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Protein kinase C phosphorylates two of the four known syndecan cytoplasmic domains in vitro.

The transmembrane heparan sulfate proteoglycans of the syndecan family are implicated to participate in several cellular reactions which are dependent on protein kinase C. We have used an in vitro assay to assess whether any of the Peptides corresponding to the complete cytoplasmic domains of rat syndecans 1 through 4 were used as substrates for the enzyme. The syndecan-2 (fibroglycan) and syndecan-3 (N-syndecan) peptides were both found to be phosphorylated by protein kinase C with Kms of 15 +/- 3 microM and 85 +/- 25 microM, respectively, while the syndecan-1 and -4 peptides were not phosphorylated under the conditions used. The sites of in vitro phosphorylation for syndecans-2 and -3 were localized to ser-197 and ser-339, respectively. Thus, among 13 available sites (serines and threonines) in the four peptides, two were selectively modified by the enzyme. The specificity and the kinetics of the reactions indicate that the cytoplasmic domains of syndecan-2 and -3 are likely to be physiological substrates for protein kinase C.

Amino Acid Sequence↗

Reg1ulatory role and molecular interactions of a cell-surface heparan sulfate proteoglycan (N-syndecan) in hippocampal long-term potentiation.

The cellular mechanisms responsible for synaptic plasticity involve interactions between neurons and the extracellular matrix. Heparan sulfates (HSs) constitute a group of glycosaminoglycans that accumulate in the beta-amyloid deposits in Alzheimer's disease and influence the development of neuron-target contacts by interacting with other cell surface and matrix molecules. However, the contribution of HSs to brain function is unknown. We found that HSs play a crucial role in long-term potentiation (LTP), a finding that is consistent with the idea that converging molecular mechanisms are used in the development of neuron-target contacts and in activity-induced synaptic plasticity in adults. Enzymatic cleavage of HS by heparitinase as well as addition of soluble heparin-type carbohydrates prevented expression of LTP in response to 100 Hz/1 sec stimulation of Schaffer collaterals in rat hippocampal slices. A prominent carrier protein for the type of glycans implicated in LTP regulation in the adult hippocampus was identified as N-syndecan (syndecan-3), a transmembrane proteoglycan that was expressed at the processes of the CA1 pyramidal neurons in an activity-dependent manner. Addition of soluble N-syndecan into the CA1 dendritic area prevented tetanus-induced LTP. A major substrate of src-type kinases, cortactin (p80/85), and the tyrosine kinase fyn copurified with N-syndecan from hippocampus. Moreover, association of both cortactin and fyn to N-syndecan was rapidly increased after induction of LTP. N-syndecan may thus act as an important regulator in the activity-dependent modulation of neuronal connectivity by transmitting signals between extracellular heparin-binding factors and the fyn signaling pathway.

Animals↗

Mapping of the syndecan genes in the mouse: linkage with members of the myc gene family.

The syndecans are a family of four cell surface heparan sulfate proteoglycans in vertebrates that mediate a variety of cell behaviors, including cell adhesion and the action of growth factors. Their core proteins contain conserved transmembrane and cytoplasmic domains but divergent extracellular regions in which only the glycosaminoglycan attachment sites are conserved. By extensive PCR analyses based on the conserved sequences, we find only four syndecan-related sequences in the mouse. These correspond to the previously described core proteins of syndecan proteoglycans from other vertebrates. We have mapped the genes for syndecan-2 to chromosome 15, syndecan-3 to chromosome 4, and syndecan-4 to chromosome 2 in the mouse. Together with the previous localization of the gene for syndecan-1 to chromosome 12, these data establish that the four syndecan genes are dispersed on different chromosomes and that each syndecan gene is located near a member of the myc gene family. Synd1 is next to Nmyc, Synd2 close to myc, Synd3 near Lmyc, and Synd4 on the same chromosome as Bmyc. The physical relationship between the members of these two gene families appears to be ancient and conserved after the two genome duplications thought to have occurred during vertebrate evolution.

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HB-GAM (heparin-binding growth-associated molecule) and heparin-type glycans in the development and plasticity of neuron-target contacts.

HB-GAM is a secretory, extracellular matrix-associated protein that was isolated by screening for factors that enhance neurite outgrowth in rat brain neurons. The HB-GAM sequence clearly (about 50%) is homologous to that of MK (midkine) sequence, a protein discovered through screening for factors that mediate retinoic acid-induced cell differentiation. These lysine- and cysteine-rich sequences define a novel family of differentiation/growth factors, which are conserved in their structures from mammals to amphibians. HB-GAM is expressed strongly along axon pathways and target regions of axons during and prior to the stage of axonal growth in tissues. These findings, together with in vitro interactions with neurons, suggest that HB-GAM is a cell matrix-associated cue for growth cone migration. N-syndecan (syndecan-3) functions as a receptor/coreceptor in HB-GAM-induced neurite outgrowth in perinatal rat brain neurons. In addition to enhancing neurite growth in a developmentally regulated manner in early neurons, HB-GAM is accumulated at the growth cone-target interphase accompanying the onset of synaptogenesis, as evidenced by its presence at the neuromuscular junction of Xenopus and rat. In vitro studies suggest that HB-GAM functions as a local, synaptic matrix-associated factor that enhances both presynaptic and postsynaptic differentiation during development. In addition, a role in adult plasticity is suggested by studies on injury-induced and activity-dependent plasticity in rat hippocampus.

Animals↗

Glypican and biglycan in the nuclei of neurons and glioma cells: presence of functional nuclear localization signals and dynamic changes in glypican during the cell cycle.

We have investigated the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, we found that a significant portion of the glypican and biglycan immunoreactivity colocalized with nuclear staining by propidium iodide and was also seen in isolated nuclei. In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals, and these were demonstrated to be functional based on their ability to target beta-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican beta-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells was greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining was seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes.

Animals↗

Interactions of neural glycosaminoglycans and proteoglycans with protein ligands: assessment of selectivity, heterogeneity and the participation of core proteins in binding.

The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth factor-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8-fold less tightly than heparin (Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of fibroblast growth factor-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400-fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.

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Syndecan-4 is a primary-response gene induced by basic fibroblast growth factor and arterial injury in vascular smooth muscle cells.

Syndecans are a family of transmembrane proteoglycans that have been implicated in cell-extracellular matrix adhesion and growth factor binding. We reported previously that syndecan-1 expression by cultured rate vascular smooth muscle cells (VSMCs) is induced by serum- or platelet-derived growth factor (PDGF). We now report that syndecan-4 mRNA is rapidly induced in cultured VSMCs in response to basic fibroblast growth factor (bFGF) or serum stimulation. In the presence of cycloheximide, induction of syndecan-4 mRNA was enhanced. These characteristics identified syndecan-4 as a primary-response gene product in VSMCs. In contrast, syndecan-1 mRNA expression in response to serum was completely blocked in the presence of cycloheximide. We also examined the expression of syndecan mRNAs in VSMCs in response to balloon catheter injury in vivo. A reverse transcriptase-polymerase chain reaction technique was developed that enabled us to amplify all four syndecan mRNAs in a single reaction tube and determine relative changes in their expression. All four syndecan mRNAs were detected in uninjured rat carotid arteries. In endothelium-denuded arteries, the medial layer (presumably VSMCs) accounted for 70% to 90% of the syndecan mRNAs in the vessel wall. The levels of syndecan-2 and syndecan-3 mRNAs were not altered significantly after balloon injury. In contrast, syndecan-4 mRNA was increased at early times after injury but then decreased to control level by 7 days. Syndecan-1 mRNA levels showed a slower but prolonged increase that reached a maximum at 7 days after injury. Immunostaining with anti-syndecan-4 antibodies demonstrated a rapid increase in syndecan-4 proteoglycan expression in the injured carotid artery.

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Divide, accumulate, differentiate: cell condensation in skeletal development revisited.

Cell condensation is a pivotal stage in skeletal development. Although prechondrogenic condensations normally exist for some 12 h, duration can vary. Variation is seen both between condensations for different cartilages (Meckel's vs. elastic ear cartilage) and within a single condensation from which more than one skeletal element will form, as in the three components of the single first arch chondrogenic condensation. Understanding how duration of the condensation phase is established--how the condensation phase is entered and exited during cell differentiation--remains a major area for future study. During chondrogenesis, cell-specific products such as collagen types II and IX and cartilage proteoglycan appear concomitant with condensation. Therefore, during chondrogenesis, condensation precedes commitment of cells as prechondroblasts. During osteogenesis, however, differentiation of preosteoblasts precedes condensation. Therefore, during osteogenesis, condensation amplifies the number of committed osteogenic cells. Further comparative analysis of skeletogenesis should provide us with a more rigorous understanding of cell commitment, when differentiation is initiated, how commitment and differentiation are measured and the relationship of condensation to onset of differentiation. Current knowledge of molecules characteristic of condensations focused attention on extracellular matrix and cell surface components on the one hand, and on growth factors homeobox genes and transcription factors on the other. We have drawn together the molecular data for pre-chondrogenic condensations in diagrammatic form in Figure 2. Three major phases of chondrogenesis are identified: (a) epithelial-mesenchymal interactions that precede condensation, (b) condensation itself, and (c) cell differentiation. Although we label the third phase differentiation, it is important to recognize that phases a and b also constitute aspects of chondroblast cell differentiation (see Dunlop and Hall, 1995 for a discussion of this point. The pre-condensation phase is characterized by expression of Hox genes, growth factors (TGF-beta and BMP-2) and the cell surface proteoglycan receptor, syndecan-1. Expression of Msx-1 and Msx-2, growth factors and syndecan continues into the condensation phase. Other molecules, such as versican, syndecan-3 and tenascin, present in low concentrations before condensation, are up-regulated during condensation. Yet other molecules--Hox genes, transcription factors, growth factors (activin, BMP-4 and -5, GDF-5), cell adhesion molecules and proteoglycans--are only expressed during the condensation phase, while the transcription factor Pax-1, fibronectin, hyaluronan and hyaladherin are expressed both during and after condensation. During condensation mRNAs for collagen types II and IX and for the core protein of cartilage proteoglycan are up-regulated. Late in condensation and increasingly thereafter, the protein products of these genes accumulate as chondroblasts differentiate (see Fig. 2 for details). Not all the molecules present before, during of after condensation can be placed into causal sequences. Some however can. In Figure 3 we summarize the causal sequences discussed in this paper as they relate to initiation of condensation and to transit from condensation to overt differentiation during chondrogenesis. Condensations form following activation of at least three pathways: (1) Initiation of epithelial-mesenchymal interactions by tenascin, BMP-2, TGF beta-1 and Msx-1 and -2. (2) Up-regulation of N-CAM by activin. (3) Up-regulation of fibronectin by TGF-beta, further enhancing N-CAM accumulation (Fig. 3). It is by these three pathways that condensations are initiated and grow. Transition from condensation to overt cell differentiation is under both positive and negative control (Fig. 3). Syndecan blocks fibronectin and so blocks N-CAM accumulation, preventing accumulation of additional cell

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Phosphorylation of recombinant N-syndecan (syndecan 3) core protein.

The cytoplasmic domain of the syndecan family of heparan sulfate proteoglycans is punctuated by the presence of four regularly spaced tyrosine residues. In this report, we explore the possibility of whether the four tyrosine residues in the cytoplasmic domain of N-syndecan (Syndecan 3) are potential substrates for phosphorylation by a tyrosine kinase. Bacterially expressed elk kinase was used to phosphorylate a series of bacterially expressed N-syndecan fusion proteins. Our results clearly demonstrate that the tyrosine residues in the cytoplasmic domain of N-syndecan can be phosphorylated by a tyrosine-specific kinase, and that all four tyrosine residues are capable of being phosphorylated.

Amino Acid Sequence↗

Embryonic expression patterns of Xenopus syndecans.

Syndecans are a family of heparan sulfate proteoglycans implicated in cell-cell and cell-matrix interactions. To investigate the roles of syndecans in early development, we identified three syndecan family members in Xenopus laevis: Xsyn-1, Xsyn-2, and Xsyn-3. Xsyn-1 and Xsyn-2 are maternal mRNAs localized to the animal pole in blastulae, and are expressed in the ectoderm of gastrulae. In neurulae, Xsyn-1 is restricted to non-neural ectoderm and Xsyn-2 is restricted to neural ectoderm. In tailbud embryos, the three syndecans are expressed in adjacent, non-overlapping patterns. Xsyn-2 is expressed in the heart while Xsyn-1 is expressed in the underlying anterior endoderm. Xsyn-3 is expressed in the hindbrain, midbrain, and forebrain, while Xsyn-2 is expressed in the intervening regions. These results suggest that different members of the syndecan family have distinct developmental roles, perhaps acting as barriers to define tissue boundaries.

Amino Acid Sequence↗

Syndecan-1 is up-regulated in ras-transformed intestinal epithelial cells.

The syndecans, a family of cell-surface heparan sulphate proteoglycans, have been proposed to mediate cellular interactions with extracellular effector molecules, such as growth factors and components of the extracellular matrix, during critical phases of development. Transcripts of all four syndecans are expressed at varying levels in the developing rat intestine and in a series of immature rat intestinal epithelial cell lines. In addition, we report the novel finding that, in the intestinal epithelial cell lines, expression of syndecan-1 transcript is up-regulated by transformation with activated H-ras. This is in contrast to other cell lines in which ras transformation is associated with a decrease in syndecan-1 levels. The observed increase in the syndecan-1 occurs as a result of increased transcription and can be correlated with the degree of transformation of the IEC-18 cells. Transformation is also associated with a decrease in apparent molecular weight and increased shedding of the proteoglycan into the culture medium. Increased shedding of syndecan-1 into the culture medium after transformation with H-ras may contribute to the disruption of proteoglycan interactions with the extracellular matrix, leading to alterations in cell adhesion and organization.

Aging↗

N-syndecan and HB-GAM (heparin-binding growth-associated molecule) associate with early axonal tracts in the rat brain.

Heparin-Binding Growth-Associated Molecule (HB-GAM)/pleiotrophin is an 18 kDa extracellular matrix- and cell-surface-associated protein shown to enhance neurite outgrowth of perinatal forebrain neurones in vitro. The heparan sulphate proteoglycan N-syndecan (Raulo et al., 1994) has been isolated as a receptor/coreceptor for the HB-GAM. We have investigated, whether HB-GAM and N-syndecan could have a similar role in neurite outgrowth and axon guidance in early axonal tracts of brain. In the present study N-syndecan was found to be spatiotemporally associated with the developing axonal tracts already on embryonic day 9 in rat, as revealed by coexpression with class III beta-tubulin, which is one of the earliest neuronal markers (Easter et al., 1993; Brittis et al., 1995). Later, N-syndecan and HB-GAM were detected in the first afferent serotonergic projections arising from the pontine raphe nuclei. The expression pattern of HB-GAM peaked in the developing rhombencephalon at embryonic stage (E) 13-14. At the same time, N-syndecan was expressed in the developing raphe neurones growing neurites towards the diencephalon along HB-GAM immunoreactive pathways. When rhombencephalic neurones were cultured on decreasing concentrations of substrate-bound HB-GAM, E13 neurones showed a significantly better neurite outgrowth response than E11, E16 or E18 neurones. The neurite outgrowth of raphe neurones in vitro was inhibited by adding soluble heparin or N-syndecan into the culture medium, whereas addition of chondroitin sulphate had no effect. In a simple pathway assay, E13 raphe neurones selectively preferred attaching and growing neurites on pathways containing HB-GAM as compared with regions containing either laminin or fibronectin alone. Our results suggest that HB-GAM may function as a developmentally regulated cue for rhombencephalic neurones that possess N-syndecan on their cell membrane.

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Syndecan 3: a member of the syndecan family of membrane-intercalated proteoglycans that is expressed in high amounts at the onset of chicken limb cartilage differentiation.

A partial cDNA that encodes a newly discovered member of the syndecan family of integral membrane proteoglycans, which we have termed syndecan 3, has been isolated from an embryonic chicken limb bud cDNA library. Syndecan 3 is distinct from but structurally related to syndecan and fibroglycan, two previously characterized members of this family of membrane-intercalated proteoglycans. Syndecan 3 contains a cytoplasmic domain potentially associated with the cytoskeleton that is 85% identical in amino acid sequence to the cytoplasmic domain of syndecan. Syndecan 3 also possesses a hydrophobic transmembrane domain and an extracellular domain containing several clustered potential glycosaminoglycan attachment sites. Like syndecan, the ectodomain of syndecan 3 has a single dibasic protease-susceptible site adjacent to the transmembrane domain, which might be involved in shedding the ectodomain from the cell surface. A striking feature of syndecan 3 is an extensive (182 amino acid) threonine, serine, and proline (T+S+P)-rich domain that closely resembles T+S+P-rich regions in several mucin-like proteins in which O-linked oligosaccharides are bound to the threonine and serine residues. Syndecan 3 is expressed in high amounts during a critical phase of chicken limb chondrogenesis in which limb mesenchymal cells condense, round up, and interact with one another before depositing a cartilage matrix. The multiple functional domains of syndecan 3 provide potential sites for mediating the adhesive cell-matrix interactions and cytoskeletal reorganization involved in this critical condensation process.

Amino Acid Sequence↗

cDNA cloning, genomic organization, and in vivo expression of rat N-syndecan.

The amino acid sequence of rat N-syndecan core protein was deduced from the cloned cDNA sequence. The sequence predicts a core protein of 442 amino acids with six structural domains: an NH2-terminal signal peptide, a membrane distal glycosaminoglycan attachment domain, a mucin homology domain, a membrane proximal glycosaminoglycan attachment domain, a single transmembrane domain, and a noncatalytic COOH-terminal cytoplasmic domain. Transfection of human 293 cells resulted in the expression of N-syndecan that was modified by heparan sulfate chain addition. Heparitinase digestion of the expressed proteoglycan produced a core protein that migrated on SDS-polyacrylamide gels at an apparent molecular weight of 120, 000, identical to N-syndecan synthesized by neonatal rat brain or Schwann cells. Rat genomic DNA coding for N-syndecan was isolated by hybridization screening. The rat N-syndecan gene is comprised of five exons. Each exon corresponds to a specific core protein structural domain, with the exception of the fifth exon, which contains the coding information for both the transmembrane and cytoplasmic domains as well as the 3'-untranslated region of the mRNA. The first intron is large, with a length of 22 kilobases. The expression of N-syndecan was investigated in late embryonic, neonatal, and adult rats by immunoblotting and Northern blotting analysis. Among the tissues and developmental stages studied, high levels of N-syndecan expression were restricted to the early postnatal nervous system. N-syndecan was expressed in all regions of the nervous system, including cortex, midbrain, spinal cord, and peripheral nerve. Immunohistochemical staining revealed high levels of N-syndecan expression in all brain regions and fiber tract areas.

Amino Acid Sequence↗

Osteoblast recruitment and bone formation enhanced by cell matrix-associated heparin-binding growth-associated molecule (HB-GAM).

Bone has an enormous capacity for growth, regeneration, and remodeling. This capacity is largely due to induction of osteoblasts that are recruited to the site of bone formation. The recruitment of osteoblasts has not been fully elucidated, though the immediate environment of the cells is likely to play a role via cell- matrix interactions. We show here that heparin-binding growth-associated molecule (HB-GAM), an extracellular matrix-associated protein that enhances migratory responses in neurons, is prominently expressed in the cell matrices that act as target substrates for bone formation. Intriguingly, N-syndecan, which acts as a receptor for HB-GAM, is expressed by osteoblasts/osteoblast precursors, whose ultrastructural phenotypes suggest active cell motility. The hypothesis that HB-GAM/N-syndecan interaction mediates osteoblast recruitment, as inferred from developmental studies, was tested using osteoblast-type cells that express N-syndecan abundantly. These cells migrate rapidly to HB-GAM in a haptotactic transfilter assay and in a migration assay where HB-GAM patterns were created on culture wells. The mechanism of migration is similar to that previously described for the HB-GAM-induced migratory response of neurons. Our hypothesis that HB-GAM/N-syndecan interaction participates in regulation of osteoblast recruitment was tested using two different in vivo models: an adjuvant-induced arthritic model and a transgenic model. In the adjuvant-induced injury model, the expression of HB-GAM and of N-syndecan is strongly upregulated in the periosteum accompanying the regenerative response of bone. In the transgenic model, the HB-GAM expression is maintained in mesenchymal tissues with the highest expression in the periosteum. The HB-GAM transgenic mice develop a phenotype characterized by an increased bone thickness. HB-GAM may thus play an important role in bone formation, probably by mediating recruitment and attachment of osteoblasts/osteoblast precursors to the appropriate substrates for deposition of new bone.

Animals↗

Nitric oxide mediates interleukin-1-induced gene expression of matrix metalloproteinases and basic fibroblast growth factor in cultured rabbit articular chondrocytes.

We recently reported that nitric oxide (NO), which is produced by chondrocytes treated with interleukin-1beta (IL-1), releases basic fibroblast growth factor (bFGF) stored in the matrix of articular chondrocytes. To clarify the mechanism of the IL-1-induced bFGF release, we investigated the production and gene expression of bFGF, matrix metalloproteinases (MMPs), syndecan 3, and inducible NO synthase (iNOS) by IL-1-treated rabbit articular chondrocytes. IL-1 stimulated not only the release of bFGF but also the production of it. Gelatin and casein zymography revealed that IL-1 stimulated the production of not only MMP-9 but also MMP-3. The increase in the production of these MMPs preceded the IL-1-stimulated bFGF release. An MMP inhibitor partially suppressed the release of bFGF, indicating that matrix degradation is at least partially involved in the IL-1-stimulated bFGF release even if increased production of bFGF is related to the release. IL-1 sequentially stimulated mRNA expression of iNOS, membrane type 1-MMP, MMP-9 and -3, and bFGF, in that order. NG-Monomethyl-L-arginine, an inhibitor of NO production, inhibited gene expression of MMP-9 and bFGF. These findings suggest that elevation of the NO level via iNOS mRNA expression stimulated by IL-1 mediates gene expression and production of MMPs and bFGF, resulting in the release of bFGF, and also reveal molecular mechanisms implicating the degradation of articular cartilage followed by angiogenesis in the synovium in arthritic joints.

Animals↗

Regulation of mRNA localization by transmembrane signalling: local interaction of HB-GAM (heparin-binding growth-associated molecule) with the cell surface localizes beta-actin mRNA.

Localization of mRNAs is currently thought to be partially responsible for molecular sorting to specific compartments within the cell. In mammalian cells the best-studied example is the beta-actin mRNA that is localized to the cell processes, and its localization is necessary in migratory responses of cells. It is reasonable to assume that mRNA localization within cells is coupled to transmembrane signalling due to extracellular factors, but little is known about such putative mechanisms. We show here that HB-GAM, an extracellular matrix-associated factor that enhances migratory responses in cells, is able to localize beta-actin mRNA when locally applied to cells via microbeads. The HB-GAM-induced mRNA localization is specifically inhibited by low concentrations of heparin and by heparitinase treatment of cells, showing that cell-surface heparin-type glycans are required for the effect. The finding that soluble N-syndecan is also inhibitory suggests that the transmembrane proteoglycan N-syndecan, previously identified as an HB-GAM receptor, is involved in the mRNA-localizing effect of HB-GAM. Inhibition of the mRNA localization by the src-kinase inhibitor PP1 is compatible with an N-syndecan-mediated effect since the receptor function of N-syndecan has been recently found to depend on the src-kinase signalling pathway. The mRNA-localizing activity of N-syndecan is also suggested by the finding that affinity-purified anti-N-syndecan antibodies coated on microbeads are able to localize beta-actin mRNA.

Actins↗

N-syndecan (syndecan 3) from neonatal rat brain binds basic fibroblast growth factor.

The cell surface proteoglycan N-syndecan (syndecan 3) was isolated from neonatal rat brain. Purified brain N-syndecan had biochemical properties very similar to N-syndecan previously identified in Schwann cells: it contained mainly, if not exclusively, heparan sulfate glycosaminoglycan chains and had a core protein with an apparent molecular mass of 120 kDa by sodium dodecyl sulfate-gel electrophoresis. We examined the interactions between purified N-syndecan and extracellular ligands using a solid phase binding assay. It was found that among all proteins tested, including a variety growth factors and extracellular matrix molecules, only basic fibroblast growth factor (bFGF) exhibited significant N-syndecan binding. N-syndecan binding to bFGF was saturable and exhibited a KD = 0.5 nM. Soluble bFGF effectively competed with immobilized bFGF for binding to N-syndecan, indicating that these two proteins also interact in solution. Heparin and heparan sulfate, but not chondroitin sulfate, inhibited N-syndecan-bFGF binding. Isolated N-syndecan core protein did not exhibit significant binding to bFGF. Thus, the heparan sulfate chains of N-syndecan, rather than its core protein, appear to be responsible for binding to bFGF. Interestingly, acidic fibroblast growth factor, which is structurally similar to bFGF, did not exhibit significant N-syndecan binding. N-syndecan also did not bind to several other heparin-binding proteins used in this study, indicating a high degree of specificity for the N-syndecan-bFGF interaction. Both N-syndecan and bFGF are abundant in neonatal brain, suggesting that N-syndecan may function as a co-receptor for bFGF during nerve tissue development.

Animals↗