Possible regulation of FGF activity by syndecan, an integral membrane heparan sulfate proteoglycan.
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Amino Acid Sequence↗
Biomedical subjects
M Bernfield
Publications and source records attributed to M Bernfield.
Developmental expression of syndecan, an integral membrane proteoglycan, correlates with cell differentiation.
Syndecan is an integral membrane proteoglycan that behaves as a matrix receptor by binding cells to interstitial matrix and associating intracellularly with the actin cytoskeleton. Using immunohistology, we have now localized this proteoglycan during the morphogenesis of various derivatives of the surface ectoderm in mouse embryos. Syndecan is expressed on ectodermal epithelia, but is selectively lost from the cells that differentiate into the localized placodes that initiate lens, nasal, otic and vibrissal development. The loss is transient on presumptive ear, nasal and vibrissal epithelia; the derivatives of the differentiating ectodermal cells that have lost syndecan subsequently re-express syndecan. In contrast, syndecan is initially absent from the mesenchyme underlying the surface ectoderm, and is transiently expressed when the surface ectoderm loses syndecan. These results demonstrate that expression of syndecan is developmentally regulated in a distinct spatiotemporal pattern. On epithelia, syndecan is lost at a time and, location that correlates with epithelial cell differentiation and, on mesenchyme, syndecan is acquired when the cells aggregate in proximity to the epithelium. This pattern of change with morphogenetic events is unique and not duplicated by other matrix molecules or adhesion receptors.
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Expression of syndecan, a putative low affinity fibroblast growth factor receptor, in the early mouse embryo.
Syndecan is an integral membrane proteoglycan that binds cells to several interstitial extracellular matrix components and binds to basic fibroblast-growth factor (bFGF) thus promoting bFGF association with its high-affinity receptor. We find that syndecan expression undergoes striking spatial and temporal changes during the period from the early cleavage through the late gastrula stages in the mouse embryo. Syndecan is detected initially at the 4-cell stage. Between the 4-cell and late morula stages, syndecan is present intracellularly and on the external surfaces of the blastomeres but is absent from regions of cell-cell contact. At the blastocyst stage, syndecan is first detected at cell-cell boundaries throughout the embryo and then, at the time of endoderm segregation, becomes restricted to the first site of matrix accumulation within the embryo, the interface between the primitive ectoderm and primitive endoderm. During gastrulation, syndecan is distributed uniformly on the basolateral cell surfaces of the embryonic ectoderm and definitive embryonic endoderm, but is expressed with an anteroposterior asymmetry on the surface of embryonic mesoderm cells, suggesting that it contributes to the process of mesoderm specification. In the extraembryonic region, syndecan is not detectable on most cells of the central core of the ectoplacental cone, but is strongly expressed by cells undergoing trophoblast giant cell differentiation and remains prominent on differentiated giant cells, suggesting a role in placental development. Immunoprecipitation studies indicate that the size of the syndecan core protein, although larger than that found in adult tissues (75 versus 69 x 10(3) Mr), does not change during peri-implantation development. The size distribution of the intact proteoglycan does change, however, indicating developmental alterations in its glycosaminoglycan composition. These results indicate potential roles for syndecan in epithelial organization of the embryonic ectoderm, in differential axial patterning of the embryonic mesoderm and in trophoblast giant cell function.
Animals↗
Syndecan, a developmentally regulated cell surface proteoglycan that binds extracellular matrix and growth factors.
Cellular behaviour during development is dictated, in part, by the insoluble extracellular matrix and the soluble growth factor peptides, the major molecules responsible for integrating cells into morphologically and functionally defined groups. These extracellular molecules influence cellular behaviour by binding at the cell surface to specific receptors that transduce intracellular signals in various ways not yet fully clear. Syndecan, a cell surface proteoglycan found predominantly on epithelia in mature tissues binds both extracellular matrix components (fibronectin, collagens I, III, V, and thrombospondin) and basic fibroblast growth factor (bFGF). Syndecan consists of chondroitin sulfate and heparan sulphate chains linked to a 31 kilodalton (kDa) integral membrane protein. Syndecan represents a family of integral membrane proteoglycans that differ in extracellular domains, but share cytoplasmic domains. Syndecan behaves as a matrix receptor: it binds selectively to components of the extracellular matrix, associates intracellularly with the actin cytoskeleton when cross-linked at the cell surface, its extracellular domain is shed upon cell rounding and it localizes solely to basolateral surfaces of simple epithelia. Mammary epithelial cells made syndecan-deficient become fibroblastic in morphology and cell behaviour, showing that syndecan maintains epithelial cell morphology. Syndecan changes in quantity, location and structure during development: it appears initially on four-cell embryos (prior to its known matrix ligands), becomes restricted in the pre-implementation embryo to the cells that will form the embryo proper, changes its expression due to epithelial-mesenchymal interactions (for example, induced in kidney mesenchyme by the ureteric bud), and with association of cells with extracellular matrix (for example, during B-cell differentiation), and ultimately, in mature tissues becomes restricted to epithelial tissues. The number and size of its glycosaminoglycan chains vary with changes in cell shape and organization yielding tissue type-specific polymorphic forms of syndecan. Its interactions with the major extracellular effector molecules that influence cell behaviour, its role in maintaining cell shape and its spatial and temporal changes in expression during development indicate that syndecan is involved in morphogenesis.
Amino Acid Sequence↗
Transient expression of a cell surface heparan sulfate proteoglycan (syndecan) during limb development.
Syndecan is an integral membrane proteoglycan that contains both heparan sulfate and chondroitin sulfate chains and that links the cytoskeleton to interstitial extracellular matrix components, including collagen and fibronectin. Immunohistochemistry with a monoclonal antibody directed to the core protein of the syndecan ectodomain has been used to analyze the distribution of this proteoglycan in the developing mouse limb bud and in high-density cultures of limb mesenchyme cells. By Day 9 of gestation when the limb buds are just apparent, syndecan is detected on cells throughout the limb region, including both ectodermal and mesenchymal components. This distribution does not change as the limb bud elongates along its proximodistal axis, except for its reduction in the apical ectodermal ridge. By Day 11, the intensity of immunofluorescence in the central core decreases relative to other regions. By Day 13 immunostaining is lost in the regions destined for chondrogenesis and myogenesis but persists in the limb ectoderm and peripheral and distal mesenchyme. In the limb mesenchyme cell cultures, syndecan is initially undetected, but is found throughout the culture by 24 hr. With further culture the antigen becomes reduced in chondrogenic foci and in association with myogenic cells. When chick limb ectoderm is placed on the high-density cultures, immunoreactivity in the mouse mesenchyme is enhanced suggesting that epithelial-mesenchymal interactions modulate syndecan expression in the limb bud. Based on analysis of 35S-labeled syndecan from the cultures, syndecan from limb mesenchyme cells contains more glycosaminoglycan chains and is larger in size than the previously described polymorphic forms of syndecan from various epithelia. The high affinity of syndecan for components of the extracellular matrix and its distribution in the early limb bud are consistent with a role in maintaining the morphologic integrity of the limb bud during the period of initiation and rapid outgrowth, and in preventing the onset of chondrogenesis.
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Endothelial heparan sulfate proteoglycan. I. Inhibitory effects on smooth muscle cell proliferation.
Proliferation of smooth muscle cells is an important component of pulmonary arterial morphogenesis, both during normal development and pathologic remodeling. However, little is known of the factors that regulate smooth muscle proliferation in these vessels. To investigate the hypothesis that factors produced by endothelial cells may regulate smooth muscle cell growth, we studied the effects of culture medium conditioned by fetal bovine pulmonary arterial endothelium on proliferation of smooth muscle cells in culture. This conditioned medium contains an inhibitor of smooth muscle proliferation that is degraded by nitrous acid, heparinase, and heparitinase, but resists degradation by protease, boiling, and chondroitin ABC lyase, indicating that the inhibitor is structurally similar to heparin. Inhibitor release occurs in both growing and confluent endothelial cell cultures and in the presence and absence of serum. A growth-inhibiting proteoglycan purified to homogeneity from endothelial cell-conditioned medium has physicochemical characteristics similar to those of the prototypic basement membrane heparan sulfate proteoglycan of the Englebreth-Holm-Swarm tumor: an overall size of approximately 10(6) D, heparan sulfate chains of 60,000 D, and a buoyant density of 1.33 g/ml. Antibody raised against the tumor basement proteoglycan recognizes this endothelial heparan sulfate proteoglycan, and Western blotting after SDS-PAGE demonstrates that the core proteins of both proteoglycans migrate as a doublet at apparent molecular weights of 450,000 and 360,000 D. Heparan sulfate glycosaminoglycan prepared from purified medium proteoglycan is a potent inhibitor of smooth muscle cell growth, exhibiting activity approximately 1,000 times greater than that of heparin. These results indicate that endothelial cells cultured from fetal bovine pulmonary arteries produce a basement membrane heparan sulfate proteoglycan that is a potent inhibitor of smooth muscle proliferation. This proteoglycan may mediate endothelial regulation of smooth muscle growth during development or pathologic pulmonary arterial remodeling.
Animals↗
Endothelial cell proteoglycans: possible mediators of vascular responses to injury.
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Endothelium, Vascular↗
Developmental biology: preventive medicine for neonatology.
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Cell Communication↗
Epithelial-mesenchymal interactions regulate the stage-specific expression of a cell surface proteoglycan, syndecan, in the developing kidney.
Morphogenesis of the kidney is regulated by reciprocal tissue interactions between the epithelial ureter bud and the metanephric mesenchyme. The differentiation of the kidney involves profound changes in the extracellular matrix, and therefore matrix receptors may have an important role in this process. We studied the expression of syndecan, a cell surface proteoglycan acting as a receptor for interstitial matrix materials, by using a monoclonal antibody against the core protein of the molecule. Syndecan was not detected in the uninduced metanephric mesenchyme. During the formation of the ureter bud from the Wolffian duct, syndecan appeared in the mesenchymal cells around the invaginating bud. Simultaneously with the first branching of the ureter bud, the whole nephric mesenchyme became syndecan positive, but a 3- to 10-cell-thick layer around the branching ureter bud, representing the presumptive tubular cells, was most intensely stained. During the assembly of the mesenchyme cells into pretubular aggregates, syndecan was detected in these aggregates and, to a lesser degree, in the morphologically undifferentiated mesenchyme. Thereafter syndecan was found only in the differentiating epithelium, from which it was gradually lost during maturation of the nephron. It was last detected in the periphery of the kidney, where tubulogenesis still continued. In transfilter cultures we showed that syndecan appeared in the nephric mesenchyme during the period when the mesenchyme becomes programmed to transform into epithelial structures. By using interspecies recombinations and a species-specific antibody we excluded the possibility that syndecan in the mesenchyme would originate from the inductor. We conclude that syndecan expression is regulated by epithelial-mesenchymal interactions. The findings that syndecan appeared as an early response to induction and that its distribution showed both spatial and temporal correlation with kidney morphogenesis suggest an important role for this molecule in development.
Animals↗
Molecular cloning of syndecan, an integral membrane proteoglycan.
We describe cDNA clones for a cell surface proteoglycan that bears both heparan sulfate and chondroitin sulfate and that links the cytoskeleton to the interstitial matrix. The cDNA encodes a unique core protein of 32,868 D that contains several structural features consistent with its role as a glycosamino-glycan-containing matrix anchor. The sequence shows discrete cytoplasmic, transmembrane, and NH2-terminal extracellular domains, indicating that the molecule is a type I integral membrane protein. The cytoplasmic domain is small and similar in size but not in sequence to that of the beta-chain of various integrins. The extracellular domain contains a single dibasic sequence adjacent to the extracellular face of the transmembrane domain, potentially serving as the protease-susceptible site involved in release of this domain from the cell surface. The extracellular domain contains two distinct types of putative glycosaminoglycan attachment sites; one type shows sequence characteristics of the sites previously described for chondroitin sulfate attachment (Bourdon, M. A., T. Krusius, S. Campbell, N. B. Schwartz, and E. Ruoslahti. 1987. Proc. Natl. Acad. Sci. USA. 84:3194-3198), but the other type has newly identified sequence characteristics that potentially correspond to heparan sulfate attachment sites. The single N-linked sugar recognition sequence is within the putative chondroitin sulfate attachment sequence, suggesting asparagine glycosylation as a mechanism for regulating chondroitin sulfate chain addition. Both 5' and 3' regions of this cDNA have sequences substantially identical to analogous regions of the human insulin receptor cDNA: a 99-bp region spanning the 5' untranslated and initial coding sequences is 67% identical and a 35-bp region in the 3' untranslated region is 81% identical in sequence. mRNA expression is tissue specific; various epithelial tissues show the same two sizes of mRNA (2.6 and 3.4 kb); in the same relative abundance (3:1), the cerebrum shows a single 4.5-kb mRNA. This core protein cDNA describes a new class of molecule, an integral membrane proteoglycan, that we propose to name syndecan (from the Greek syndein, to bind together).
Amino Acid Sequence↗
B lymphocytes express and lose syndecan at specific stages of differentiation.
Lymphopoietic cells require interactions with bone marrow stroma for normal maturation and show changes in adhesion to matrix during their differentiation. Syndecan, a heparan sulfate-rich integral membrane proteoglycan, functions as a matrix receptor by binding cells to interstitial collagens, fibronectin, and thrombospondin. Therefore, we asked whether syndecan was present on the surface of lymphopoietic cells. In bone marrow, we find syndecan only on precursor B cells. Expression changes with pre-B cell maturation in the marrow and with B-lymphocyte differentiation to plasma cells in interstitial matrices. Syndecan on B cell precursors is more heterogeneous and slightly larger than on plasma cells. Syndecan 1) is lost immediately before maturation and release of B lymphocytes into the circulation, 2) is absent on circulating and peripheral B lymphocytes, and 3) is reexpressed upon their differentiation into immobilized plasma cells. Thus, syndecan is expressed only when and where B lymphocytes associate with extracellular matrix. These results indicate that B cells differentiating in vivo alter their matrix receptor expression and suggest a role for syndecan in B cell stage-specific adhesion.
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The differential effect of prenatal care on the incidence of low birth weight among blacks and whites in a prepaid health care plan.
Black infants are born with low birth weights (less than 2500 g) and very low birth weights (less than 1500 g) at twice the rate of white infants. We investigated the effect of prenatal care delivered in a health maintenance organization on the birth weights of black and white infants at normal risk for low birth weight. Using birth certificates for all children born in 1978 in the California Kaiser-Permanente hospitals, we studied data on more than 31,000 black and white newborns whose mothers' ages and levels of education were comparable. The data show that black mothers used prenatal care less extensively and had a higher incidence of infants with low birth weights (8.4 vs. 3.6 percent) and very low birth weights (2.0 vs. 0.7 percent) than white mothers. The difference in the use of prenatal care, however, accounted for less than 15 percent of the difference in the incidence of low birth weight. The rates of low birth weight, very low birth weight, and preterm birth (less than 260 days' gestation) decreased with increasing levels of prenatal care for both blacks and whites. However, increasing levels of care were associated with a greater reduction among black infants than among white infants in low birth weight, very low birth weight, and low birth weight at term (greater than or equal to 260 days' gestation). When we compared mothers who received adequate care with those who received inadequate care, the relative risk of giving birth to a very-low-birth-weight infant was reduced 3.6-fold (95 percent confidence interval, 2.0 to 6.6) for black mothers and 2.1-fold (confidence interval, 1.3 to 3.4) for white mothers; the relative risk of giving birth to a low-birth-weight infant at term was reduced 3.4-fold (95 percent confidence interval, 2.2 to 5.4) for black mothers and 1.6-fold (confidence interval, 1.1 to 2.3) for white mothers. We conclude that even in a population of women at low risk for giving birth to low-birth-weight infants, prenatal care is more beneficial for blacks than for whites.
Black or African American↗
The cell surface proteoglycan of mouse mammary epithelial cells. The extracellular domain contains N terminus and a peptide sequence present in a conditioned medium proteoglycan.
The cell surface proteoglycan of mouse mammary epithelial (NMuMG) cells behaves as a receptor for interstitial matrix materials and consists of a membrane-associated domain and an extracellular domain (ectodomain). The ectodomain can be released intact from the cell surface by mild trypsin treatment and appears to be shed from the cells into the culture medium by cleavage from the membrane-associated domain. We have examined the chemical relationship between the trypsin-released proteoglycan and shed proteoglycan to assess their relationship to each other and to the cell surface. Purification and amino acid sequencing of the ectodomain released by mild trypsin treatment resulted in no clear signal until the protein was cleaved by CNBr treatment, suggesting that its N terminus is blocked and oriented extracellularly. The amino acid sequence identified in the trypsin-released ectodomain is present near the N terminus of the shed proteoglycan purified from conditioned medium, indicating that both forms possess closely related (if not identical) core proteins. The sequence reveals a pentapeptide identical to one near the C terminus of the rat hepatic lectin (RHL-1, rat asialoglycoprotein receptor). The medium proteoglycan, which migrates as a smear on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (between 93 and 200 kDa), is heterogeneous due to varying amounts of glycosaminoglycan and substituted O-linked oligosaccharide present on an approximately 46-kDa polypeptide.
Amino Acid Sequence↗
Glycosaminoglycans vary in accumulation along the neuraxis during spinal neurulation in the mouse embryo.
We have utilized the method of whole embryo culture for metabolic labeling of mouse embryos with [3H]glucosamine during closure of neural folds at the posterior neuropore (27- to 29-somite stage). Accumulations of newly synthesized glycopeptides, lactosaminoglycans, hyaluronate, and sulfated glycosaminoglycans (GAG) were assessed by ion-exchange chromatography of glycoconjugates isolated from labeled embryos. Accumulation of hyaluronate and sulfated GAG was greatest in the posterior neuropore and decreased progressively toward the hindbrain where neurulation was already complete. Hyaluronate comprised a progressively smaller proportion of total newly synthesized glycoconjugate from the posterior neuropore toward the cranial region and glycopeptides showed the opposite trend. Sulfated GAG and lactosaminoglycans showed no consistent differences in relative abundance along the neuraxis. Autoradiographic analysis of newly synthesized glycoconjugates revealed especially heavy incorporation into developing basement membranes, beneath the neuroepithelium and around the notochord, in the posterior neuropore and recently closed neural tube regions, but not at more cranial levels of the neuraxis. Predigestion of sections with a specific hyaluronidase showed a significant quantity of this glycoconjugate to be hyaluronate. These results are consistent with a role for neuroepithelial and notochordal basement membrane hyaluronate in spinal neurulation.
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Accumulation of basement membrane-associated hyaluronate is reduced in the posterior neuropore region of mutant (curly tail) mouse embryos developing spinal neural tube defects.
We investigated the accumulation of newly synthesized glycoconjugates during spinal neurulation in mutant curly tail mouse embryos, a proportion of which develop lower spinal neural-tube defects (NTD). Embryos undergoing closure of the posterior neuropore (27- to 29-somite stage) were labeled in vitro with [3H]glucosamine, and [3H]glycoconjugates were analyzed by ion-exchange chromatography. Mutant embryos undergoing normal spinal neurulation exhibited a pattern of glycoconjugate accumulation closely similar to that observed for nonmutant embryos (Copp and Bernfield, 1988, Dev. Biol. 130, 573-582). Mutant embryos developing spinal NTD accumulated reduced amounts of [3H]hyaluronate specifically in the posterior neuropore region. Other embryonic regions and other glycoconjugates appeared unaffected by the developmental abnormality. Autoradiographic analysis of labeled curly tail embryos confirmed that [3H]hyaluronate accumulates in reduced amounts in the posterior neuropore region and indicated that this reduction is mainly localized to the site of developing basement membranes, beneath the neuroepithelium and around the notochord. Accumulation of [3H]hyaluronate in the interstitial mesenchymal matrix of the posterior neuropore region is not consistently affected in embryos developing spinal NTD. These results provide support for a role for basement-membrane hyaluronate in lower spinal neurulation.
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Cell surface proteoglycan expression correlates with epithelial-mesenchymal interaction during tooth morphogenesis.
Tooth morphogenesis and differentiation of the dental cells are guided by interactions between epithelial and mesenchymal tissues. Because the extracellular matrix is involved in these interactions, the expression of matrix receptors located at the cell surface may change during this developmental sequence. We have examined the distribution of an epithelial cell surface proteoglycan antigen, known to behave as a receptor for interstitial matrix, during tooth morphogenesis. Intense staining was seen around the cells of the embryonic oral epithelium as well as the dental epithelium at the early bud stage. With development, expression was greatly reduced in the enamel organ. Differentiation of these cells into ameloblasts was associated with the loss of expression, while the epithelial cells remaining in the stratum intermedium and stellate reticulum regained intense staining. The PG antigen was weakly expressed in the loose neural crest-derived jaw mesenchyme but it became strongly reactive in the condensed dental papilla mesenchyme when extensive morphogenetic movements took place. With development, the PG antigen disappeared from the advanced dental papilla mesenchyme but persisted in the dental sac mesenchyme, which gives rise to periodontal tissues. The PG antigen was not expressed by odontoblasts. Hence, the expression of the PG antigen changes during the epithelial-mesenchymal interactions of tooth development and is lost during terminal cell differentiation. The expression follows morphogenetic rather than histologic boundaries. The acquisition and loss of expression in epithelial and mesenchymal tissues during tooth development suggest that this proteoglycan has specific functions in the epithelial-mesenchymal interactions that guide morphogenesis.
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Molecular polymorphism of a cell surface proteoglycan: distinct structures on simple and stratified epithelia.
Epithelial cells are organized into either a single layer (simple epithelia) or multiple layers (stratified epithelia). Maintenance of these cellular organizations requires distinct adhesive mechanisms involving many cell surface molecules. One such molecule is a cell surface proteoglycan, named syndecan, that contains both heparan sulfate and chondroitin sulfate chains. This proteoglycan binds cells to fibrillar collagens and fibronectin and thus acts as a receptor for interstitial matrix. The proteoglycan is restricted to the basolateral surface of simple epithelial cells, but is located over the entire surface of stratified epithelial cells, even those surfaces not contacting matrix. We now show that the distinct localization in simple and stratified epithelia correlates with a distinct proteoglycan structure. The proteoglycan from simple epithelia (modal molecular size, 160 kDa) is larger than that from stratified epithelia (modal molecular size, 92 kDa), but their core proteins are identical in size and immunoreactivity. The proteoglycan from simple epithelia has more and larger heparan sulfate and chondroitin sulfate chains than the proteoglycan from stratified epithelia. Thus, the cell surface proteoglycan shows a tissue-specific structural polymorphism due to distinct posttranslational modifications. This polymorphism likely reflects distinct proteoglycan functions in simple and stratified epithelia, potentially meeting the different adhesive requirements of the cells in these different organizations.
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Cell surface proteoglycan binds mouse mammary epithelial cells to fibronectin and behaves as a receptor for interstitial matrix.
The proteoglycan (PG) on the surface of NMuMG mouse mammary epithelial cells consists of at least two functional domains, a membrane-intercalated domain which anchors the PG to the plasma membrane, and a trypsin-releasable ectodomain which bears both heparan and chondroitin sulfate chains. The ectodomain binds cells to collagen types I, III, and V, but not IV, and has been proposed to be a matrix receptor. Because heparin binds to the adhesive glycoproteins fibronectin, an interstitial matrix component, and laminin, a basal lamina component, we asked whether the cell surface PG also binds these molecules. Cells harvested with either trypsin or EDTA bound to fibronectin; binding of trypsin-released cells was inhibited by the peptide GRGDS but not by heparin, whereas binding of EDTA-released cells was inhibited only by a combination of GRDS and heparin, suggesting two distinct cell binding mechanisms. In the presence of GRGDS, the EDTA-released cells bound to fibronectin via the cell surface PG. Binding via the cell surface PG was to the COOH-terminal heparin binding domain of fibronectin. In contrast with the binding to fibronectin, EDTA-released cells did not bind to laminin under identical assay conditions. Liposomes containing the isolated intact cell surface PG mimic the binding of whole cells. These results indicate that the mammary epithelial cells have at least two distinct cell surface receptors for fibronectin: a trypsin-resistant molecule that binds cells to the sequence RGD and a trypsin-labile, heparan sulfate-rich PG that binds cells to the COOH-terminal heparin binding domain. Because the cell surface PG binds cells to the interstitial collagens (types I, III, and V) and to fibronectin, but not to basal lamina collagen (type IV) or laminin, we conclude that the cell surface PG is a receptor on epithelial cells specific for interstitial matrix components.
Animals↗