Alan C. Rapraeger

Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation.

Basic fibroblast growth factor (bFGF) binds to heparan sulfate proteoglycans at the cell surface and to receptors with tyrosine kinase activity. Prevention of binding between cell surface heparan sulfate and bFGF (i) substantially reduces binding of fibroblast growth factor to its cell-surface receptors, (ii) blocks the ability of bFGF to support the growth of Swiss 3T3 fibroblasts, and (iii) induces terminal differentiation of MM14 skeletal muscle cells, which is normally repressed by fibroblast growth factor. These results indicate that cell surface heparan sulfate is directly involved in bFGF cell signaling.

The syndecan-1 ectodomain regulates αvβ3 integrin activity in human mammary carcinoma cells

The αvβ3 integrin participates in cell morphogenesis, growth factor signaling, and cell survival. Activation of the integrin is central to these processes and is influenced by specific ECM components, which engage both integrins and syndecans. This paper demonstrates that the αvβ3 integrin and syndecan-1 (S1) are functionally coupled. The integrin is dependent on the syndecan to become activated and to mediate signals required for MDA-MB-231 and MDA-MB-435 human mammary carcinoma cell spreading on vitronectin or S1-specific antibody. Coupling of the syndecan to αvβ3 requires the S1 ectodomain (ED), as ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances αvβ3 recognition of vitronectin; and treatments that target this domain, including competition with recombinant S1ED protein or anti-S1ED antibodies, mutation of the S1ED, or down-regulation of S1 expression by small-interfering RNAs, disrupt αvβ3-dependent cell spreading and migration. Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated αvβ3 integrins.

Syndecan-1 regulates αvβ3 and αvβ5 integrin activation during angiogenesis and is blocked by synstatin, a novel peptide inhibitor

Syndecan-1 (Sdc1) is a matrix receptor shown to associate via its extracellular domain with the αvβ3 and αvβ5 integrins, potentially regulating cell adhesion, spreading, and invasion of cells expressing these integrins. Using Sdc1 deletion mutants expressed in human mammary carcinoma cells, we identified the active site within the Sdc1 core protein and derived a peptide inhibitor called synstatin (SSTN) that disrupts Sdc1s interaction with these integrins. Because the αvβ3 and αvβ5 integrins are critical in angiogenesis, a process in which a role for Sdc1 has been uncertain, we used human vascular endothelial cells in vitro to show that the Sdc1 regulatory mechanism is also required for integrin activation on these cells. We found Sdc1 expressed in the vascular endothelium during microvessel outgrowth from aortic explants in vitro and in mouse mammary tumors in vivo. Moreover, we show that SSTN blocks angiogenesis in vitro or when delivered systemically in a mouse model of angiogenesis in vivo, and impairs mammary tumor growth in an orthotopic mouse tumor model. Thus, Sdc1 is a critical regulator of these two important integrins during angiogenesis and tumorigenesis, and is inhibited by the novel SSTN peptide.

Spatial and temporal expression of heparan sulfate in mouse development regulates FGF and FGF receptor assembly

Heparan sulfate (HS) interacts with diverse growth factors, including Wnt, Hh, BMP, VEGF, EGF, and FGF family members, and is a necessary component for their signaling. These proteins regulate multiple cellular processes that are critical during development. However, a major question is whether developmental changes occur in HS that regulate the activity of these factors. Using a ligand and carbohydrate engagement assay, and focusing on FGF1 and FGF8b interactions with FGF receptor (FR)2c and FR3c, this paper reveals global changes in HS expression in mouse embryos during development that regulate FGF and FR complex assembly. Furthermore, distinct HS requirements are identified for both complex formation and signaling for each FGF and FR pair. Overall, these results suggest that changes in HS act as critical temporal regulators of growth factor and morphogen signaling during embryogenesis.

Heparanase-enhanced shedding of syndecan-1 by myeloma cells promotes endothelial invasion and angiogenesis.

Heparanase enhances shedding of syndecan-1 (CD138), and high levels of heparanase and shed syndecan-1 in the tumor microenvironment are associated with elevated angiogenesis and poor prognosis in myeloma and other cancers. To explore how the heparanase/syndecan-1 axis regulates angiogenesis, we used myeloma cells expressing either high or low levels of heparanase and examined their impact on endothelial cell invasion and angiogenesis. Medium conditioned by heparanase-high cells significantly stimulated endothelial invasion in vitro compared with medium from heparanase-low cells. The stimulatory activity was traced to elevated levels of vascular endothelial growth factor (VEGF) and syndecan-1 in the medium. We discovered that the heparan sulfate chains of syndecan-1 captured VEGF and also attached the syndecan-1/VEGF complex to the extracellular matrix where it then stimulated endothelial invasion. In addition to its heparan sulfate chains, the core protein of syndecan-1 was also required because endothelial invasion was blocked by addition of synstatin, a peptide mimic of the integrin activating region present on the syndecan-1 core protein. These results reveal a novel mechanistic pathway driven by heparanase expression in myeloma cells whereby elevated levels of VEGF and shed syndecan-1 form matrix-anchored complexes that together activate integrin and VEGF receptors on adjacent endothelial cells thereby stimulating tumor angiogenesis.

Syndecan-1-mediated cell spreading requires signaling by αvβ3 integrins in human breast carcinoma cells

Abstract Syndecans are cell surface heparan sulfate proteoglycans with regulatory roles in cell adhesion, proliferation, and differentiation [Annu. Rev. Biochem. 68 (1999) 729]. While the syndecan heparan sulfate chains are essential for matrix binding, less is known about the signaling role of their core proteins. To mimic syndecan-specific adhesion, MDA-MB-231 mammary carcinoma cells were plated on antibodies against syndecan-4 or syndecan-1. While cells adherent via syndecan-4 spread, cells adherent via syndecan-1 do not. However, cells adherent via syndecan-1 can be induced to spread by Mn2+, suggesting that activation of a β1 or β3 integrin partner is required. Surprisingly, pretreatment of cells with a function-activating β1 antibody does not induce spreading, whereas function-blocking β1 integrin antibodies do, suggesting involvement of a β1-to-β3 integrin cross-talk. Indeed, blockade of β1 integrin activation induces αvβ3 integrin activation detectable by soluble fibrinogen binding. Spreading in response to syndecan-1 is independent of integrin-ligand binding. Furthermore, competition with soluble murine syndecan-1 ectodomain, which does not disrupt cell adhesion, nonetheless blocks the spreading mechanism. These data suggest that the ectodomain of the syndecan-1 core protein directly participates in the formation of a signaling complex that signals in cooperation with αvβ3 integrins; signaling via this complex is negatively regulated by β1 integrins.

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.

THE COORDINATED REGULATION OF HEPARAN SULFATE, SYNDECANS AND CELL BEHAVIOR

Recent advances in our understanding of heparan sulfate proteoglycans at the cell periphery implicate them as important participants in cell surface signaling. Regulation of the expression of discrete core proteins and of the specificity inherent in their heparan sulfate chains is thus emerging as a critical means of controlling cell behavior.

HSulf-1 and HSulf-2 Are Potent Inhibitors of Myeloma Tumor Growth in Vivo

To participate as co-receptor in growth factor signaling, heparan sulfate must have specific structural features. Recent studies show that when the levels of 6-O-sulfation of heparan sulfate are diminished by the activity of extracellular heparan sulfate 6-O-endosulfatases (Sulfs), fibroblast growth factor 2-, heparin binding epidermal growth factor-, and hepatocyte growth factor-mediated signaling are attenuated. This represents a novel mechanism for regulating cell growth, particularly within the tumor microenvironment where the Sulfs are known to be misregulated. To directly test the role of Sulfs in tumor growth control in vivo, a human myeloma cell line was transfected with cDNAs encoding either of the two known human endosulfatases, HSulf-1 or HSulf-2. When implanted into severe combined immunodeficient (SCID) mice, the growth of these tumors was dramatically reduced on the order of 5- to 10-fold as compared with controls. In addition to an inhibition of tumor growth, these studies revealed the following. (i) HSulf-1 and HSulf-2 have similar functions in vivo. (ii) The extracellular activity of Sulfs is restricted to the local tumor cell surface. (iii) The Sulfs promote a marked increase in extracellular matrix deposition within tumors that may, along with attenuated growth factor signaling, contribute to the reduction in tumor growth. These findings demonstrate that dynamic regulation of heparan sulfate structure by Sulfs present within the tumor microenvironment can have a dramatic impact on the growth and progression of malignant cells in vivo.

Role of heparan sulfate as a tissue-specific regulator of FGF-4 and FGF receptor recognition

FGF signaling uses receptor tyrosine kinases that form high-affinity complexes with FGFs and heparan sulfate (HS) proteoglycans at the cell surface. It is hypothesized that assembly of these complexes requires simultaneous recognition of distinct sulfation patterns within the HS chain by FGF and the FGF receptor (FR), suggesting that tissue-specific HS synthesis may regulate FGF signaling. To address this, FGF-2 and FGF-4, and extracellular domain constructs of FR1-IIIc (FR1c) and FR2-IIIc (FR2c), were used to probe for tissue-specific HS in embryonic day 18 mouse embryos. Whereas FGF-2 binds HS ubiquitously, FGF-4 exhibits a restricted pattern, failing to bind HS in the heart and blood vessels and failing to activate signaling in mouse aortic endothelial cells. This suggests that FGF-4 seeks a specific HS sulfation pattern, distinct from that of FGF-2, which is not expressed in most vascular tissues. Additionally, whereas FR2c binds all FGF-4–HS complexes, FR1c fails to bind FGF-4–HS in most tissues, as well as in Raji-S1 cells expressing syndecan-1. Proliferation assays using BaF3 cells expressing either FR1c or FR2c support these results. This suggests that FGF and FR recognition of specific HS sulfation patterns is critical for the activation of FGF signaling, and that synthesis of these patterns is regulated during embryonic development.