LTBP-2 competes with tropoelastin for binding to fibulin-5 and heparin,and is a negative modulator of elastinogenesis

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of ill-defined function associated with elastic fibers during elastinogenesis. Although LTBP-2 binds fibrillin-1, fibulin-5, and heparin/heparan sulfate, molecules critical for normal elastic fiber assembly, it does not interact directly with elastin or its precursor, tropoelastin. We investigated the modulating effect of LTBP-2 on two key interactions of tropoelastin during elastinogenesis a) with fibulin-5 and b) with heparan sulfate (using heparin). Firstly, using solid phase assays we showed that LTBP-2 bound fibulin-5 (Kd = 26.47 ± 5.68 nM) with an affinity similar to that of the tropoelastin-fibulin-5 interaction (Kd = 24.66 ± 5.64 nM). Then using a competitive binding assay we showed that LTBP-2 inhibited the tropoelastin-fibulin-5 interaction in a dose dependent manner with almost complete inhibition obtained with 5-fold molar excess of LTBP-2. Interestingly, a fragment of LTBP-2 containing the fibulin-5 binding sequence only partially inhibited the tropoelasin-fibulin-5 interaction suggesting that LTBP-2 was directly blocking only the C-terminal tropoelastin binding site on fibulin-5 and indirectly blocking tropoelastin binding to the N-terminal region. In parallel experiments heparin was shown to have minor inhibitory effects on fibulin-5 interactions with tropoelastin and LTBP-2. However, LTBP-2 was shown to significantly inhibit the binding of heparin to tropoelastin with 50% inhibition achieved with 10 fold molar excess of LTBP-2. Confocal microscopy of fibroblast matrix showed strong co-distribution of LTBP-2 with fibulin-5 and fibrillin-1 and partial co-distribution with heparan sulfate proteoglycans, perlecan and syndecan-4. Also addition of exogenous LTBP-2 to ear cartilage chondrocyte cultures blocked elastinogenesis in a concentration-dependent manner. Overall the results indicate that LTBP-2 may have a negative regulatory role during elastic fiber assembly, perhaps in displacing elastin microassemblies from complexes with fibulin-5 and/or cell surface heparan sulfate proteoglycans.     

Inducible expression of the cell surface heparan sulfate proteoglycan syndecan-2 (fibroglycan) on human activated macrophages can regulate fibroblast growth factor action.

Monocyte/macrophages play important roles in regulating tissue growth and angiogenesis through the controlled release of heparin-binding growth factors such as fibroblast growth factor (FGF), vascular endothelial growth factor, and heparin binding epidermal growth factor. The action of these potent growth mediators is known to be regulated by adsorption to heparan sulfate proteoglycans (HSPGs) on the surface and within the extracellular matrix of other neighboring cells, which respectively promote or restrict interactions with their signal-transducing receptors on target cells. Here we report on the nature of HSPGs inducibly expressed on the surface of macrophages that confer these cells with the capacity to regulate endogenous growth factor activity. We reveal that activated human macrophages express only a single major 48-kDa cell surface HSPG, syndecan-2 (fibroglycan) as the result of de novo RNA and protein synthesis. In addition, we demonstrate this macrophage HSPG selectively binds the macrophage-derived growth factors FGF-2, vascular endothelial growth factor and heparin binding EGF and can present FGF-2 in a form that transactivates receptor-bearing BaF32 cells. These results define a novel and unique proteoglycan profile for macrophages and imply a key role for syndecan-2 in the delivery of sequestered growth factors by inflammatory macrophages for productive binding to their appropriate target cells in vivo.     

Different heparan sulfate proteoglycans serve as cellular receptors for human papillomaviruses

Papillomaviruses replicate in stratified epithelia of skin and mucosa. Infection with certain human papillomavirus (HPV) types is the main cause of anogenital neoplasia, in particular cervical cancer. Early events of papillomavirus infectivity are poorly understood. While heparan sulfate proteoglycans (HSPGs) mediate initial binding to the cell surface, the class of proteins carrying heparan sulfates has not been defined. Here we examined two processes of papillomavirus infection, attachment of virus-like particles (VLP) to cells and infection with authentic HPV type 11 (HPV11) virions. Of the HSPGs, syndecan-1 is the major epithelial form and is strongly upregulated in wound edge keratinocytes. We employed K562 cells, which lack HSPGs except minor amounts of endogenous betaglycan, and stable clones that express cDNAs of syndecan-1, syndecan-4, or glypican-1. Binding of VLP correlated with levels of heparan sulfate on the cell surface. Parental K562 bound HPV16 VLP weakly, whereas all three K562 transfectants demonstrated enhanced binding, with the highest binding capacity observed for syndecan-1-transfected cells, which also expressed the most HSPG. For HPV11 infectivity assays, a high virion inoculum was required to infect K562 cells, whereas ectopic expression of syndecan-1 increased permissiveness eightfold and expression of syndecan-4 or glypican-1 fourfold. Infection of keratinocytes was eliminated by treatment with heparitinase, but not phospholipase C, further implicating the syndecan family of integral membrane proteins as receptor proteins. Human keratinocytes with a homozygous deletion of alpha6 integrin are permissive for HPV11 infection. These results indicate that several HSPGs can serve as HPV receptors and support a putative role for syndecan-1, rather than alpha6 integrin, as a primary receptor protein in natural HPV infection of keratinocytes.     

Syndecans reside in sphingomyelin-enriched low-density fractions of the plasma membrane isolated from a parathyroid cell line

Background

Heparan sulfate proteoglycans (HSPGs) are one of the basic constituents of plasma membranes. Specific molecular interactions between HSPGs and a number of extracellular ligands have been reported. Mechanisms involved in controlling the localization and abundance of HSPG on specific domains on the cell surface, such as membrane rafts, could play important regulatory roles in signal transduction.

Methodology/Principal Findings

Using metabolic radiolabeling and sucrose-density gradient ultracentrifugation techniques, we identified [³⁵S]sulfate-labeled macromolecules associated with detergent-resistant membranes (DRMs) isolated from a rat parathyroid cell line. DRM fractions showed high specific radioactivity ([³⁵S]sulfate/mg protein), implying the specific recruitment of HSPGs to the membrane rafts. Identity of DRM-associated [³⁵S]sulfate-labeled molecules as HSPGs was confirmed by Western blotting with antibodies that recognize heparan sulfate (HS)-derived epitope. Analyses of core proteins by SDS-PAGE revealed bands with an apparent MW of syndecan-4 (30–33 kDa) and syndecan-1 (70 kDa) suggesting the presence of rafts with various HSPG species. DRM fractions enriched with HSPGs were characterized by high sphingomyelin content and found to only partially overlap with the fractions enriched in ganglioside GM1. HSPGs could be also detected in DRMs even after prior treatment of cells with heparitinase.

Conclusions/Significance

Both syndecan-1 and syndecan-4 have been found to specifically associate with membrane rafts and their association seemed independent of intact HS chains. Membrane rafts in which HSPGs reside were also enriched with sphingomyelin, suggesting their possible involvement in FGF signaling. Further studies, involving proteomic characterization of membrane domains containing HSPGs might improve our knowledge on the nature of HSPG-ligand interactions and their role in different signaling platforms.     

The cell surface proteoglycan syndecan-1 mediates fibroblast growth factor-2 binding and activity

Binding of fibroblast growth factors (FGFs) to receptor tyrosine kinases (FGFRs) and signaling is facilitated by binding of FGF to heparan sulfate proteoglycans (HSPGs). There are multiple families of HSPGs, including extracellular and cell surface forms. An important and potentially controversial question is whether cell surface forms of HSPGs act as positive or negative regulators of FGF signaling. This study examines the ability of the cell surface HSPG syndecan-1 to regulate FGF binding and signaling. HSPG-deficient Raji lymphoma cells, expressing a transfected syndecan-1 cDNA (Raji S1 cells), were used as HSPG “donor” cells. BaF3 cells, expressing an FGFR1 cDNA (FR1C-11 cells), were used as FGFR “reporter” cells. Using Raji S1 cells preincubated with FGF, it was found that they formed heterotypic aggregates with FR1C-11 cells in the presence of FGF-2, but not FGF-1. In addition, the FR1C-11 cells demonstrated FGF-2, but not FGF-1, dependent survival when cultured on fixed Raji S1 cells. Thus, Raji syndecan-1 (1) differentially regulates the binding and signaling of FGFs 1 and 2 and (2) acts as a positive regulator of FGF-2 signaling. J. Cell. Physiol. 174:310–321, 1998. © 1998 Wiley-Liss, Inc.     

HIV-1 p17 matrix protein interacts with heparan sulfate side chain of CD44v3, syndecan-2, and syndecan-4 proteoglycans expressed on human activated CD4+ T cells affecting tumor necrosis factor alpha and interleukin 2 production

HIV-1 p17 contains C- and N-terminal sequences with positively charged residues and a consensus cluster for heparin binding. We have previously demonstrated by affinity chromatography that HIV-1 p17 binds strongly to heparin-agarose at physiological pH and to human activated CD4(+) T cells. In this study we demonstrated that the viral protein binds to heparan sulfate side chains of syndecan-2, syndecan-4, and CD44v3 purified from HeLa cells and that these heparan sulfate proteoglycans (HSPGs) co-localize with HIV-1 p17 on activated human CD4(+) T cells by confocal fluorescence analysis. Moreover, we observed a stimulatory or inhibitory activity when CD4(+) T cells were activated with mitogens together with nanomolar or micromolar concentrations of the matrix protein.     

Heparin increases the infectivity of Human Papillomavirus Type 16 independent of cell surface proteoglycans and induces L1 epitope exposure

Human Papillomaviruses (HPVs) are the etiological agents of cervical cancer, and HPV‐16 is the most prevalent type. Several HPVs require heparan sulfate proteoglycans (HSPGs) for cell binding. Here, we analyse the phenomenon that preincubation of HPV‐16 with increasing concentrations of heparin results in partial restoration rather than more efficient inhibition of infection. While corroborating that the HSPGs are cell‐binding receptors for HPV‐16, heparin‐preincubated virus bound to the extracellular matrix (ECM) via laminin‐332. Furthermore, the interaction of virions with heparin, a representative of the highly sulfated S‐domains of heparan sulfate (HS) chains of HSPGs, allowed HPV‐16 infection in the absence of cell surface HSPGs. Therefore, we concluded that specific glycan moieties but not specific HSPG protein backbones are required for infection. The increased binding of an epitope‐specific antibody to the viral capsid after heparin binding suggested that initial conformational changes in the HPV‐16 virion occur during infection by interaction with‘heparin‐like’ domains of cellular HSPGs. We propose that HS sequences with specific sulfation patterns are required to facilitate HPV‐16 infection.     

Further evidence that papillomavirus capsids exist in two distinct conformations

Cell surface heparan sulfate proteoglycans (HSPGs) serve as primary attachment receptors for human papillomaviruses (HPVs). To demonstrate that a biologically functional HPV-receptor interaction is restricted to a specific subset of HSPGs, we first explored the role of HSPG glucosaminoglycan side chain modifications. We demonstrate that HSPG O sulfation is essential for HPV binding and infection, whereas de-N-sulfated heparin interfered with VLP binding but not with HPV pseudoinfection. This points to differences in VLP-HSPG and pseudovirion-HSPG interactions. Interestingly, internalization kinetics of VLPs and pseudovirions, as measured by fluorescence-activated cell sorting analysis, also differ significantly with approximate half times of 3.5 and 7.5 h, respectively. These data suggest that differences in HSPG binding significantly influence postbinding events. We also present evidence that pseudovirions undergo a conformational change after cell attachment. A monoclonal antibody (H33.J3), which displays negligible effectiveness in preattachment neutralization assays, efficiently neutralizes cell-bound virions. However, no difference in H33.J3 binding to pseudovirions and VLPs was observed in enzyme-linked immunosorbent assay and virus capture assays. In contrast to antibody H33.B6, which displays equal efficiencies in pre- and postattachment neutralization assays, H33.J3 does not block VLP binding to heparin, demonstrating that it interferes with steps subsequent to virus binding. Our data strongly suggest that H33.J3 recognizes a conformation-dependent epitope in capsid protein L1, which undergoes a structural change after cell attachment.     

Blockage of undesirable endocytosis of recombinant human growth/differentiation factor-5 in Chinese hamster ovary cell cultures requires heparin analogs with specific chain lengths

Cell surface heparan sulfate proteoglycan (HSPG)-mediated endocytosis lowers the yield of recombinant human bone morphogenetic proteins (rhBMPs), such as rhBMP-2 and rhBMP-4, from Chinese hamster ovary (CHO) cell cultures. Exogenous recombinant human growth/differentiation factor-5 (rhGDF-5), a member of the BMP family, bound to cell surface HSPGs and was actively internalized into CHO cells. Knockdown of heparan sulfate (HS) synthesis enzymes in CHO cells revealed that the chain length and N-sulfation of HS affected the binding of rhGDF-5 to HSPGs and subsequent rhGDF-5 internalization. To increase product yield by minimizing rhGDF-5 internalization in recombinant CHO (rCHO) cell cultures, heparin, and dextran sulfate (DS) of various polysaccharide chain lengths, which are structural analogs of HS, were examined for blockage of rhGDF-5 internalization. Heparin fragments of four monosaccharides (MW of 1.2 kDa) and DS (MW of 15 kDa) did not inhibit rhGDF-5 internalization whereas unfractionated heparin and DS of 200 kDa could significantly inhibit it. Compared to the control cultures, supplementation with unfractionated heparin or DS of 200 kDa at 1 g L^-1 resulted in more than a 10-fold increase in the maximum rhGDF-5 concentration. Taken together, the supplementation of structural HS analogs improved rhGDF-5 production in rCHO cell cultures by inhibiting rhGDF-5 internalization.     

NTS2-selective neurotensin mimetics with tetrahydrofuran amino acids

Stimulation of the NTS2 neurotensin receptor causes antipsychotic effects and leads to a promotion of the μ-opioid-independent antinociception, which is important in the modulation of tonic pain sensitivity. We report the synthesis and properties of a small library of peptidic agonists based on the active neurotensin fragment NT(8–13). Two tetrahydrofuran amino acid derivatives were synthesized to replace Tyr¹¹ in NT(8–13). Additionally, Arg⁸, Arg⁹, and Ile¹² of the lead peptide were exchanged by Lys, Lys, and Gly, respectively. The new compounds showed substantial NTS2 binding affinity and up to 1000-fold selectivity over NTS1. The highest selectivity (K_i(NTS2): 29 nM, K_i(NTS1): 35,000 nM) was observed for the peptide analog 17R^trans.     

Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration.

Myogenesis in the embryo and the adult mammal consists of a highly organized and regulated sequence of cellular processes to form or repair muscle tissue that include cell proliferation, migration, and differentiation. Data from cell culture and in vivo experiments implicate both FGFs and HGF as critical regulators of these processes. Both factors require heparan sulfate glycosaminoglycans for signaling from their respective receptors. Since syndecans, a family of cell-surface transmembrane heparan sulfate proteoglycans (HSPGs) are implicated in FGF signaling and skeletal muscle differentiation, we examined the expression of syndecans 1-4 in embryonic, fetal, postnatal, and adult muscle tissue, as well as on primary adult muscle fiber cultures. We show that syndecan-1, -3, and -4 are expressed in developing skeletal muscle tissue and that syndecan-3 and -4 expression is highly restricted in adult skeletal muscle to cells retaining myogenic capacity. These two HSPGs appear to be expressed exclusively and universally on quiescent adult satellite cells in adult skeletal muscle tissue, suggesting a role for HSPGs in satellite cell maintenance or activation. Once activated, all satellite cells maintain expression of syndecan-3 and syndecan-4 for at least 96 h, also implicating these HSPGs in muscle regeneration. Inhibition of HSPG sulfation by treatment of intact myofibers with chlorate results in delayed proliferation and altered MyoD expression, demonstrating that heparan sulfate is required for proper progression of the early satellite cell myogenic program. These data suggest that, in addition to providing potentially useful new markers for satellite cells, syndecan-3 and syndecan-4 may play important regulatory roles in satellite cell maintenance, activation, proliferation, and differentiation during skeletal muscle regeneration.     

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.      

Heparin–protein interactions: From affinity and kinetics to biological roles. Application to an interaction network regulating angiogenesis

Numerous extracellular proteins, growth factors, chemokines, cytokines, enzymes, lipoproteins, involved in a variety of biological processes, interact with heparin and/or heparan sulfate at the cell surface and in the extracellular matrix (ECM). The goal of this study is to investigate the relationship(s) between affinity and kinetics of heparin–protein interactions and the localization of the proteins, their intrinsic disorder and their biological roles. Most proteins bind to heparin with a higher affinity than their fragments and form more stable complexes with heparin than with heparan sulfate. Lipoproteins and matrisome-associated proteins (e.g. growth factors and cytokines) bind to heparin with very high affinity. Matrisome-associated proteins form transient complexes with heparin. However they bind to this glycosaminoglycan with a higher affinity than the proteins of the core matrisome, which contribute to ECM assembly and organization, and than the secreted proteins which are not associated with the ECM. The association rate of proteins with heparin is related to the intrinsic disorder of heparin-binding sites. Enzyme inhibitor activity, protein dimerization, skeletal system development and pathways in cancer are functionally associated with proteins displaying a high or very high affinity for heparin (K_D < 100 nM). Besides their use in investigating molecular recognition and functions, kinetics and affinity are essential to prioritize interactions in networks and to build network models as discussed for the interaction network established at the surface of endothelial cells by endostatin, a heparin-binding protein regulating angiogenesis.     

Latent TGF-β-binding proteins

The LTBPs (or latent transforming growth factor β binding proteins) are important components of the extracellular matrix (ECM) that interact with fibrillin microfibrils and have a number of different roles in microfibril biology. There are four LTBPs isoforms in the human genome (LTBP-1, − 2, − 3, and − 4), all of which appear to associate with fibrillin and the biology of each isoform is reviewed here.The LTBPs were first identified as forming latent complexes with TGFβ by covalently binding the TGFβ propeptide (LAP) via disulfide bonds in the endoplasmic reticulum. LAP in turn is cleaved from the mature TGFβ precursor in the trans-golgi network but LAP and TGFβ remain strongly bound through non-covalent interactions. LAP, TGFβ, and LTBP together form the large latent complex (LLC). LTBPs were originally thought to primarily play a role in maintaining TGFβ latency and targeting the latent growth factor to the extracellular matrix (ECM), but it has also been shown that LTBP-1 participates in TGFβ activation by integrins and may also regulate activation by proteases and other factors. LTBP-3 appears to have a role in skeletal formation including tooth development. As well as having important functions in TGFβ regulation, TGFβ-independent activities have recently been identified for LTBP-2 and LTBP-4 in stabilizing microfibril bundles and regulating elastic fiber assembly.     

Association of cell surface heparan sulfate proteoglycans of Schwann cells with extracellular matrix proteins

The terminal differentiation of Schwann cells is dependent on contact with basement membrane. The present study was undertaken to investigate the role of cell surface heparan sulfate proteoglycans (HSPGs) in mediating Schwann cell responses to extracellular matrix contact. Phosphatidylinositol-specific phospholipase C-releasable cell surface HSPGs purified from cultures of neonatal rat Schwann cells were subjected to affinity chromatography on immobilized laminin and fibronectin. Binding of the HSPG to both affinity matrices was observed. The strength of the association, however, was sensitive to the ionic strength of the buffer. In 0.1 M Tris-HCl, HSPG binding was essentially irreversible whereas in physiological ionic strength buffer (e.g. 0.142 M NaCl, 10 mM Tris), weaker binding was detected as a delay in elution of the HSPG from the affinity columns. Further studies of HSPG-laminin binding suggested that the binding was mediated by the glycosaminoglycan chains of the proteoglycans. Results of equilibrium gel filtration chromatography provided additional evidence for a reversible association of the HSPG and laminin with a Kd of approximately 1 x 10(-6) M. When Schwann cells were plated on plastic dishes coated with laminin, the cells attached and extended long slender processes. Inclusion of heparin, but not chondroitin sulfate, in the assay medium resulted in partial inhibition of process extension, but at concentrations of heparin which were higher than that needed to disrupt laminin-HSPG association in vitro. Addition of anti-integrin receptor antibodies resulted in more extensive inhibition of laminin-dependent process extension. Anti-integrin antibodies plus heparin essentially totally inhibited laminin-dependent process extension. These results demonstrate that cell surface HSPGs are capable of reversible association with extracellular matrix molecules and suggest that HSPG-laminin interactions play a role in laminin-dependent Schwann cell spreading.     

Growth modulation and proteoglycan turnover in cultured mesangial cells

Proliferation of mesangial cells is a common feature of renal disease, and conditioned media from glomerular epithelial and endothelial cells have been found to contain heparin-like molecules that suppress proliferation of rat mesangial cells (RMC). We have partially characterized the glycosaminoglycans that are labeled with ³⁵SO₄^2? by RMC in culture at early passage and examined their ability to inhibit mitogenic stimulation of the cells. Four chondroitin/dermatan sulfate proteoglycans (CS/DSPG) were identified, the largest and smallest of which (K_d of 0.04 and 0.26 on Superose 6) were retained in the cell layer while the other two (K_d = 0.17 and 0.22) were secreted into the medium. Heparan sulfate proteoglycans (HSPG) with K_d values of 0.09, 0.13, and 0.39 were minor components of the cell layer, while a single heparan sulfate (K_d = 0.17) was recovered from the medium. After 16 h of labeling in serum-free medium, about 60% of macromolecular ³⁵S was cell-associated and 40% was in the medium. Cell-associated label consisted of 7% CS/DSPG, 9% HSPG, and 84% free glycosaminoglycan chains (mostly CS/DS), whereas the medium contained 52% CS/DSPG, 17% HSPG, and approximately equal amounts of free HS and CS/DS chains. Bovine lung heparin (1 μg/ml) decreased by 45% the incorporation of [³H]-thymidine into DNA after release of serum-starved RMC from growth arrest. Heparin acted prior to the G₁/S interface; arrest of the cells in early S phase with aphidicolin abrogated the heparin response. The endogenous HSPGs had a slight antimitogenic effect on the RMC, but heparan sulfate chains from both the medium and cell layer had a potent effect. On an equivalent mass basis, only the free glycosaminoglycan chains were more potent than heparin in this regard, decreasing thymidine incorporation by over 90% when present at 1 μg/ml. These results demonstrate that heparan sulfate glycosaminoglycans derived from mesangial proteoglycans are potential negative autocrine growth regulators. Proteoglycan metabolism releases these soluble heparan sulfate chains, determining the level of this activity. © 1994 wiley-Liss, Inc.     

Repression of myogenic differentiation by aFGF, bFGF, and K-FGF is dependent on cellular heparan sulfate

We have proposed a model in which fibroblast growth factor (FGF) signalling requires the interaction of FGF with at least two FGF receptors, a heparan sulfate proteoglycan (HSPG) and a tyrosine kinase. Since FGF may be a key mediator of skeletal muscle differentiation, we examined the synthesis of glycosaminoglycans in MM14 skeletal muscle myoblasts and their participation in FGF signalling. Proliferating and differentiated MM14 cells exhibit similar levels of HSPG, while differentiated cells exhibit reduced levels of chondroitin sulfate proteoglycans and heparan sulfate chains. HSPGs, including syndecan, present in proliferating cells bind bFGF, while the majority of chondroitin sulfate and heparan sulfate chains do not. Treatment of skeletal muscle cells with chlorate, a reversible inhibitor of glycosaminoglycan sulfation, was used to examine the requirement of sulfated proteoglycans for FGF signalling. Chlorate treatment reduced glycosaminoglycan sulfation by 90% and binding of FGF to high affinity sites by 80%. Chlorate treatment of MM14 myoblasts abrogated the biological activity of acidic, basic, and Kaposi's sarcoma FGFs resulting in terminal differentiation. Chlorate inhibition of FGF signalling was reversed by the simultaneous addition of sodium sulfate or heparin. Further support for a direct role of heparan sulfate proteoglycans in fibroblast growth factor signal transduction was demonstrated by the ability of heparitinase to inhibit basic FGF binding and biological activity. These results suggest that activation of FGF receptors by acidic, basic or Kaposi's sarcoma FGF requires simultaneous binding to a HSPG and the tyrosine kinase receptor. Skeletal muscle differentiation in vivo may be dependent on FGFs, FGF tyrosine kinase receptors, and HSPGs. The regulation of these molecules may then be expected to have important implications for skeletal muscle development and regeneration.     

Heparan sulfate proteoglycans as regulators of fibroblast growth factor-2 signaling in brain endothelial cells. Specific role for glypican-1 in glioma angiogenesis

Fibroblast growth factor-2 (FGF2) is a potent angiogenic factor in gliomas. Heparan sulfate promotes ligand binding to receptor tyrosine kinase and regulates signaling. The goal of this study was to examine the contribution of heparan sulfate proteoglycans (HSPGs) to glioma angiogenesis. Here we show that all brain endothelial cell HSPGs carry heparan sulfate chains similarly capable of forming a ternary complex with FGF2 and fibroblast growth factor receptor-1c and of promoting a mitogenic signal. Immunohistochemical analysis revealed that glypican-1 was overexpressed in glioma vessel endothelial cells, whereas this cell-surface HSPG was consistently undetectable in normal brain vessels. To determine the effect of increased glypican-1 expression on FGF2 signaling, we transfected normal brain endothelial cells, which express low base-line levels of glypican-1, with this proteoglycan. Glypican-1 expression enhanced growth of brain endothelial cells and sensitized them to FGF2-induced mitogenesis despite the fact that glypican-1 remained a minor proteoglycan. In contrast, overexpression of syndecan-1 had no effect on growth or FGF2 sensitivity. We conclude that the glypican-1 core protein has a specific role in FGF2 signaling. Glypican-1 overexpression may contribute to angiogenesis and the radiation resistance characteristic of this malignancy.     

Regulation of ureteric bud branching morphogenesis by sulfated proteoglycans in the developing kidney

Glycosaminoglycans in the form of heparan sulfate proteoglycans (HSPG) and chondroitin sulfate proteoglycans (CSPG) are required for normal kidney organogenesis. The specific roles of HSPGs and CSPGs on ureteric bud (UB) branching morphogenesis are unclear, and past reports have obtained differing results. Here we employ in vitro systems, including isolated UB culture, to clarify the roles of HSPGs and CSPGs on this process. Microarray analysis revealed that many proteoglycan core proteins change during kidney development (syndecan-1,2,4, glypican-1,2,3, versican, decorin, biglycan). Moreover, syndecan-1, syndecan-4, glypican-3, and versican are differentially expressed during isolated UB culture, while decorin is dynamically regulated in cultured isolated metanephric mesenchyme (MM). Biochemical analysis indicated that while both heparan sulfate (HS) and chondroitin sulfate (CS) are present, CS accounts for approximately 75% of the glycosaminoglycans (GAG) in the embryonic kidney. Selective perturbation of HS in whole kidney rudiments and in the isolated UB resulted in a significant reduction in the number of UB branch tips, while CS perturbation has much less impressive effects on branching morphogenesis. Disruption of endogenous HS sulfation with chlorate resulted in diminished FGF2 binding and proliferation, which markedly altered kidney area but did not have a statistically significant effect on patterning of the ureteric tree. Furthermore, perturbation of GAGs did not have a detectable effect on FGFR2 expression or epithelial marker localization, suggesting the expression of these molecules is largely independent of HS function. Taken together, the data suggests that nonselective perturbation of HSPG function results in a general proliferation defect; selective perturbation of specific core proteins and/or GAG microstructure may result in branching pattern defects. Despite CS being the major GAG synthesized in the whole developing kidney, it appears to play a lesser role in UB branching; however, CS is likely to be integral to other developmental processes during nephrogenesis, possibly involving the MM. A model is presented of how, together with growth factors, heterogeneity of proteoglycan core proteins and glycosaminoglycan sulfation act as a switching mechanism to regulate different stages of the branching process. In this model, specific growth factor-HSPG combinations play key roles in the transitioning between stages and their maintenance.