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PubMed1998

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↗
PubMed1996

Heparan sulfate proteoglycan expression in chronic cholestatic human liver diseases.

The mechanism of bile ductular reaction and accompanying fibrogenesis depends on interactions of ductular cells with the matrix and growth factors. Heparan sulfate proteoglycans (HSPGs) are essential cofactors in cell-matrix adhesion processes, in cell-cell recognition systems, and in receptor-growth factor interactions. We used monoclonal antibodies specific for the cell surface HSPGs (syndecans, glypican), for matrix HSPG (perlecan), and for heparan sulfate carbohydrate (HS) to investigate their immunohistochemical expression in 20 specimens with chronic cholestatic liver disease and in five normal human liver specimens. Because activated hepatic stellate cells (HSC are a major source of fibrosis in the liver, we also examined HSPG expression in primary cultures of human activated HSC using immunocytochemistry and Western blotting and for syndecan-1 also Northern blotting. In comparison with bile ductular cells of normal liver, reactive ductules in chronic cholestasis were marked by an elevated expression of syndecan-1, surrounded by an increased perlecan expression. In acinar zone 1, large stimulated macrophages and HSC, present in increased numbers, were strongly positive for syndecan-3. Cultured HSC showed a membranous staining pattern for syndecan-1, syndecan-3, and heparan sulfate, and in addition intracellular staining for syndecan-2, -3, and 4. Perlecan immunoreactivity was detected as intercellular strings. Western blotting revealed positive bands with all antibodies and Northern blotting for syndecan-1 was also positive. These results show that cultured human HSC can synthesize all four syndecans, glypican, and perlecan. These data reveal changes in the expression of syndecan-1, syndecan-3, and perlecan in human chronic cholestatic liver disease, that may be important in the deposition of matrix components and activation of growth factors that support ductular reaction and accompanying fibrogenesis.

Antibodies, Monoclonal↗
PubMed1994

Syndecans, cell surface heparan sulfate proteoglycans, are induced by a proline-rich antimicrobial peptide from wounds.

Cell surface heparan sulfate proteoglycans, such as the syndecans, are required for cellular responses to heparin-binding growth factors and extracellular matrix components. Expression of syndecan-1 and -4 is induced in mesenchymal cells during wound repair in the mouse, consistent with a role for syndecans in regulating cell proliferation and migration in response to these effectors. Here we show that wound fluid contains inductive activity that mimics the in vivo induction in time of appearance, specificity for mesenchymal cells, and selectivity for syndecan-1 and -4. We have purified and synthesized a 4.8-kDa proline-rich protein from wound fluid that reproduces this induction of syndecan-1 and -4 in cultured cells. This peptide, identical to the antibacterial peptide PR-39, is released into the wound by the cellular infiltrate and induces syndecan expression at the same peptide concentrations that lyse bacteria. These results indicate that wounds contain a multifunctional protein that induces mammalian cells to express cell surface heparan sulfate proteoglycans as part of the wound repair process and that kills bacteria as part of a nonimmune defense mechanism.

3T3 Cells↗
PubMed1996

Human ryudocan from endothelium-like cells binds basic fibroblast growth factor, midkine, and tissue factor pathway inhibitor.

Ryudocan, a heparan sulfate proteoglycan, was isolated from human endothelium-like EAhy926 cells by a combination of ion-exchange and immunoaffinity chromatography. Purified human ryudocan has biochemical properties similar to those of rat ryudocan isolated from microvascular endothelial cells. Human ryudocan contains only heparan sulfate (HS) glycosaminoglycan chains along with a core protein with an apparent molecular mass of 30 kDa. We evaluated the interactions between purified human ryudocan and several extracellular ligands by using a solid-phase binding assay. We found that basic fibroblast growth factor (bFGF), midkine (MK), and tissue factor pathway inhibitor (TFPI) exhibit significant ryudocan binding. Heparitinase (but not chondroitin ABC lyase) treatment destroyed the ability of ryudocan binding to bFGF, MK, and TFPI. Heparin and HS, but not chondroitin sulfate, inhibited such ryudocan binding. Thus, the HS chains of ryudocan appear to be responsible for its binding to bFGF, MK, and TFPI. The apparent dissociation constants for purified ryudocan were as follows: bFGF, 0.50 nM; MK, 0.30 nM; and TFPI, 0.74 nM. Immunohistochemical analysis revealed that ryudocan was expressed in fibrous connective tissues, peripheral nerve tissues, and placental trophoblasts. These findings suggest that ryudocan may possess multiple biological functions, such as bFGF modulation, neurite growth promotion, and anticoagulation, via HS-binding effectors in the cellular microenvironment.

Amino Acid Sequence↗
PubMed1997

Regulated shedding of syndecan-1 and -4 ectodomains by thrombin and growth factor receptor activation.

The syndecan family of transmembrane heparan sulfate proteoglycans is abundant on the surface of all adherent mammalian cells. Syndecans bind and modify the action of various growth factors/cytokines, proteases/antiproteases, cell adhesion molecules, and extracellular matrix components. Syndecan expression is highly regulated during wound repair, a process orchestrated by many of these effectors. Each syndecan ectodomain is shed constitutively by cultured cells, but the mechanism and significance of this shedding are not understood. Therefore, we examined (i) whether physiological agents active during wound repair influence syndecan shedding, and (ii) whether wound fluids contain shed syndecan ectodomains. Using SVEC4-10 endothelial cells we find that certain proteases and growth factors accelerate shedding of the syndecan-1 and -4 ectodomains. Protease-accelerated shedding is completely inhibited by serum-containing media. Thrombin activity is duplicated by the 14-amino acid thrombin receptor agonist peptide that directly activates the thrombin receptor and is not inhibited by serum. Epidermal growth factor family members accelerate shedding but FGF-2, platelet-derived growth factor-AB, transforming growth factor-beta, tumor necrosis factor-alpha, and vascular endothelial cell growth factor 165 do not. Shed ectodomains are soluble, stable in the conditioned medium, have the same size core proteins regardless whether shed at a basal rate, or accelerated by thrombin or epidermal growth factor-family members and are found in acute human dermal wound fluids. Thus, shedding is accelerated by activation of at least two distinct receptor classes, G protein-coupled (thrombin) and protein tyrosine kinase (epidermal growth factor). Proteases and growth factors active during wound repair can accelerate syndecan shedding from cell surfaces. Regulated shedding of syndecans suggests physiological roles for the soluble proteoglycan ectodomains.

Amino Acid Sequence↗
PubMed1998

Tyrosine phosphorylation of syndecan-1 and -4 cytoplasmic domains in adherent B82 fibroblasts.

The syndecans, a family of cell surface proteoglycans, have highly conserved cytoplasmic domains that bind proteins containing PDZ domains and co-localize with the actin cytoskeleton. The syndecan cytoplasmic domains contain four conserved tyrosine residues, two of which are located within favorable sequences for phosphorylation. Endogenous tyrosine phosphorylation of syndecans-1 and -4 is detected in adherent B82 fibroblasts. Approximately 1.5% of total syndecan is endogenously phosphorylated, while most, if not all, cell surface syndecan is phosphorylated following treatment with the tyrosine phosphatase inhibitor pervanadate. Syndecan phosphorylation is also detected in Raji-S1 and NMuMG cells, but only following treatment with vanadate or pervanadate, suggesting that endogenous phosphorylation is maintained in an "off" state in these cells. Endogenous syndecan phosphorylation in B82 cells is rapidly blocked by genistein (IC50 < 10 microM) confirming the presence of a constitutively active kinase and a corresponding tyrosine phosphatase. Phosphorylation is also inhibited by herbimycin A (IC50 < 1.0 microM) and staurosporine (IC50 < 1.0 nM), suggesting a role for Src family kinases in regulating syndecan phosphorylation. Together, these data suggest an important role for tyrosine phosphorylation of the syndecan cytoplasmic domains in regulating downstream signaling events in response to cell adhesion and/or growth factor activity.

Amino Acid Sequence↗
PubMed1999

Elastase-mediated release of heparan sulfate proteoglycans from pulmonary fibroblast cultures. A mechanism for basic fibroblast growth factor (bFGF) release and attenuation of bfgf binding following elastase-induced injury.

We have investigated elastase-mediated alterations in the expression of basic fibroblast growth factor (bFGF) receptors and proteoglycan co-receptors and characterized the subsequent effects on bFGF receptor binding profiles. For these studies, pulmonary fibroblast cultures were treated with porcine pancreatic elastase, and elastase-mediated changes in bFGF receptor expression and binding profiles were assessed. Quantitation of [(35)S]sulfate-labeled proteoglycan and total glycosaminoglycan release from fibroblast matrices indicated that elastase treatment released sulfated proteoglycan from the cell surface in a time- and dose-dependent fashion that correlated strongly with elastase-mediated bFGF release. Ligand binding studies indicated that elastase treatment decreased total binding of (125)I-bFGF to the cell surface and affected both fibroblast growth factor receptor and heparan sulfate proteoglycan (HSPG) binding sites. Western blot analyses indicated that elastase treatment did not release significant amounts of fibroblast growth factor receptor protein. These findings indicate that elastase-mediated HSPG release from fibroblast matrices reduces the effective affinity of bFGF for its receptor. Collectively, these studies suggest that HSPG co-receptors are important mediators of the pulmonary fibroblast response to elastase treatment and that bFGF, HSPG, and other elastase-released entities play an important role in the response of the lung to chronic injury.

Animals↗
PubMed1992

Molecular cloning of amphiglycan, a novel integral membrane heparan sulfate proteoglycan expressed by epithelial and fibroblastic cells.

We have synthesized an antisense oligonucleotide primer that matches a supposedly conserved sequence in messages for heparan sulfate proteoglycans with transmembrane orientations. With the aid of this primer we have amplified partial and selected full-length copies of a message from human lung fibroblasts that codes for a novel integral membrane heparan sulfate proteoglycan. The encoded protein is 198 amino-acids long, with discrete cytoplasmic, transmembrane, and amino-terminal extracellular domains. Except for the sequences that represent putative heparan sulfate chain attachment sites, the extracellular domain of this protein has a unique structure. The transmembrane and cytoplasmic domains, in contrast, are highly similar to the corresponding domains of fibroglycan and syndecan, the two cell surface proteoglycans that figured as models for the design of the antisense primer. This similarity includes the conservation of four tyrosine residues, one immediately in front of the stop transfer sequence and three in the cytoplasmic segment, and of the most proximal and most distal cytoplasmic sequences. The cDNA detects a single 2.6-kb message in cultured human lung fibroblasts and in a variety of human epithelial and fibroblastic cell lines. Polyclonal and monoclonal antibodies raised against the encoded peptide after expression as a beta-galactosidase fusion protein react with the 35-kD coreprotein of a cell surface heparan sulfate proteoglycan of human lung fibroblasts and decorate the surface of many cell types. We propose to name this proteoglycan "amphiglycan" (from the Greek words amphi, "around, on both sides of" and amphoo, "both") referring to its domain structure which extends on both sides of the plasmamembrane, and to its localization around cells of both epithelial and fibroblastic origin.

Amino Acid Sequence↗
PubMed1994

Syndecan 4 heparan sulfate proteoglycan is a selectively enriched and widespread focal adhesion component.

Focal adhesion formation in fibroblasts results from complex transmembrane signaling processes initiated by extracellular matrix molecules. Although a role for integrins with attendant tyrosine kinases has been established, there is evidence that cell surface heparan sulfate proteoglycans (HSPGs) are also involved with an associated role of protein kinase C. The identity of the proteoglycan has remained elusive, but we now report that syndecan 4 (ryudocan/amphiglycan) is present in focal adhesions of a number of cell types. Affinity-purified antibodies raised against a unique portion of the cytoplasmic domain of syndecan 4 core protein recognized an HSPG of similar characteristics to those of syndecan 4. These antibodies stained focal adhesions only after cell permeabilization and recognized differing mammalian species. Syndecan 4 was associated with focal adhesions that contained either beta 1 or beta 3 integrin subunits and those that formed on substrates of fibronectin, laminin, vitronectin, or type I collagen. No focal adhesions were found that were vinculin-containing but lacked syndecan 4. In contrast, syndecan 2, whose cytoplasmic domain is closely homologous to syndecan 4, does not appear to be a focal adhesion component. Thus, syndecan 4 represents a new transmembrane focal adhesion component, probably involved in their assembly.

Amino Acid Sequence↗
PubMed1997

Molecular cloning, genomic organization, promoter activity, and tissue-specific expression of the mouse ryudocan gene.

Ryudocan, a ubiquitous heparan sulfate proteoglycan, is a member of the syndecan family of cell surface proteoglycans. The full-length cDNA encoding the murine ryudocan core protein has now been cloned and sequenced. The deduced primary structure of mouse ryudocan, including the three glycosaminoglycan attachment sites in the extracellular domain as well as the transmembrane and cytoplasmic regions, is highly similar to those of the rat, human, and chicken proteins. Northern analysis detected a 2.7-kb transcript in all mouse tissues examined, with the highest concentrations apparent in liver, kidney, and lung. The mouse ryudocan gene was shown to span approximately 19.7 kb of genomic DNA and to contain five exons, with an intron-exon organization identical to that of the human gene. The promoter region of the mouse gene contains various cis-acting elements, including a TATA-like box and a GC box as well as potential binding sites for the transcription factors NF-IL6, MyoD, GATA, C/EBP, AP-2, NF-kappaB, AP-1, and Sp1. Transient transfection experiments with a construct containing the 690 bp upstream of the transcription start site fused to a luciferase reporter gene showed functional promoter activity. Deletion analysis suggested that the proximal promoter region including the TATA-like box, the GC box, and other Sp1 binding sites was required for full transcriptional activity. These findings will be useful for the study of ryudocan gene regulation and the generation of mice with targeted disruption of the gene.

Amino Acid Sequence↗
PubMed1993

Isolation and characterization of ryudocan and syndecan heparan sulfate proteoglycans, core proteins, and cDNAs from a rat endothelial cell line.

We have isolated heparan sulfate proteoglycans (HSPGs) from cloned rat microvascular endothelial cells using a combination of ion-exchange chromatography, affinity fractionation with antithrombin III (AT III), and gel filtration in denaturing solvents. The anticoagulantly active heparan sulfate proteoglycans (HSPGact) which bind tightly to AT III bear mainly anticoagulantly active heparan sulfate (HSact) whereas the anticoagulantly inactive heparan sulfate proteoglycans (HSPGinact) possess mainly anticoagulantly inactive heparan sulfate (HSinact). The core proteins of HSPGact and HSPGinact were isolated by treatment with Flavobacterium heparitinase and purification by ion-exchange chromatography. SDS-PAGE showed that both sets of core proteins exhibited three major components with M(r) of 25-, 30-, and 50-kD, respectively. Peptide mapping revealed that HSPGact and HSPGinact possess extremely similar core proteins. The primary sequences of internal peptides obtained from HSPGinact core proteins and the NH2-terminal sequence analyses of the 25-kD component from the HSPGinact core proteins demonstrate that the 30-kD component is a previously unidentified species--designated as ryudocan--with the 25-kD component representing a proteolytic degradation product; while the 50-kD component is the rat homolog of syndecan [Saunders S, Jalkanen M, O'Farrell S, Bernfield M: J Cell Biol 1989; 108:1547-1556]. Specific oligonucleotide probes were obtained for ryudocan and syndecan by PCR, and the corresponding cDNAs were isolated from a RFP-EC library. The cDNAs encode type I integral membrane proteins of 202 and 313 amino acids, respectively, which have homologous transmembrane and intracellular domains but very distinct extracellular regions. In particular, ryudocan exhibits only 3 potential glycosaminoglycan (GAG) attachment sites within the extracellular region while syndecan has 5 GAG attachment sites within the same domain. The levels of ryudocan and syndecan mRNA were measured by quantitative PCR in primary microvascular endothelial cells and associated non-endothelial cells isolated by cell sorting. Ryudocan and syndecan mRNAs were abundantly expressed in both populations representing about 0.1-0.5% of mRNA.

Amino Acid Sequence↗
PubMed1999

Heparan sulfate proteoglycan expression is induced during early erythroid differentiation of multipotent hematopoietic stem cells.

Heparan sulfate (HS) proteoglycans of bone marrow (BM) stromal cells and their extracellular matrix are important components of the microenvironment of hematopoietic tissues and are involved in the interaction of hematopoietic stem and stromal cells. Although previous studies have emphasized the role of HS proteoglycan synthesis by BM stromal cells, we have recently shown that the human hematopoietic progenitor cell line TF-1 also expressed an HS proteoglycan. Immunochemical, reverse transcriptase-polymerase chain reaction (RT-PCR), and Northern blot analysis of this HS proteoglycan showed that it was not related to the syndecan family of HS proteoglycans or to glypican. To answer the question of whether the expression of HS proteoglycans is associated with the differentiation state of hematopoietic progenitor cells, we have analyzed the proteoglycan synthesis of several murine and human hematopoietic progenitor cell lines. Proteoglycans were isolated from metabolically labeled cells and purified by several chromatographic steps. Isolation and characterization of proteoglycans from the cell lines HEL and ELM-D, which like TF-1 cells have an immature erythroid phenotype, showed that these cells synthesize the same HS proteoglycan, previously detected in TF-1 cells, as a major proteoglycan. In contrast, cell lines of the myeloid lineage, like the myeloblastic/promyelocytic cell lines B1 and B2, do not express HS proteoglycans. Taken together, our data strongly suggest that expression of this HS proteoglycan in hematopoietic progenitor cell lines is associated with the erythroid lineage. To prove this association we have analyzed the proteoglycan expression in the nonleukemic multipotent stem cell line FDCP-Mix-A4 after induction of erythroid or granulocytic differentiation. Our data show that HS proteoglycan expression is induced during early erythroid differentiation of multipotent hematopoietic stem cells. In contrast, during granulocytic differentiation, no expression of HS proteoglycans was observed.

Cell Differentiation↗
PubMed1992

Molecular cloning and expression of two distinct cDNA-encoding heparan sulfate proteoglycan core proteins from a rat endothelial cell line.

The cloned rat fat pad endothelial cell (RFP-EC) line synthesizes anticoagulantly active heparan sulfate proteoglycans (HSPGact) and anticoagulantly inactive heparan sulfate proteoglycans (HSPGinact), both of which exhibit 25-, 30-, and 50-kDa core proteins of extremely similar structure. The primary sequences of internal peptides obtained from HSPGinact core proteins and the NH2-terminal sequence analyses of the 25-kDa component from the HSPGinact core proteins demonstrate that the 30-kDa component is a previously unidentified species, designated as ryudocan, with the 25-kDa component representing a proteolytic degradation product, while the 50-kDa component is the rat homolog of syndecan (Saunders, S. Jalkanen, M., O'Farrell, S., and Bernfield, M. (1989) J. Cell Biol. 108, 1547-1556). Specific oligonucleotide probes were obtained for ryudocan and syndecan by polymerase chain reaction, and the corresponding cDNAs were isolated from a RFP-EC library. The cDNAs encode type I integral membrane proteins of 202 and 313 amino acids, respectively, which have homologous transmembrane and intracellular domains but very distinct extracellular regions. In particular, ryudocan exhibits only three potential glycosaminoglycan attachment sites within the extracellular region while syndecan has five glycosaminoglycan attachment sites within the same domain. Both species are expressed in RFP-EC lines, primary rat aortic smooth muscle cells and primary rat skin fibroblast cells. The levels of ryudocan and syndecan mRNA were measured by quantitative polymerase chain reaction in primary microvascular endothelial cells and closely associated non-endothelial cells isolated by cell sorting. Ryudocan and syndecan mRNAs were abundantly expressed in both populations representing about 0.1-0.5% of mRNA.

Amino Acid Sequence↗
PubMed1994

Characterization of ryudocan glycosaminoglycan acceptor sites.

The specificity of the glycosaminoglycan (GAG) acceptor sites of ryudocan was examined by stably expressing epitope-tagged ryudocan cDNA constructs in mouse L cells, which normally produce this proteoglycan. Immunopurified ryudocan was glycanated with both heparan sulfate (HS) and chondroitin sulfate (CS). The attachment of GAGs to ryudocan was prevented by creating Ser-->Thr mutations in all possible combinations at positions 44, 65, and 67. The resulting ryudocan of exogenous origin was immunopurified and evaluated with regard to attached GAG chains. The data reveal that ryudocan possesses three functional GAG attachment sites, that the sites are always occupied with GAG chains, and that each site is capable of bearing either HS or CS. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of GAG lyase digests of intact ryudocan reveal the production of the following multiple isoforms: pure HS-ryudocan, various HS/CS-hybrids, and pure CS-ryudocan. The data suggest that the occupancy bias of each site for HS or CS is slight and that each site functions in a relatively independent fashion. The GAG lyase analysis of partially purified L cell proteoglycans shows two pure CS-homoglycans with core proteins of M(r) = 130,000 and 52,000, respectively. A similar analysis of immunopurified L cell glypican demonstrates that this species only exists as a pure HS-homoglycan. The production of pure homoglycans by this clonal cell line strongly suggests that the functional promiscuity of GAG attachment sites of ryudocan must be encoded in the core protein structure. This property of ryudocan is not peculiar to L cells, as ryudocan synthesized by early passage human endothelial cells also bears both HS and CS. The production of multiple isoforms of ryudocan may serve to expand the functional versatility of this cell surface component and allow it to participate in many different biologic processes.

Amino Acid Sequence↗
PubMed1994

Pathway-specific regulation of the synthesis of anticoagulantly active heparan sulfate.

L cells and endothelial cells synthesize a heparan sulfate (HS) subpopulation, HSact, that exhibits anticoagulant activity due to a specific monosaccharide sequence; the remaining heparan sulfate, HSinact, lacks this region of defined structure and is anticoagulantly inactive. HSact biosynthesis was examined in these two cell types by stably expressing epitope-tagged rat ryudocan (ryudocan12CA5), which possesses three glycosaminoglycan (GAG) acceptor sites. Both HSact and HSinact were present on ryudocan12CA5 isolated from L cells and endothelial cells; thus, a core protein with a unique primary sequence initiates the synthesis of both GAGs. The expression in L cells of ryudocan12CA5 variants containing a single functional GAG acceptor site demonstrated that each of the three acceptor regions initiates the synthesis of both types of GAGs to a similar extent. Most importantly, in both cell types total HSact generation declined as a function of ryudocan12CA5 overexpression even though HSinact production increased linearly as a function of this variable. This discordant relationship is a general property of the biosynthetic machinery since in both cell types HSact production was reduced to an equal extent on protein cores of either exogenous or endogenous origins. The suppression of HSact generation was also observed with a secreted form of core protein lacking transmembrane and cytoplasmic domains or by a GAG acceptor site mutated form of core protein incapable of augmenting GAG synthesis. These results suggest that elevated intracellular levels of core protein saturate the capacity of a critical component of the HSact biosynthetic machinery. This critical component is not a member of the common set of biosynthetic enzymes involved in the production of HSact and HSinact since no structural changes were observed in either GAG during overexpression of core protein. Based upon the above data, we conclude that increased intracellular levels of ryudocan probably act by saturating the capacity of components which regulate HSact production by coordinating the function of biosynthetic enzymes.

Amino Acid Sequence↗
PubMed1994

Characterization of a cell mutant specifically defective in the synthesis of anticoagulantly active heparan sulfate.

We have investigated the characteristics of clonal L cell mutant VI-7 that has previously been demonstrated to exhibit reduced levels of cell surface proteoglycans containing heparan sulfate (HS) with regions of defined monosaccharide sequence that interact with antithrombin (HSact) (de Agostini, A. L., Lau, H. K., Leone, C., Youssoufian, H., and Rosenberg, R. D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9784-9788). Pulse labeling revealed that the synthesis of HSact in mutant cells, as compared to wild-type cells, was reduced by 5-7-fold, which is identical to the previously observed reduction in cell surface-bound antithrombin. This alteration is independent of growth state since production of HSact by mutant cells, as compared to wild-type cells, is decreased to a similar extent in exponentially growing and post-confluent cultures. The synthetic defect was specific for HSact since production of total HS and chondroitin sulfate by mutant cells, as compared to wild-type cells, was identical in magnitude. The synthetic abnormality is not due to an alteration in core protein since complementation was not observed when mutant cells were stably transfected with the epitope-tagged ryudocan cDNA which can initiate HSact production. Structural analyses revealed that HS from mutant cells, as compared to wild-type cells, exhibited normal molecular weight, extent and distribution of sulfate, and disaccharide composition, which indicate that the mutation did not affect HS biosynthetic enzymes. Together, the data suggest that mutant VI-7 is defective in a regulatory component which directly or indirectly coordinates HS biosynthetic enzymes to specifically generate the defined monosaccharide sequence of HSact.

Animals↗
PubMed1994

Smooth muscle cell expression of extracellular matrix genes after arterial injury.

Accumulation of extracellular matrix (ECM) after arterial injury is an important event in the development of intimal thickening and is modulated by heparin. To investigate the regulation of matrix protein expression, we have analyzed messenger RNA levels by Northern blotting for various ECM proteins in the rat carotid artery balloon injury model. RNA was extracted from normal arteries and from intima-medial preparations at 2 days, 1 week, 2 weeks, and 4 weeks after balloon injury of arteries in animals receiving either saline or heparin infusion. Transcripts for the heparan sulfate proteoglycans perlecan, syndecan, and ryudocan; the chondroitin sulfate proteoglycan versican; the dermatan sulfate proteoglycan biglycan; type I procollagen; and tropoelastin all were increased on Northern blots beginning at 1 week after injury. By in situ hybridization, the transcripts for elastin nd biglycan were primarily localized to smooth muscle cells in the intima and were diminished by heparin in proportion to the decrease in intimal mass. Other matrix genes (perlecan, ryudocan) were expressed in the intima and media and were not affected by heparin. The results support the conclusion that ECM gene expression is a relatively late event in the response of the carotid artery, and that some of the genes are expressed only in the intima whereas others are expressed in both the intima and media.

Animals↗