Syndecan-4 binding to the high affinity heparin-binding domain of fibronectin drives focal adhesion formation in fibroblasts

Cell adhesion to extracellular matrix involves signaling mechanisms which control attachment, spreading and the formation of focal adhesions and stress fibers. Fibronectin can provide sufficient signals for all three processes, even when protein synthesis is prevented by cycloheximide. Primary fibroblasts attach and spread following integrin ligation, but do not form focal adhesions unless treated with a heparin-binding fragment of fibronectin (HepII), a peptide from this domain, or phorbol esters to activate protein kinase C. Syndecan-4 heparan sulfate proteoglycan is a transmembrane component present together with integrins in focal adhesions. Syndecan-4 binds and activates protein kinase Calpha, whose activity is needed for focal adhesion formation. We now report that the glycosaminoglycan chains of syndecan-4 bind recombinant HepII and it is incorporated into forming focal adhesions.     

Heparan sulfate chains from glypican and syndecans bind the Hep II domain of fibronectin similarly despite minor structural differences

Numerous functions of heparan sulfate proteoglycans are mediated through interactions between their heparan sulfate glycosaminoglycan chains and extracellular ligands. Ligand binding specificity for some molecules, including many growth factors, is determined by complex heparan sulfate fine structure, where highly sulfated, iduronate-rich domains alternate with N-acetylated domains. Syndecan-4, a cell surface heparan sulfate proteoglycan, has a distinct role in cell adhesion, suggesting its chains may differ from those of other cell surface proteoglycans. To determine whether the specific role of syndecan-4 correlates with a distinct heparan sulfate structure, we have analyzed heparan sulfate chains from the different surface proteoglycans of a single fibroblast strain and compared their ability to bind the Hep II domain of fibronectin, a ligand known to promote focal adhesion formation through syndecan-4. Despite distinct molecular masses of glypican and syndecan glycosaminoglycans and minor differences in disaccharide composition and sulfation pattern, the overall proportion and distribution of sulfated regions and the affinity for the Hep II domain were similar. Therefore, adhesion regulation requires core protein determinants of syndecan-4.     

Syndecan-4 deficiency impairs focal adhesion formation only under restricted conditions

Two domains of fibronectin deliver two different but cooperative signals required for focal adhesion formation. The signal from the cell-binding domain is mediated by integrins, whereas the signal from the heparin-binding domain is recognized by heparan sulfate proteoglycans, of which syndecan-4 has been hypothesized to be involved in focal adhesion formation. We generated mice deficient in syndecan-4 to study its role directly. Even in fibroblasts from syndecan-4-deficient mice, focal adhesions were formed, and actin fibers terminated normally at focal adhesions when they were cultured on coverslips coated with fibronectin or with a mixture of its cell-binding and heparin-binding fragments. However, when the cells were cultured on the cell-binding fragment and the heparin-binding fragment was added to the medium, focal adhesion formation was impaired in the syndecan-4 null fibroblasts as compared with that in wild-type cells. Therefore, syndecan-4 is essential for promoting focal adhesion formation only when the signal of the heparin-binding domain of fibronectin is delivered as a soluble form, most probably from the apical surface. When the signal is delivered as a substratum-bound form, other molecule(s) also participate(s) in the signal reception.     

A Shared Mechanism of Adhesion Modulation for Tenascin-C and Fibulin-1

Adhesion modulatory proteins are important effectors of cell–matrix interactions during tissue remodeling and regeneration. They comprise a diverse group of matricellular proteins that confer antiadhesive properties to the extracellular matrix (ECM). We compared the inhibitory effects of two adhesion modulatory proteins, fibulin-1 and tenascin-C, both of which bind to the C-terminal heparin-binding (HepII) domain of fibronectin (FN) but are structurally distinct. Here, we report that, like tenascin-C, fibulin-1 inhibits fibroblast spreading and cell-mediated contraction of a fibrin–FN matrix. These proteins act by modulation of focal adhesion kinase and extracellular signal-regulated kinase signaling. The inhibitory effects were bypassed by lysophosphatidic acid, an activator of RhoA GTPase. Fibroblast response to fibulin-1, similar to tenascin-C, was dependent on expression of the heparan sulfate proteoglycan syndecan-4, which also binds to the HepII domain. Therefore, blockade of HepII-mediated signaling by competitive binding of fibulin-1 or tenascin-C represents a shared mechanism of adhesion modulation among disparate modulatory proteins.     

Syndecans promote integrin-mediated adhesion of mesenchymal cells in two distinct pathways

Syndecans are transmembrane proteoglycans that support integrin-mediated adhesion. Well documented is the contribution of syndecan-4 that interacts through its heparan sulphate chains to promote focal adhesion formation in response to fibronectin domains. This process has requirements for integrin and signaling through the cytoplasmic domain of syndecan-4. Here an alternate pathway mediated by the extracellular domains of syndecans-2 and -4 is characterized that is independent of both heparan sulphate and syndecan signaling. This pathway is restricted to mesenchymal cells and was not seen in any epithelial cell line tested, apart from vascular endothelia. The syndecan ectodomains coated as substrates promoted integrin-dependent attachment, spreading and focal adhesion formation. Syndecan-4 null cells were competent, as were fibroblasts compromised in heparan sulphate synthesis that were unable to form focal adhesions in response to fibronectin. Consistent with actin cytoskeleton organization, the process required Rho-GTP and Rho kinase. While syndecan-2 and -4 ectodomains could both promote integrin-mediated adhesion, their pathways were distinct, as shown by competition assays. Evidence for an indirect interaction of beta1 integrin with both syndecan ectodomains was obtained, all of which suggests a distinct mechanism of integrin-mediated adhesion.     

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 mediates cell spreading in transfected human lymphoblastoid (Raji) cells

Syndecan-1 is a cell surface proteoglycan containing a highly conserved transmembrane and cytoplasmic domain, and an extracellular domain bearing heparan sulfate glycosaminoglycans. Through these domains, syndecan-1 is proposed to have roles in growth factor action, extracellular matrix adhesion, and cytoskeletal organization that controls cell morphology. To study the role of syndecan-1 in cell adhesion and cytoskeleton reorganization, mouse syndecan-1 cDNA was transfected into human Raji cells, a lymphoblastoid cell line that grows as suspended cells and exhibits little or no endogenous cell surface heparan sulfate. High expressing transfectants (Raji-Sl cells) bind to and spread on immobilized thrombospondin or fibronectin, which are ligands for the heparan sulfate chains of the proteoglycan. This binding and spreading as not dependent on the cytoplasmic domain of the core protein, is mutants expressing core proteins with cytoplasmic deletions maintain the ability to spread. The spreading is mediated through engagement of the syndecan-1 core protein, as the Raji-S 1 cells also bind to and spread on immobilized mAb 281.2, an antibody specific for the ectodomain of the syndecan-1 core protein. Spreading on the antibody is independent of the heparan sulfate glycosaminoglycan chains and can be inhibited by competition with soluble mAb 281.2. The spreading can be inhibited by treatment with cytochalasin D or colchicine. These data suggest that the core protein of syndecan-1 mediates spreading through the formation of a multimolecular signaling complex at the cell surface that signals cytoskeleton reorganization. This complex may form via intramembrane or extracellular interactions with the syndecan core protein.     

PR-39 coordinates changes in vascular smooth muscle cell adhesive strength and locomotion by modulating cell surface heparan sulfate-matrix interactions

PR-39 is proline-rich peptide produced at sites of tissue injury. While the functional properties of this peptide have not been fully defined, PR-39 may be an important regulator of processes related to cell-matrix adhesion since it reportedly upregulates syndecan-4, which is a critical determinant of focal adhesion formation. The ability of PR-39 to modulate the adhesion and chemokinetic migration behavior of arterial smooth muscle cells (SMCs) in a fashion coordinated with syndecan-4 expression was investigated. Treatment of SMCs with PR-39 did not alter syndecan-1 mRNA, but did induce a two-fold increase in syndecan-4 mRNA (P < 0.0001) and significantly enhanced cell surface expression of both syndecan-4 (P < 0.01) and heparan sulfate (HS) (P < 0.05). These observations were consistent with an observed increase in cell-matrix adhesive strength (P < 0.05) and a reduction in cell speed (P < 0.01) on fibronectin-coated substrates. Incubation of PR-39 treated cells with a soluble fibronectin derived heparin-binding peptide, as a competitive inhibitor of heparan sulfate/matrix interactions, abolished these effects. These data suggest that PR-39 mediated alterations of cell adhesion and motility may be related, in part, to the increased expression of heparan sulfate glycosaminoglycans (GAGs) that accompany the upregulation of cell surface syndecan-4. Furthermore, this investigation supports the notion that factors which control syndecan-4 expression may play an important role in regulating adhesion related cell processes.     

Heparin II domain of fibronectin mediates contractility through an α4β1 co-signaling pathway

In the trabecular meshwork (TM) of the eye, regulation of tissue contractility by the PPRARI sequence within the Heparin II (HepII) domain of fibronectin is believed to control the movement of aqueous humor and dictate the level of intraocular pressure. This study shows that the HepII domain utilizes activated α4β1 integrin and collagen to mediate a co-signaling pathway that down-regulates contractility in TM cells. siRNA silencing of α4β1 integrin blocked the actin disrupting effects of both PPRARI and the HepII domain. The down-regulation of the actin cytoskeleton and contractility did not involve syndecan-4 or other heparan sulfate proteoglycans (HSPGs) since siRNA silencing of syndecan-4 expression or heparitinase removal of cell surface HSPGs did not prevent the HepII-mediated disruption of the actin cytoskeleton. HepII-mediated disruption of the cytoskeleton depended upon the presence of collagen in the extracellular matrix, and cell binding studies indicated that HepII signaling involved cross-talk between α4β1 and α1/α2β1 integrins. This is the first time that the PPRARI sequence in the HepII domain has been shown to serve as a physiological α4β1 ligand, suggesting that α4β1 integrin may be a key regulator of tissue contractility.     

Coregulation of fibronectin signaling and matrix contraction by tenascin-C and syndecan-4

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. Here, we report that 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.     

Structural and cell adhesion properties of zebrafish syndecan-4 are shared with higher vertebrates

The syndecan proteoglycans are an ancient class of receptor, bearing heparan sulfate chains that interact with numerous potential ligands including growth factors, morphogens, and extracellular matrix molecules. The single syndecan of invertebrates appears not to have cell adhesion roles, but these have been described for mammalian paralogues, especially syndecan-4. This member is best understood in terms of interactions, signaling, and structure of its cytoplasmic domain. The zebrafish homologue of syndecan-4 has been genetically linked to cell adhesion and migration in zebrafish embryos, but no molecular and cellular studies have been reported. Here it is demonstrated that key functional attributes of syndecan-4 are common to both zebrafish and mammalian homologues. These include glycosaminoglycan substitution, a NXIP motif in the extracellular domain that promotes integrin-mediated cell adhesion, and a transmembrane GXXXG motif that promotes dimer formation. In addition, despite some amino acid substitutions in the cytoplasmic domain, its ability to form twisted clamp dimers is preserved, as revealed by nuclear magnetic resonance spectroscopy. This technique also showed that phosphatidylinositol 4,5-bisphosphate can interact with the zebrafish syndecan-4 cytoplasmic domain, and that the molecule in its entirety supports focal adhesion formation, and complements the murine null cells to restore a normal actin cytoskeleton identically to the rat homologue. Therefore, the cell adhesion properties of syndecan-4 are consistent across the vertebrate spectrum and reflect an early acquisition of specialization after syndecan gene duplication events at the invertebrate/early chordate boundary.     

Hierarchy between the transmembrane and cytoplasmic domains in the regulation of syndecan-4 functions

Syndecan-4, a transmembrane heparan sulfate proteoglycan, plays a critical role in cell adhesion. Both the transmembrane and cytoplasmic domains of syndecan-4 are known to contribute to its functions, but the regulatory mechanisms underlying the functional interplay between the two domains were previously unclear. Here, we examined the functional relationship between these two domains. Fluorescence resonance energy transfer (FRET)-based assays showed that syndecan-4 expression enhanced RhoA activation. Furthermore, rat embryonic fibroblasts (REFs) plated on fibronectin fragments lacking the heparin-binding domain that interacts with syndecan-4 showed much lower RhoA activation than that in cells plated on full-length fibronectin, indicating that RhoA is involved in syndecan-4-mediated cell adhesion signaling. Syndecan-4 mutants defective in transmembrane domain-induced oligomerization and syndecan-4 phosphorylation-mimicking cytoplasmic domain mutants showed decreases in RhoA activation and RhoA-related functions, such as adhesion, spreading and focal adhesion formation, and subsequent increase in cell migration, but the inhibitory effect was much higher in cells expressing the transmembrane domain mutants. The cytoplasmic domain mutants (but not the transmembrane domain mutants) retained the capacity to form SDS-resistant dimers, and the cytoplasmic mutants showed less inhibition of syndecan-4-mediated protein kinase C activation compared to the transmembrane domain mutants. Finally, cytoplasmic domain activation failed to overcome the inhibition conferred by mutation of the transmembrane domain. Taken together, these data suggest that the transmembrane domain plays a major role in regulating syndecan-4 functions, and further show that a domain hierarchy exists in the regulation of syndecan-4.     

Fibronectin-tissue transglutaminase matrix rescues RGD-impaired cell adhesion through syndecan-4 and beta1 integrin co-signaling

Heterotropic association of tissue transglutaminase (TG2) with extracellular matrix-associated fibronectin (FN) can restore the adhesion of fibroblasts when the integrin-mediated direct binding to FN is impaired using RGD-containing peptide. We demonstrate that the compensatory effect of the TG-FN complex in the presence of RGD-containing peptides is mediated by TG2 binding to the heparan sulfate chains of the syndecan-4 cell surface receptor. This binding mediates activation of protein kinase Calpha (PKCalpha) and its subsequent interaction with beta(1) integrin since disruption of PKCalpha binding to beta(1) integrins with a cell-permeant competitive peptide inhibits cell adhesion and the associated actin stress fiber formation. Cell signaling by this process leads to the activation of focal adhesion kinase and ERK1/2 mitogen-activated protein kinases. Fibroblasts deficient in Raf-1 do not respond fully to the TG-FN complex unless either the full-length kinase competent Raf-1 or the kinase-inactive domain of Raf-1 is reintroduced, indicating the involvement of the Raf-1 protein in the signaling mechanism. We propose a model for a novel RGD-independent cell adhesion process that could be important during tissue injury and/or remodeling whereby TG-FN binding to syndecan-4 activates PKCalpha leading to its association with beta(1) integrin, reinforcement of actin-stress fiber organization, and MAPK pathway activation.     

Syndecan-4 cytoplasmic domain regulation of turkey satellite cell focal adhesions and apoptosis

Syndecan-4 is a cell membrane proteoglycan composed of a transmembrane core protein and substituted glycosaminoglycan (GAG) and N-linked glycosylated (N-glycosylated) chains. The core protein has three domains: extracellular, transmembrane and cytoplasmic domains. The GAG and N-glycosylated chains and the cytoplasmic domain of syndecan-4, especially the amino acids: Ser(178) and Tyr(187) are critical in regulation of turkey satellite cell growth and development. How these processes are regulated is still unknown. The objective of the current study was to determine whether the syndecan-4 GAG and N-glycosylated chains and the cytoplasmic domain functions through modulating focal adhesion formation and apoptosis. Twelve mutant clones were generated: a truncated syndecan-4 without the cytoplasmic domain with or without GAG and N-glycosylated chains, and Ser(178) and Tyr(187) mutants with or without GAG and N-glycosylated chains. The wild type syndecan-4 and all of the syndecan-4 mutants were transfected into turkey myogenic satellite cells after which cell apoptosis and focal adhesion formation were measured. Syndecan-4 increased cell membrane localization of β1 integrin and the activity of focal adhesion kinase (FAK) whereas the cytoplasmic domain mutation decreased the phosphorylation of FAK. However, syndecan-4 and syndecan-4 mutants did not influence cell apoptosis. They also had no effect on vinculin or paxillin-containing focal adhesion formation. These results suggested that the syndecan-4 cytoplasmic domain plays an important role in regulating FAK activity and β1 integrin cell membrane localization but not cell apoptosis and vinculin or paxillin-containing focal adhesion formation.     

Role of syndecan-4 side chains in turkey satellite cell apoptosis and focal adhesion formation

S4 (syndecan-4) is a cell membrane heparan sulfate proteoglycan that functions in muscle growth and development. It is composed of a central core protein and two types of side chains: GAGs (glycosaminoglycans) and N-glycosylated (N-linked glycosylated) chains. The N-glycosylated chains and GAG chains are required for S4 to regulate turkey myogenic satellite cell proliferation. The objective of the current study was to determine whether the S4 side chains regulate cell proliferation through muscle cell focal adhesion formation and apoptosis. S4 mutants with only one or without any N-glycosylated chains attached to the core protein with or without GAG chains were generated to study the function of N-glycosylated chains and the interaction between N-glycosylated chains and GAG chains. The wild-type S4 and all of the S4 side chain mutants were transfected into turkey myogenic satellite cells. Cell apoptosis and focal adhesion formation were measured, and PKCα (protein kinase Cα) cell membrane localization was investigated. S4 increased FAK (focal adhesion kinase) activity and the deletion of the side chains decreased this effect. S4 and the S4 mutants had no effect on β1-integrin expression, but increased the cell membrane localization of β1-integrin and PKCα. Furthermore, cell apoptosis and vinculin containing focal adhesions were not affected by S4 and its mutants. The results suggest that S4 and its side chains play important roles in regulating FAK activity, and PKCα and β1-integrin cell membrane localization, but not cell apoptosis and vinculin-containing focal adhesion formation.     

Syndecan-4 distribution during the differentiation of satellite cells isolated from soleus muscle treated by phorbol ester and calphostin C

It was shown that syndecans have a potential role in muscle development. We focused this study on the role of syndecan-4 distribution and phosphorylation during the differentiation of satellite cells isolated from Soleus muscle. Syndecans are cell surface heparan sulfate proteoglycans (HSPGs) that bind numerous ligands through their HS glycosaminoglycan chains (GAG). They play a role in cell-extracellular matrix and cell-cell adhesion, signal transduction and the targeting of growth factors and other molecules to the cell surface. Syndecan-4 acts as a co-receptor or, along with integrins, is localized to the cell membrane of focal contacts. Syndecan-4 participates in the organization of the structure of focal contacts reacting with extracellular matrix molecules. The interaction of syndecan-4 with protein kinase C (PKC) isoforms is the main mechanism regulating its distribution in cells. Our current study focused on the role of the distribution of syndecan-4, and its interactions with PKC isoforms during the differentiation of activated satellite cells. We used the PKC activator TPA (12-O-tetradecanoyl phorbol 13-acetate) and the PKC inhibitor Calphostin C (Cal C). We concluded that syndecan-4 was important not only in the activation of satellite cells, but also in myoblast differentiation. During our research, we observed the presence of syndecan-4 and changes in its location over the course of that process. We also showed that TPA and Cal C treatment had an influence on the subcellular distribution of syndecan-4, but there was no influence on myoblast differentiation. We speculated that the reason for changes after TPA treatment was the interactions with activated PKC alpha, which provoked syndecan-4/PKC alpha complex translocation to integrins. We also supposed that Cal C treatment inhibited PKC delta activity and probably induced PKC lpha association to syndecan-4, and syndecan-4 translocation to integrins.     

Characterization of heparin-binding site of tissue transglutaminase: its importance in cell surface targeting, matrix deposition, and cell signaling

Tissue transglutaminase (TG2) is a multifunctional Ca(2+)-activated protein cross-linking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease, and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a nontransamidating mechanism via its association with fibronectin, heparan sulfates (HS), and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modeling and mutagenesis, we have identified the HS-binding site of TG2 (202)KFLKNAGRDCSRRSSPVYVGR(222). We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS-binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate for the RGD-induced loss of cell adhesion on fibronectin via binding to syndecan-4, leading to activation of PKCα, pFAK-397, and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed.     

Characterization of the structure of antithrombin-binding heparan sulfate generated by heparan sulfate 3-O-sulfotransferase 5

The 3-O-sulfation of glucosamine is a key modification step during the biosynthesis of anticoagulant heparan sulfate (HS). Both heparan sulfate 3-O-sulfotransferase -1 (3-OST-1) and 3-O-sulfotransferase-5 (3-OST-5) transfer sulfate to the 3-OH group of glucosamine to generate antithrombin-binding heparan sulfate (HS(act)). Here, we reported the isolation and characterization of the antithrombin-binding HS oligosaccharides generated by 3-OST-5 (3-OST-5 oligo(act)). (3)H-labeled HS of Chinese hamster ovary cells was exhaustively modified by 3-OST-1 to remove the 3-OST-1 modification sites followed by antithrombin-affinity fractionation. The non-antithrombin-binding fraction of 3-OST-1 pretreated HS was further modified by 3-OST-5 to generate additional antithrombin-binding HS, which was designated as 3-OST-5 HS(act). Structural analysis of 3-OST-5 HS(act) revealed that the antithrombin-binding site of 3-OST-5 HS(act) is located within a domain clustered with N-sulfated glucosamine units. We also isolated 3-OST-5 antithrombin-binding oligosaccharides (3-OST-5 oligo(act)) from high pH nitrous acid degraded 3-OST-5 HS(act). A disaccharide analysis revealed that 3-OST-5 oligo(act) were composed of multiple 3-O-sulfated glucosamine units. Our results provide additional insights on the relationship between the anticoagulant activity and structure of HS.     

Syndecans in cell adhesion and differentiation

Syndecans are transmembrane proteoglycans expressed on adherent cells. They are a family of four proteins, which participate in cell-matrix adhesion, the regulation of growth factors (FGFs, VEGF, HGF) binding and signaling. The extracellular domain of syndecans contains heparan sulfate and chondroitin sulfate glycosaminoglycan chains. Syndecans have transmembrane region and a short cytoplasmic domain. The cytoplasmic domain attaches activated protein kinase Calpha, phosphatidyl-inositol-4,5-bisphosphate, syntenin, beta-catenin and many others molecules. Syndecans bind numerous ligands, which are present in extracellular matrix: growth factors, enzymes, extracellular matrix molecules (fibronectin, laminin). They form connections with actin cytoskeleton. The changes in syndecan expression influence on cell adhesion and migration, structure of focal contacts and cytoskeleton. Syndecans participate in cell differentiation and tissue regeneration.     

Acquisition of Anoikis Resistance Up-Regulates Syndecan-4 Expression in Endothelial Cells

Anoikis is a programmed cell death induced upon cell detachment from extracellular matrix, behaving as a critical mechanism in preventing adherent-independent cell growth and attachment to an inappropriate matrix, thus avoiding colonization of distant organs. Cell adhesion plays an important role in neoplastic transformation. Tumors produce several molecules that facilitate their proliferation, invasion and maintenance, especially proteoglycans. The syndecan-4, a heparan sulfate proteoglycan, can act as a co-receptor of growth factors and proteins of the extracellular matrix by increasing the affinity of adhesion molecules to their specific receptors. It participates together with integrins in cell adhesion at focal contacts connecting the extracellular matrix to the cytoskeleton. Changes in the expression of syndecan-4 have been observed in tumor cells, indicating its involvement in cancer. This study investigates the role of syndecan-4 in the process of anoikis and cell transformation. Endothelial cells were submitted to sequential cycles of forced anchorage impediment and distinct lineages were obtained. Anoikis-resistant endothelial cells display morphological alterations, high rate of proliferation, poor adhesion to fibronectin, laminin and collagen IV and deregulation of the cell cycle, becoming less serum-dependent. Furthermore, anoikis-resistant cell lines display a high invasive potential and a low rate of apoptosis. This is accompanied by an increase in the levels of heparan sulfate and chondroitin sulfate as well as by changes in the expression of syndecan-4 and heparanase. These results indicate that syndecan-4 plays a important role in acquisition of anoikis resistance and that the conferral of anoikis resistance may suffice to transform endothelial cells.