to interact with heparin sulfate proteoglycans in order to bind to and activate their specific cell surface receptors. Syndecan-3 is an integral membrane heparin sulfate proteoglycan that is highly expressed by the distal mesodermal cells of the chick limb bud that are undergoing proliferation and outgrowth in response to the AER. Maintenance of high-level syndecan-3 expression by the subridge mesoderm of the chick limb bud is directly or indirectly dependent on the AER, since its expression is severely impaired in the distal mesoderm of the limb buds of limbless and wingless mutant embryos, which lack functional AERs capable of directing the outgrowth of limb mesoderm. Exogenous FGF-2 maintains a domain of high-level syndecan-3 expression in the outgrowing mesodermal cells of explants of the posterior mesoderm of normal limb buds cultured in the absence of the AER and in the outgrowing subapical mesoderm of explants of limbless mutant limb buds, which lack a functional AER. These results suggest that the domain of high-level syndecan-3 expression in the subridge mesoderm of normal limb buds is maintained by FGFs produced by the AER. Polyclonal antibodies against a syndecan-3 fusion protein inhibit the ability of FGF-2 to promote the proliferation and outgrowth of the posterior subridge mesoderm of limb buds cultured in the absence of the AER. These results suggest that syndecan-3 plays an essential role in limb outgrowth by mediating the interaction of FGFs produced by the AER with the underlying mesoderm of the limb bud (Dealy, 1997).
Syndecan-4 is a heparan sulfate-carrying core protein that has been directly implicated in fibroblast growth factor 2 (FGF2) signaling. Recent studies have suggested that many signaling proteins localize to the raft compartment of the plasma cell membrane. To establish whether syndecan-4 is present in the raft compartment, the distribution has been studied of the core protein and an Fc receptor (FcR)-syndecan-4 chimera prior to and following clustering with FGF2 or antibodies. Whereas unclustered syndecan-4 was present predominantly in the non-raft membrane compartment, clustering induced extensive syndecan-4 redistribution to the rafts, as demonstrated by the sucrose gradient centrifugation and live confocal microscopy. Although syndecan-4 and caveolin-1 moved in tandem, syndecan-4 was not present in caveolae, a major subset of raft compartments. It is concluded that syndecan-4 clustering induces its redistribution to the non-caveolae raft compartment. This process may play an important role in syndecan-4-mediation of FGF2 signaling (Tkachenko, 2002).
Full activity of fibroblast growth factors (FGFs) requires their internalization in addition to the interaction with cell surface receptors. Recent studies have suggested that the transmembrane proteoglycan syndecan-4 functions as a FGF2 receptor. The molecular basis of syndecan endocytosis and its role in FGF2 internalization was investigated in endothelial cells. Syndecan-4 uptake, induced either by treatment with FGF2 or by antibody clustering, requires the integrity of plasma membrane lipid rafts for its initiation, occurs in a non-clathrin-, non-dynamin-dependent manner and involves Rac1, which is activated by syndecan-4 clustering. FGF2 was internalized in a complex with syndecan-4 in 70 kDa dextran-containing endocytic vesicles. FGF2 and syndecan-4 but not dextran endocytosis were blocked by the dominant negative Rac1, while amiloride and the dominant-negative Cdc42 blocked internalization of dextran in addition to FGF2 and syndecan-4. Taken together, these results demonstrate that FGF2 endocytosis requires syndecan-4 clustering-dependent activation of Rac1 and the intact CDC42-dependent macropinocytic pathway (Tkachenko, 2004).
The alpha(v)beta(3) integrin participates in cell morphogenesis, growth factor signaling, and cell survival. Activation of the integrin is central to these processes and is influenced by specific ECM components, which engage both integrins and syndecans. The alpha(v)beta(3) integrin and syndecan-1 (S1) are functionally coupled. The integrin is dependent on the syndecan to become activated and to mediate signals required for MDA-MB-231 and MDA-MB-435 human mammary carcinoma cell spreading on vitronectin or S1-specific antibody. Coupling of the syndecan to alpha(v)beta(3) requires the S1 ectodomain (ED); ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances alpha(v)beta(3) recognition of vitronectin, and treatments that target this domain, including competition with recombinant S1ED protein or anti-S1ED antibodies, mutation of the S1ED, or down-regulation of S1 expression by small-interfering RNAs, disrupt alpha(v)beta(3)-dependent cell spreading and migration. Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated alpha(v)beta(3) integrins (Beuvais, 2004).
TGF-beta has multiple functions including increasing extracellular matrix deposition in fibrosis. It functions through a complex family of cell surface receptors that mediate downstream signaling. A transmembrane heparan sulfate proteoglycan, syndecan-2 (S2), can regulate TGF-beta signaling. S2 protein increases in the renal interstitium in diabetes and regulates TGF-beta-mediated increased matrix deposition in vitro. Transfection of renal papillary fibroblasts with S2 or a S2 construct that has a truncated cytoplasmic domain (S2DeltaS) promotes TGF-beta binding and S2 core protein ectodomain directly binds TGF-beta. Transfection with S2 increased the amounts of type I and type II TGF-beta receptors (TbetaRI and TbetaRII), whereas S2DeltaS was much less effective. In contrast, S2DeltaS dramatically increases the level of type III TGF-beta receptor (TbetaRIII), betaglycan, whereas S2 results in a decrease. Syndecan-2 specifically co-immunoprecipitates with betaglycan but not with TbetaRI or TbetaRII. This is a novel mechanism of control of TGF-beta action that may be important in fibrosis (Chen, 2004).
N-syndecan (syndecan-3) is a cell surface receptor for heparin-binding growth-associated molecule (HB-GAM) and is suggested to mediate the neurite growth-promoting signal from cell matrix-bound HB-GAM to the cytoskeleton of neurites. However, it is unclear whether N-syndecan would possess independent signaling capacity in neurite growth or in related cell differentiation phenomena. N18 neuroblastoma cells were transfected with a rat N-syndecan cDNA and it was shown that N-syndecan transfection clearly enhances HB-GAM-dependent neurite growth and that the transfected N-syndecan distributes to the growth cones and the filopodia of the neurites. The N-syndecan-dependent neurite outgrowth is inhibited by the tyrosine kinase inhibitors herbimycin A and PP1. Biochemical studies show that a kinase activity, together with its substrate(s), binds specifically to the cytosolic moiety of N-syndecan immobilized to an affinity column. Western blotting reveals both c-Src and Fyn in the active fractions. In addition, cortactin, tubulin, and a 30-kDa protein are identified in the kinase-active fractions that bind to the cytosolic moiety of N-syndecan. Ligation of N-syndecan in the transfected cells by HB-GAM increases phosphorylation of c-Src and cortactin. It is suggested that N-syndecan binds a protein complex containing Src family tyrosine kinases and their substrates and that N-syndecan acts as a neurite outgrowth receptor via the Src kinase-cortactin pathway (Kinnunen, 1998).
Using an affinity matrix in which a recombinant glypican-Fc fusion protein expressed in 293 cells was coupled to protein A-Sepharose, at least two proteins were isolated from rat brain that were detected by SDS-polyacrylamide gel electrophoresis as a single 200-kDa silver-stained band, from which 16 partial peptide sequences were obtained by nano-electrospray tandem mass spectrometry. Mouse expressed sequence tags containing two of these peptides were employed for oligonucleotide design and synthesis of probes by polymerase chain reaction and enabled the isolation from a rat brain cDNA library a 4.1-kilobase clone that encoded two of the peptide sequences and represented the N-terminal portion of a protein containing a signal peptide and three leucine-rich repeats. Comparisons with recently published sequences also showed that these peptides were derived from proteins that are members of the Slit/MEGF protein family, which share a number of structural features such as N-terminal leucine-rich repeats and C-terminal epidermal growth factor-like motifs, and in Drosophila Slit is necessary for the development of midline glia and commissural axon pathways. All of the five known rat and human Slit proteins contain 1523-1534 amino acids, and the peptide sequences correspond best to those present in human Slit-1 and Slit-2. Binding of these ligands to the glypican-Fc fusion protein requires the presence of the heparan sulfate chains, but the interaction appears to be relatively specific for glypican-1 insofar as no other identified heparin-binding proteins were isolated using the affinity matrix. Northern analysis demonstrated the presence of two mRNA species of 8.6 and 7.5 kilobase pairs using probes based on both N- and C-terminal sequences, and in situ hybridization histochemistry showed that these glypican-1 ligands are synthesized by neurons, such as hippocampal pyramidal cells and cerebellar granule cells, where glypican-1 mRNA and immunoreactivity are found. These results therefore indicate that Slit family proteins are functional ligands of glypican-1 in nervous tissue and suggest that their interactions may be critical for certain stages of central nervous system histogenesis (Liang, 1999).
Slit proteins are a family of secreted guidance proteins that can repel neuronal migration and axon growth via interaction with their cellular roundabout receptors (Robos). Slit2-Robo-1 interactions were enhanced by cell-surface heparan sulfate. Removal of heparan sulfate decreases the affinity of Slit for Robo by about threefold. In addition, removal of cell-surface heparan sulfate by heparinase III abolishes the chemorepulsive response to Slit2 normally shown by both the migrating neurons and growing axons. These results indicate essential roles for cell-surface heparan sulfate in the repulsive activities of Slit2 (Hu, 2001).
Syndecan-4 is a transmembrane heparan sulfate proteoglycan that acts as a coreceptor with integrins in focal adhesion formation. The central region of syndecan-4 cytoplasmic domain (4V; LGKKPIYKK) binds phosphatidylinositol 4,5-bisphosphate, and together they regulate protein kinase C alpha (PKC alpha) activity. Syndecan 4V peptide directly potentiates PKC alpha activity, leading to 'superactivation' of the enzyme, apparently through an interaction with its catalytic domain. Yeast two-hybrid and in vitro binding assays have been performed to determine the interaction sites between 4V and PKC alpha. Full-length PKC alpha weakly interacted with 4V by yeast two-hybrid assays, but PKC alpha constructs that lack the pseudosubstrate region or constructs of the whole catalytic domain interacted more strongly. A mutated 4V sequence [4V(YF): LGKKPIFKK] did not interact with PKC alpha, indicating that tyrosine 192 in the syndecan-4 cytoplasmic domain might be critical for this interaction. Further assays identified a novel interaction site in the C terminus of the catalytic domain of PKC alpha (amino acid sequence 513-672). This encompasses the autophosphorylation sites, which are implicated in activation and stability. Yeast two-hybrid data were confirmed by in vitro binding and coimmunoprecipitation assays. The interaction of syndecan-4 with PKC alpha appears unique since PKC delta and epsilon did not interact with 4V in yeast two-hybrid assays or coimmunoprecipitate with syndecan-4. Finally, overexpression of syndecan-4 in rat embryo fibroblast cells, but not expression of the YF mutant, increased PKC alpha localization to focal adhesions. The data support a mechanism where syndecan-4 binds PKC alpha and localizes it to focal adhesions, whose assembly may be regulated by the kinase (Lim, 2003).
During cell-matrix adhesion, syndecan-4 transmembrane heparan sulphate proteoglycan plays a critical role in the formation of focal adhesions and stress fibers. The syndecan-4 cytoplasmic domain directly binds to and activates PKC-alpha (protein kinase C-alpha) in vitro. However, whether syndecan-4 has the same activity in vivo needs to be addressed. Using mammalian two-hybrid assays, it has been shown that syndecan-4 interacts with PKC-alpha in vivo and that this interaction is mediated through syndecan-4 cytoplasmic domain. Furthermore, the activation of PKC increases the extent of interaction between syndecan-4 and PKC-alpha. Overexpression of syndecan-4, but not a mutant lacking its cytoplasmic domain, specifically increases the level of endogenous PKC-alpha and enhances the translocation of PKC-alpha into both detergent-insoluble and membrane fractions. In addition, rat embryo fibroblasts overexpressing syndecan-4 exhibit a slowed down-regulation of PKC-alpha in response either to a prolonged treatment with PMA or to maintaining cells in suspension culture. PKC-alpha immunocomplex kinase assays also showed that syndecan-4 overexpression increases the activity of membrane PKC-alpha. Taken together, these results suggest that syndecan-4 interacts with PKC-alpha in vivo and regulates its localization, activity and stability (Keum, 2004).
Basal keratinocytes of the epidermis adhere to their underlying basement membrane through a specific interaction with laminin-5, which is composed by the association of alpha3, beta3, and gamma2 chains. Laminin-5 has the ability to induce either stable cell adhesion or migration depending on specific processing of different parts of the molecule. One event results in the cleavage of the carboxyl-terminal globular domains 4 and 5 (LG4/5) of the alpha3 chain. In this study, the human alpha3LG4/5 fragment was recombinantly expressed in mammalian cells, and this fragment was shown to induce adhesion of normal human keratinocytes and fibrosarcoma-derived HT1080 cells in a heparan- and chondroitin-sulfate-dependent manner. Immunoprecipitation experiments with Na2 35SO4-labeled keratinocyte and HT1080 cell lysates as well as immunoblotting experiments revealed that the major proteoglycan receptor for the alpha3LG4/5 fragment is syndecan-1. Syndecan-4 from keratinocytes also bound to alpha3LG4/5. Furthermore unprocessed laminin-5 specifically binds syndecan-1, while processed laminin-5 does not. These results demonstrate that the LG4/5 modules within unprocessed laminin-5 permit its cell binding activity through heparan and chondroitin sulfate chains of syndecan-1 and reinforce previous data suggesting specific properties for the precursor molecule (Okamoto, 2003).
The laminin alpha1 chain G domain has multiple biological activities. Cell binding sequences in the laminin alpha1 chain G domain have been identified by screening 113 synthetic peptide-polystyrene beads for cell attachment activity. A recombinant protein of the laminin alpha1 G domain (rec-alpha1G) and a large set of synthetic peptides were used to further identify and characterize heparin, cell, and syndecan-4 binding sites in the laminin alpha1 chain G domain. The rec-alpha1G protein promotes both cell attachment and heparin binding. Cell attachment to the rec-alpha1G protein was inhibited 60% by heparin and 30% by EDTA. The heparin binding sites were identified by competing heparin binding to the rec-alpha1G protein with 110 synthetic peptides in solution. Only two peptides, AG73 and AG75, inhibited heparin binding to rec-alpha1G. When the peptides were compared in a solid-phase heparin binding assay, AG73 showed more heparin binding than AG75. AG73 also inhibited fibroblast attachment to the rec-alpha1G protein, but AG75 did not. Cell attachment to the peptides was studied using peptide-coated plates and peptide-conjugated sepharose beads. AG73 promoted cell attachment in both assays, but AG75 only showed cell attachment activity in the bead assay. Additionally, AG73, but not AG75, inhibited branching morphogenesis of mouse submandibular glands in organ culture. Furthermore, the rec-alpha1G protein bound syndecan-4, and both AG73 and AG75, inhibited this binding. These results suggest that the AG73 and AG75 sites are important for heparin and syndecan-4 binding in the laminin alpha1 chain G domain. These sites may play a critical role in the diverse biological activities involving heparin and syndecan-4 binding (Suzuki, 2003).
The syndecans play critical roles in several signal transduction pathways. The core proteins of these heparan sulfate proteoglycans are characterized by highly conserved transmembrane and intracellular domains which are required for signaling across the membrane and for interaction with cytosolic proteins. However, regulatory mechanisms controlling these functions remain largely unknown. upon ligand-induced primary proteolytic cleavage within the ectodomain, the intracellular domain of syndecan 3 is released by regulated intramembrane proteolysis. The cleavage is mediated by presenilin/gamma-secretase complex and negatively regulates the plasma membrane targeting of the transcriptional cofactor CASK (Schulz, 2004).
Hedgehog proteins exert critical roles in embryogenesis and require heparan sulfate proteoglycans (HS-PGs) for action. Indian hedgehog (Ihh) is produced by prehypertrophic chondrocytes in developing long bones and regulates chondrocyte proliferation and other events, but it is not known whether it requires HS-PGs for function. Because the HS-PG syndecan-3 is preferentially expressed by proliferating chondrocytes, whether it mediates Ihh action was tested. Primary chick chondrocyte cultures were treated with recombinant Ihh (rIhh-N) in absence or presence of heparinase I or syndecan-3 neutralizing antibodies. While rIhh-N stimulated proliferation in control cultures, it failed to do so in heparinase- or antibody-treated cultures. In reciprocal gain-of-function studies, chondrocytes were made to overexpress syndecan-3 by an RCAS viral vector. Cells became more responsive to rIhh-N, but even this response was counteracted by heparinase or antibody treatment. To complement the in vitro data, RCAS viral particles were microinjected in day 4-5 chick wing buds and effects of syndecan-3 misexpression were monitored over time. Syndecan-3 misexpression led to widespread chondrocyte proliferation and, interestingly, broader expression and distribution of Ihh. In addition, the syndecan-3 misexpressing skeletal elements were short, remained cartilaginous, lacked osteogenesis, and exhibited a markedly reduced expression of collagen X and osteopontin, products characteristic of hypertrophic chondrocytes and bone cells. The data are the first to indicate that Ihh action in chondrocyte proliferation involves syndecan-3 and to identify a specific member of the syndecan family as mediator of hedgehog function (Shimo, 2004).
Syndecan-4 is a transmembrane heparan sulfate proteoglycan that co-operates with integrins during cell-matrix interactions for the assembly of focal adhesions and actin stress fibers and in the phosphorylation of focal adhesion kinase (FAK) on Tyr397. These cellular events are regulated by the small GTPase Rho, and in the absence of syndecan-4 ligation, cellular levels of GTP-bound Rho are decreased, implicating syndecan-4 in the regulation of the small GTPases. Compared with wild type cells, fibronectin-adherent syndecan-4-null fibroblasts showed enhanced lamellipodia and increased Rac1 activity that is down-regulated by re-expression of syndecan-4 in the mutant cells. Consistent with the role for Rac1 in activating p38 and JNK signaling, syndecan-4-null cells display higher levels of active p38 MAPK and JNK that are abolished by the expression of a dominant-negative RacN17 mutant. Since p38 and JNK regulate gene expression by phosphorylating and activating transcription factors, both the phosphorylation state and the transcriptional activity of the ATF-2 transcription factor were compared, as a direct p38 and JNK target in syndecan-4-null and wild type cells. In the absence of syndecan-4, both ATF-2 phosphorylation and transcriptional activity are significantly more elevated compared with wild type cells, and both activities are decreased either by the re-expression of syndecan-4 or by the expression of RacN17. These results reveal a novel function for syndecan-4 in modulating nuclear transcriptional activity and indicate an underlying mechanism that acts at the level of Rac1-p38/JNK signaling (Saoncella, 2004).
In Caenorhabditis elegans, the identification of many enzymes involved in the synthesis and modification of glycosaminoglycans (GAGs), essential components of proteoglycans, has attained special attention in recent years. Mutations in all the genes that encode for GAG biosynthetic enzymes show defects in the development of the vulva, specifically in the invagination of the vulval epithelium. Mutants for certain heparan sulfate modifying enzymes present axonal and cellular guidance defects in specific neuronal classes. Although most of the enzymes involved in the biosynthesis and modification of heparan sulfate have been characterized in C. elegans, little is known regarding the core proteins to which these GAGs covalently bind in proteoglycans. A single syndecan homologue (sdn-1) has been identified in the C. elegans genome through sequence analysis. C. elegans synthesizes sulfated proteoglycans, seen as three distinct species in Western blot analysis. In the sdn-1 deletion mutant allele the lack of one species, which corresponds to a 50 kDa product after heparitinase treatment, is seen. The expression of sdn-1 mRNA and sequencing revealed that sdn-1 deletion mutants lack two glycosylation sites. Hence, the missing protein in the Western blot analysis probably corresponds to SDN-1. In addition, SDN-1 localizes to the C. elegans nerve ring, nerve cords and to the vulva. SDN-1 is found specifically phosphorylated in nerve ring neurons and in the vulva, in both wild-type worms and sdn-1 deletion mutants. These mutants show a defective egg-laying phenotype. The results show the identification, localization and some functional aspects of syndecan in the nematode C. elegans (Minniti, 2004).
During nervous system development, axons that grow out simultaneously in the same extracellular environment are often sorted to different target destinations. Since there is only a restricted set of guidance cues known, regulatory mechanisms are likely to play a crucial role in controlling cell migration and axonal pathfinding. Heparan sulfate proteoglycans (HSPGs) carry long chains of differentially modified sugar residues that have been proposed to encode specific information for nervous system development. This study shows that the cell surface proteoglycan syndecan SDN-1 functions autonomously in neurons to control the neural migration and guidance choices of outgrowing axons. Epistasis analysis suggests that heparan sulfate (HS) attached to SDN-1 can regulate guidance signaling by the Slit/Robo pathway. Furthermore, SDN-1 acts in parallel with other HSPG core proteins whose HS side chains are modified by the C5-epimerase HSE-5, and/or the 2O-sulfotransferase HST-2, depending on the cellular context. Taken together, these experiments show that distinct HS modification patterns on SDN-1 are involved in regulating axon guidance and cell migration in C. elegans (Rhiner, 2005).
Loss of SDN-1 function interferes with the migration of the HSN, CAN and ALM neurons, which, together with Q neuroblasts, are the only groups of neurons that migrate long distances in C. elegans. Because similar ALM migration defects have been reported for mutations in sax-3/Robo, SDN-1 might also modulate Slit/Robo signaling in cell migration, in addition to its role in the regulation of Slit signaling in midline guidance indicated by the data. However, lack of SDN-1 does not perturb cell migration in general; for example, sex myoblast migration is normal in sdn-1(zh20) animals. Furthermore, sdn-1 null mutants exhibit no circumferential distal tip cell (DTC) migration defects. Mutations in the gene encoding perlecan/UNC-52, a basement membrane HSPG, enhance the DTC migration defects of UNC-6/netrin signaling mutants -- an effect that can be partially suppressed by mutations disrupting growth factor-like signaling. Whether SDN-1 also contributes to signaling by EGL-20/WNT, UNC-129/TGF-þ or EGL-17/FGF still needs to be determined (Rhiner, 2005).
The cell surface proteoglycan syndecan-2 can induce dendritic spine formation in hippocampal neurons. The EphB2 receptor tyrosine kinase phosphorylates syndecan-2 and this phosphorylation event is crucial for syndecan-2 clustering and spine formation. Syndecan-2 is tyrosine phosphorylated and forms a complex with EphB2 in mouse brain. Dominant-negative inhibition of endogenous EphB receptor activities blocks clustering of endogenous syndecan-2 and normal spine formation in cultured hippocampal neurons. This is the first evidence that Eph receptors play a physiological role in dendritic spine morphogenesis. These observations suggest that spine morphogenesis is triggered by the activation of Eph receptors: this causes tyrosine phosphorylation of target molecules, such as syndecan-2, in presumptive spines (Ethell, 2001).
Dendritic spines are the principal postsynaptic targets for excitatory synapses. In recent years, these small protrusions on the surface of dendrites have attracted significant interest because changes in their morphology are implicated in synaptic plasticity and long-term memory. Dendritic spines undergo morphological changes in a developmentally regulated and activity-dependent manner. Abnormal spine morphologies have been reported in several neurodevelopmental disorders, including fragile X syndrome. The molecular mechanisms that govern spine morphogenesis are not completely understood, but several different physiological and molecular factors have been shown to affect spine morphology. These factors include synaptic activity and plasticity, actin filament reorganization, calcium dynamics, and protein phosphorylation (Ethell, 2001 and references therein).
Dendritic spine formation occurs during the late stages of development after neuronal connectivity has been established. Before the appearance of mature spines, dendrites exhibit long, thin filopodia-like protrusions without a bulbous head. As the brain matures, these dendritic filopodia disappear, and spines, which typically have mushroom-like and stubby shapes, begin to appear. Primary cultures of rat hippocampal neurons provide an excellent system in which the process of spine formation can be studied in vitro. At 1 week in vitro, these neurons possess predominantly filopodia-like protrusions. Over the next few weeks, these dendritic filopodia gradually decrease in number and are progressively replaced by protrusions that have mushroom-like and stubby shapes. After 3-4 weeks, the majority of the protrusions exhibit mature spine morphologies. The cell surface proteoglycan syndecan-2 has been shown to play a role in spine formation (Ethell, 2001 and references therein).
Syndecan-2 is a member of the syndecan family of transmembrane heparan sulfate proteoglycans. There is increasing evidence that syndecans are involved in transmembrane signaling by interacting with cytoskeletal and signaling molecules. The cytoplasmic domain of syndecans can be subdivided into a highly conserved juxtamembrane segment (C1 region), another conserved segment at the C terminus (C2 region), and a variable segment (V region) located between the C1 and C2 regions. The EFYA (Glu-Phe-Tyr-Ala) sequence at the C terminus serves as the binding site for at least four cytoplasmic proteins, namely syntenin, CASK, synectin, and synbindin. Moreover, the cytoplasmic domain contains 4 tyrosine residues that are conserved among all syndecans. Some of these tyrosine residues are phosphorylated in vitro, and tyrosine phosphorylation has been speculated to play important roles in syndecan-mediated signal transduction (Ethell, 2001 and references therein).
Potential functional roles of syndecan-2 in synapses were first suggested by its interaction with the synaptic PDZ domain protein CASK. Syndecan-2 is clustered at dendritic spines of mature hippocampal neurons in culture and its accumulation occurs concomitant with the morphological maturation of spines. More importantly, transfection of syndecan-2 induces the formation of morphologically mature dendritic spines in immature (8 days in vitro [DIV]) hippocampal neurons. Deletion studies have demonstrated that the C1 and V regions of the syndecan-2 cytoplasmic domain (which contains two potential tyrosine phosphorylation sites) are required for syndecan-2 clustering on dendrites and the induction of mature spines. Based on these data, it is hypothesized that tyrosine phosphorylation of syndecan-2 is the crucial upstream event that leads to dendritic spine formation. This premise then suggests that tyrosine kinase(s) present in dendritic spines play a role in spine formation by phosphorylating syndecan-2 (Ethell, 2001 and references therein).
The Eph family is a large family of receptor tyrosine kinases. Upon stimulation by ephrin ligands, Eph receptors activate signaling cascades in various biological systems. While Eph receptors have been studied primarily in the context of axon guidance during development, there have been suggestions that they may play some roles in synapses in the adult brain. It is speculated that Eph receptors are the kinases involved in the syndecan-2-induced spine formation for several reasons.: (1) syndecan-3, another member of the syndecan family, is tyrosine phosphorylated by recombinant EphB1 in vitro; (2) some Eph receptors interact with syntenin, a syndecan-2 binding PDZ domain protein, and (3) most importantly, some Eph receptors, including EphB2, are present in dendritic spines. These observations have led to this investigation of the possibility that Eph receptors are involved in syndecan-2 phosphorylation during dendritic spine formation (Ethell, 2001 and references therein).
In this paper, it is demonstrated that EphB2 is a crucial tyrosine kinase that phosphorylates syndecan-2 during dendritic spine formation. Furthermore, inhibition of endogenous EphB receptor activities by dominant-negative EphB2 blocks endogenous syndecan-2 clustering and normal spine formation. These results demonstrate a physiological role for EphB2/syndecan-2 signaling in dendritic spine morphogenesis. These findings provide a basis for the role of cell surface ligand-receptor interactions in spine morphogenesis and suggest that the signaling cascade leading to the formation of mature spines is triggered by the activation of Eph receptors by their extracellular ligands (Ethell, 2001).
Skeletal muscle regeneration is a highly complex and regulated process that involves muscle precursor proliferation and differentiation and probably requires the participation of heparin binding growth factors such as FGFs, HGF and TGFbeta. Heparan sulfate proteoglycans, key components of cell-surfaces and ECM, modulate growth factor activities and influence cell growth and differentiation. Their expression in forming muscle masses during development and in cell culture, suggest their participation in the regulation of myogenesis. In the present study, heparan sulfate proteoglycan expression in skeletal muscle regeneration induced by barium chloride injection was evaluated. Expression of muscle differentiation markers and neuromuscular junction (NMJ) components was characterized. Immunoblots with anti-Delta-heparan sulfate antibody showed that four major species -- perlecan, glypican, syndecan-3 and syndecan-4 -- are transiently up-regulated. The first three were detected at the surface or basement membranes of newly formed myotubes by specific indirect immunofluorescence. Syndecan-3, a satellite cell marker, showed the earliest and most significant increase. Experiments involving myoblast grafting into regenerating muscle showed that C2C12 cell clones, with inhibited syndecan-3 expression resulting from antisense transfection, presented a normal proliferation rate but an impaired capacity to fuse and form skeletal muscle fibers. These data constitute the first in vivo evidence suggesting the requirement of a specific heparan sulfate proteoglycan for successful skeletal muscle regeneration (Casar, 2004).
Syndecan-3 and syndecan-4 function as coreceptors for tyrosine kinases and in cell adhesion. Syndecan-3-/- mice exhibit a novel form of muscular dystrophy characterized by impaired locomotion, fibrosis, and hyperplasia of myonuclei and satellite cells. Explanted syndecan-3-/- satellite cells mislocalize MyoD, differentiate aberrantly, and exhibit a general increase in overall tyrosine phosphorylation. Following induced regeneration, the hyperplastic phenotype is recapitulated. While there are fewer apparent defects in syndecan-4-/- muscle, explanted satellite cells are deficient in activation, proliferation, MyoD expression, myotube fusion, and differentiation. Further, syndecan-4-/- satellite cells fail to reconstitute damaged muscle, suggesting a unique requirement for syndecan-4 in satellite cell function (Cornelison, 2004).
Raji cells expressing syndecan-1 (Raji-S1) adhere and spread when plated on heparan sulfate-binding extracellular matrix ligands or monoclonal antibody 281.2, an antibody directed against the syndecan-1 extracellular domain. Cells plated on monoclonal antibody 281.2 initially extend a broad lamellipodium, a response accompanied by membrane ruffling at the cell margin. Membrane ruffling then becomes polarized, leading to an elongated cell morphology. The syndecan-1 cytoplasmic domain is not required for these activities, suggesting important roles for the syndecan-1 transmembrane and/or extracellular domains in the assembly of a signaling complex necessary for spreading. Work described here demonstrates that truncation of the syndecan-1 extracellular domain does not affect the initial lamellipodial extension in the Raji-S1 cells but does inhibit the active membrane ruffling that is necessary for cell polarization. Replacement of the entire syndecan-1 transmembrane domain with leucine residues completely blocks the cell spreading. These data demonstrate that the syndecan-1 transmembrane and extracellular domains have important but distinct roles in Raji-S1 cell spreading; the extracellular domain mediates an interaction that is necessary for dynamic cytoskeletal rearrangements whereas an interaction of the transmembrane domain is required for the initial spreading response (McQuade, 2003).
Wound repair is a tightly regulated process stimulated by proteases, growth factors, and chemokines, which are modulated by heparan sulfate. To characterize further the role of the heparan sulfate proteoglycan syndecan-1 in wound repair, mice were generated overexpressing syndecan-1 (Snd/Snd) and dermal wound repair was studied. Wound closure, reepithelialization, granulation tissue formation, and remodeling were delayed in Snd/Snd mice. Soluble syndecan-1 was increased, and shedding was prolonged in wounds from Snd/Snd mice. Excess syndecan-1 increased the elastolytic activity of wound fluids. Additionally, cells in the granulation tissue and keratinocytes at wound edges showed markedly reduced proliferation rates in Snd/Snd mice. Skin grafting experiments between Snd/Snd and control mice indicated that the slower growth rate was mainly due to a soluble factor in the Snd/Snd mouse skin. Syndecan-1 immunodepletion and further degradation experiments identified syndecan-1 ectodomain as a dominant negative inhibitor of cell proliferation. These studies indicate that shed syndecan-1 ectodomain may enhance proteolytic activity and inhibit cell proliferation during wound repair (Elenius, 2004).
Syndecan-4 is a ubiquitously expressed heparan sulfate proteoglycan that modulates cell interactions with the extracellular matrix. It is transiently up-regulated during tissue repair by cells that mediate wound healing. Syndecan-4 is essential for optimal fibroblast response to the three-dimensional fibrin-fibronectin provisional matrix that is deposited upon tissue injury. Interference with syndecan-4 function inhibits matrix contraction by preventing cell spreading, actin stress fiber formation, and activation of focal adhesion kinase and RhoA mediated-intracellular signaling pathways. Tenascin-C is an extracellular matrix protein that regulates cell response to fibronectin within the provisional matrix. Syndecan-4 is also required for tenascin-C action. Inhibition of syndecan-4 function suppresses tenascin-C activity and overexpression of syndecan-4 circumvents the effects of tenascin-C. In this way, tenascin-C and syndecan-4 work together to control fibroblast morphology and signaling and regulate events such as matrix contraction that are essential for efficient tissue repair (Midwood, 2004).
Syndecan-1 was overexpressed in T47D, MCF-7, or Hs578t human breast carcinoma cell lines, mimicking overexpression observed in carcinomas in vivo. Overexpression of syndecan-1, or its ectodomain alone fused to a glycosylphosphatidylinositol anchor (GPI-mS1ED), promotes cell rounding in 2D culture. Deletions within the syndecan-1 ectodomain (S1ED) implicate an active site within the core protein between the glycosaminoglycan attachment region and the transmembrane domain. Polyclonal antibodies directed against the ectodomain, or treatment with the tyrosine kinase inhibitor genistein, block activity and revert GPI-mS1ED overexpressing cells to a normal morphology. Extracellular matrix (ECM)-dependent signaling appears to be targeted, since GPI-mS1ED cells attach and spread similarly to control cells in response to E-cadherin engagement, but fail to spread on integrin-dependent ligands. However, integrin-dependent cell attachment, and integrin activation and subsequent FAK phosphorylation are unaffected, suggesting that the syndecan regulates the integration of signaling following matrix adhesion. In 3D culture, where syndecan-1 may have a more critical role in cell behavior, the disrupted signaling leads to poorly cohesive, invasive colonies. Thus, altered matrix-dependent signaling due to increased levels of cell surface syndecan-1 may lead to epithelial cell invasion during early stages of tumorigenesis (Burbach, 2004).
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. Cell surface syndecan-1 has been shown to inhibit invasion of tumor cells into three dimensional gels composed of type I collagen. Inhibition of invasion is dependent on syndecan's heparan sulfate chains, but a role for the syndecan-1 ectodomain core protein is 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 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 (aa 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 domains on the cell surface. 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 (Langford, 2004).
Heparanase (HPSE-1) is involved in the degradation of both cell-surface and extracellular matrix (ECM) heparan sulfate (HS) in normal and neoplastic tissues. Degradation of heparan sulfate proteoglycans (HSPG) in mammalian cells is dependent upon the enzymatic activity of HPSE-1, an endo-beta-d-glucuronidase, which cleaves HS using a specific endoglycosidic hydrolysis rather than an eliminase type of action. Elevated HPSE-1 levels are associated with metastatic cancers, directly implicating HPSE-1 in tumor progression. The mechanism of HPSE-1 action to promote tumor progression may involve multiple substrates because HS is present on both cell-surface and ECM proteoglycans. However, the specific targets of HPSE-1 action are not known. Of particular interest is the relationship between HPSE-1 and HSPG, known for their involvement in tumor progression. Syndecan-1, an HSPG, is ubiquitously expressed at the cell surface, and its role in cancer progression may depend upon its degradation. Conversely, another HSPG, perlecan, is an important component of basement membranes and ECM, which can promote invasive behavior. Down-regulation of perlecan expression suppresses the invasive behavior of neoplastic cells in vitro and inhibits tumor growth and angiogenesis in vivo. This work demonstrates the following: (1) HPSE-1 cleaves HS present on the cell surface of metastatic melanoma cells; (2) HPSE-1 specifically degrades HS chains of purified syndecan-1 or perlecan HS; (3) syndecan-1 does not directly inhibit HPSE-1 enzymatic activity; (4) the presence of exogenous syndecan-1 inhibits HPSE-1-mediated invasive behavior of melanoma cells by in vitro chemoinvasion assays; (5) inhibition of HPSE-1-induced invasion requires syndecan-1 HS chains. These results demonstrate that cell-surface syndecan-1 and ECM perlecan are degradative targets of HPSE-1, and syndecan-1 regulates HPSE-1 biological activity. This suggests that expression of syndecan-1 on the melanoma cell surface and its degradation by HPSE-1 are important determinants in the control of tumor cell invasion and metastasis (Reiland, 2004).
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