[Show abstract][Hide abstract] ABSTRACT: Syndecans comprise a major family of cell surface heparan sulfate proteoglycans (HSPGs). Syndecans bind and modulate a wide variety of biological molecules through their heparan sulfate (HS) moiety. Although all syndecans contain the ligand binding HS chains, they likely perform specific functions in vivo because their temporal and spatial expression patterns are different. However, how syndecan expression is regulated has yet to be clearly defined. In this study, we examined how syndecan-1 expression is regulated in epithelial cells. Our results showed that among several bioactive agents tested, only forskolin and three isoforms of TGFbeta (TGFbeta1-TGFbeta3) significantly induced syndecan-1, but not syndecan-4, expression on various epithelial cells. Steady-state syndecan-1 mRNA was not increased by TGFbeta treatment and cycloheximide did not inhibit syndecan-1 induction by TGFbeta, indicating that TGFbeta induces syndecan-1 in a post-translational manner. However, TGFbeta induction of syndecan-1 was inhibited by transient expression of a dominant-negative construct of protein kinase A (PKA) and by specific inhibitors of PKA. Further (i) syndecan-1 cytoplasmic domains were Ser-phosphorylated when cells were treated with TGFbeta and this was inhibited by a PKA inhibitor, (ii) PKA was co-immunoprecipitated from cell lysates by anti-syndecan-1 antibodies, (iii) PKA phosphorylated recombinant syndecan-1 cytoplasmic domains in vitro, and (iv) expression of a syndecan-1 construct with its invariant Ser(286) replaced with a Gly was not induced by TGFbeta. Together, these findings define a regulatory mechanism where TGFbeta signals through PKA to phosphorylate the syndecan-1 cytoplasmic domain and increases syndecan-1 expression on epithelial cells.
[Show abstract][Hide abstract] ABSTRACT: Syndecans are a family of four transmembrane heparan sulfate proteoglycans that act as coreceptors for a variety of cell-surface ligands and receptors. Receptor activation in several cell types leads to shedding of syndecan-1 and syndecan-4 ectodomains into the extracellular space by metalloproteinase-mediated cleavage of the syndecan core protein. We have found that 3T3-L1 adipocytes express syndecan-1 and syndecan-4 and that their ectodomains are shed in response to insulin in a dose-, time-, and metalloproteinase-dependent manner. Insulin responsive shedding is not seen in 3T3-L1 fibroblasts. This shedding involves both Ras-MAP kinase and phosphatidylinositol 3-kinase pathways. In response to insulin, adipocytes are known to secrete active lipoprotein lipase, an enzyme that binds to heparan sulfate on the luminal surface of capillary endothelia. Lipoprotein lipase is transported as a stable enzyme from its site of synthesis to its site of action, but the transport mechanism is unknown. Our studies indicate that shed adipocyte syndecans associate with lipoprotein lipase. The shed syndecan ectodomain can stabilize active lipoprotein lipase. These data suggest that syndecan ectodomains, shed by adipocytes in response to insulin, are physiological extracellular chaperones for lipoprotein lipase as it translocates from its site of synthesis to its site of action.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Many of the biological functions attributed to cell surface heparan sulfate (HS) proteoglycans, including the Syndecan family, are elicited through the interaction of their HS chains with soluble extracellular molecules. Tightly controlled, cell-specific sulfation and epimerization of HS precursors endows these chains with highly sulfated, iduronate-rich regions, which are major determinants of cytokine and matrix-protein binding and which are interspersed by N-acetylated, poorly sulfated regions. Until this study, there have been no comprehensive structural comparisons made on HS chains decorating simultaneously expressed, but different, syndecan core proteins. In this paper we demonstrate that the HS chains on affinity-purified syndecan-1 and -4 from murine mammary gland cells are essentially identical by a number of parameters. Size determination, disaccharide analyses, enzymatic and chemical scission methods, and affinity co-electrophoresis all failed to reveal any significant differences in fine structure, domain organization, or ligand-binding properties of these HS species. These findings lead us to suggest that the imposition of the fine structure onto HS occurs independently of the core protein to which it is attached and that these core proteins, in addition to the HS chains, may play a pivotal role in the various biological functions ascribed to these macromolecules.
[Show abstract][Hide abstract] ABSTRACT: Mice lacking syndecan-1 are viable, fertile and have morphologically normal skin, hair and ocular surface epithelia. While studying the response of these mice to corneal epithelial and skin wounding, we identified defects in epithelial cell proliferation and regulation of integrin expression. mRNA profiling of corneal epithelial tissues obtained from wild-type and syndecan-1(-/-) mice suggest that these defects result from differences in overall gene transcription. In the cornea, syndecan-1(-/-) epithelial cells migrate more slowly, show reduced localization of alpha9 integrin during closure of wounds and fail to increase their proliferation rate 24 hours after wounding. In the skin, we did not document a migration defect after full thickness wounds but did observe cell proliferation delays and reduced localization of alpha9 integrin in the syndecan-1(-/-) epidermis after dermabrasion. Despite increased cell proliferation rates in the uninjured syndecan-1(-/-) epidermis and the corneal epithelium, morphologically normal epithelial thickness is maintained prior to injury; however, wounding is accompanied by prolonged hypoplasia in both tissues. Analyses of integrin protein levels in extracts from full thickness skin, revealed increased levels of alpha3 and alpha9 integrins both prior to injury and after hair removal in syndecan-1(-/-) mice but no increase 2 days after dermabrasion. These data for the first time show involvement of alpha9 integrin in skin wound healing and demonstrate essential roles for syndecan-1 in mediating cell proliferation and regulation of integrin expression in normal and wounded epithelial tissues.
[Show abstract][Hide abstract] ABSTRACT: Transgenic expression in the hypothalamus of syndecan-1, a cell surface heparan sulfate proteoglycan (HSPG) and modulator of ligand-receptor encounters, produces mice with hyperphagia and maturity-onset obesity resembling mice with reduced action of alpha melanocyte stimulating hormone (alphaMSH). Via their HS chains, syndecans potentiate the action of agouti-related protein and agouti signaling protein, endogenous inhibitors of alphaMSH. In wild-type mice, syndecan-3, the predominantly neural syndecan, is expressed in hypothalamic regions that control energy balance. Food deprivation increases hypothalamic syndecan-3 levels several-fold. Syndecan-3 null mice, otherwise apparently normal, respond to food deprivation with markedly reduced reflex hyperphagia. We propose that oscillation of hypothalamic syndecan-3 levels physiologically modulates feeding behavior.
[Show abstract][Hide abstract] ABSTRACT: The syndecans are a gene family of four transmembrane heparan sulfate proteoglycans that bind, via their HS chains, diverse components of the cellular microenvironment. To evaluate the expression of the individual syndecans, we prepared cDNA probes to compare mRNA levels in various adult mouse tissues and cultured mouse cells representing various epithelial, fibroblastic, endothelial, and neural cell types and B cells at various stages of differentiation. We also prepared antibody probes to assess whether the extracellular domains of the individual syndecans are shed into the conditioned media of cultured cells. Our results show that all cells and tissues studied, except B-stem cells, express at least one syndecan family member; most cells and tissues express multiple syndecans. However, each syndecan family member is expressed selectively in cell-, tissue-, and development-specific patterns. The extracellular domain of all syndecan family members is shed as an intact proteoglycan. Thus, most, if not all, cells acquire a distinctive repertoire of the four syndecan family members as they differentiate, resulting in selective patterns of expression that likely reflect distinct functions.
Molecular Biology of the Cell 08/1994; 5(7):797-805. DOI:10.1091/mbc.5.7.797 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Syndecan-1, the prototype of a family of heparan sulfate-containing integral membrane proteoglycans, associates extracellularly with a variety of matrix molecules and growth factors and intracellularly with the actin cytoskeleton. Expressed constitutively on epithelia in mature tissues and in a developmentally regulated manner on epithelial and induced mesenchymal cells during embryogenesis, syndecan-1 appears to be involved in controlling the shape and organization of cells and tissues. To better understand the function and regulation of syndecan-1, we determined the structure of the mouse syndecan-1 gene (Synd-1). Synd-1 is approximately 19.5 kilobases in size and is organized into five exons that appear conserved in other family members. Exon 1 encodes the signal peptide; exon 2, the N-terminal glycosaminoglycan attachment region; exon 3, the bulk of the extracellular domain; exon 4, the protease-susceptible site; and exon 5, the transmembrane and cytoplasmic domains which are highly homologous between syndecan family members. Synd-1 has three transcriptional start sites, two polyadenylation sites, and is not alternatively spliced to produce its 2.6- and 3.4-kilobase mRNA species. Upstream sequences have promoter activity and contain TATA and CAAT boxes as well as a variety of other potential binding sites for transcription factors, including Sp1 (GC box), NF-kappa B, MyoD (E box), and Antennapedia. The structure of the promoter region suggests that control of Synd-1 expression is both constitutive and developmentally regulated. Because Synd-1 exons encode discrete functional domains of the syndecan-1 protein that are conserved throughout the syndecan family, all syndecan genes are likely derived from a common ancestor.
Journal of Biological Chemistry 06/1993; 268(15):11440-8. · 4.57 Impact Factor