University of Wisconsin–Madison
Alan Rapraeger, PhD

Alan Rapraeger, PhD

Professor

Department of Human Oncology

I am a professor in the Department of Human Oncology and the executive director of the UW–Madison Office of Postdoctoral Studies. Members of my laboratory seek to discover novel signaling mechanisms through which cell adhesion regulates the activation and signaling of receptor tyrosine kinases in cancer and to develop new therapeutics that target these mechanisms. Our focus is on the syndecan (Sdc) family of cell-matrix receptors. The syndecans act as organizers of receptor complexes containing integrins and receptor tyrosine kinases (EGFR, IGF1R, VEGFR2 and others) that drive tumorigenesis and tumor angiogenesis. We are developing synstatins (SSTNs), therapeutic peptides that are highly effective inhibitors of these processes in vitro and in vivo and block tumor growth, survival and invasion and the angiogenesis upon which tumors depend.

Education

Postdoctoral Scholar, Stanford University, Developmental Biology (1985)

PhD, University of California–Berkeley, Zoology/Developmental Biology (1978)

BS, University of Oregon, Biology (1972)

Academic Appointments

Professor, Human Oncology (2010)

Professor, Pathology and Laboratory Medicine (1996)

Associate Professor, Pathology and Laboratory Medicine (1991)

Assistant Professor, Pathology and Laboratory Medicine (1985)

Boards, Advisory Committees and Professional Organizations

Executive Director, UW-Madison Office of Postdoctoral Studies (2012–present)

Co-Organizer, International syndecan meeting: “Syndecans in Cell Regulation and Disease," Leuven, Belgium

Vice Chair (2004) and Chair (2006) Proteoglycan Gordon Conference

Steering committee, American Society for Matrix Biology (2005–2008)

Program Leader, Cell Signaling and Growth Control, Carbone Comprehensive Cancer (1997–2001)

Research Focus

Breast Cancer, Multiple Myeloma, Head and Neck Cancer, Immunotherapy

The Rapraeger Lab seeks to develop new therapeutics that target receptor tyrosine kinases linked to cell adhesion signaling mechanisms in cancer.

Our focus is on the syndecans (Sdc) family of cell-matrix receptors that act as organizers of receptor complexes containing integrins and receptor tyrosine kinases (EGFR, IGF1R, VEGFR2 and others) that drive tumorigenesis and angiogenesis. We are developing synstatins (SSTNs)—therapeutic peptides that disrupt these signaling mechanisms and block tumor growth, survival and invasion, as well as tumor-induced angiogenesis.

 

The Rapraeger Lab was the first to demonstrate that syndecans contain “cell binding” motifs in their extracellular domains. The importance of this finding soon became clear when it was realized that such sites are critical for the signaling by growth factor receptor tyrosine kinases (RTKs) in tumor cells. RTKs and integrins have well-established roles in tumor cell proliferation, invasion and survival, often functioning in a coordinated fashion at sites of cell-matrix adhesion. Work from the Rapraeger Lab has shown that syndecans, a four-member class of heparan-sulfate-decorated matrix receptors (Sdc1, Sdc2, Sdc3 and Sdc4), utilize docking motifs in their extracellular domains to organize RTKs and integrins into functional units. This work, combined with prior work from the laboratory that demonstrated as essential role for heparan sulfate (HS) glycosaminoglycans in the activity of growth factors, paints the picture of syndecans as “organizers” of RTK signaling at the cell surface. At present, the laboratory has identified docking motifs that organize signaling apparatus containing EGFR, IGF-1R, VEGFR2 or HER2 and the avb3, avb5, a4b1, a3b1, or a6b4 integrins and the signaling mechanisms activated by these receptor complexes. Peptide mimetics of the docking motifs in the syndecans, called “synstatins” (SSTNIGF1R, SSTNVEGFR2, SSTNVLA-4, SSTNEGFR and SSTNHER2) prevent assembly of the receptor apparatus, block their signaling activities and are highly effective against tumor cell proliferation, survival and invasion. They are also effective against angiogenesis upon which tumors depend. Patents for these peptides have been granted or are pending and work is proceeding to advance them for FDA approval and clinical trials.

Regulation of IGF-1R signaling by Sdc1.

Expression of the avb3 or avb5 integrin, usually in response to malignant transformation of epithelial cells or activation of endothelial cells during angiogenesis, results in integrin docking to the extracellular domain of human Sdc1 (amino acids 89-120), which is followed by capture of the type 1 insulin-like growth factor receptor (IGF-1R) at the same docking site. Once formed, constitutive or matrix-induced clustering of the ternary receptor complex activates IGF-1R by autophosphorylation independent of IGF-1 growth factor ligand. Activated endothelial cells or tumor cells bearing the ternary receptor complex rely on the syndecan-activated IGF-1R to phosphorylate and suppress the activity of apoptosis signal-regulating kinase-1 (ASK-1) engaged with its cytoplasmic domain, preventing ASK-1-mediated activation of Jun-N-terminal kinase (JNK) and blocking entry into apoptosis, thus sustaining tumor cell survival. In a second activity, downstream signaling from IGF-1R activates the avb3 or avb5 integrin via an inside-out signaling pathway that targets the integrin-activating protein talin, resulting in endothelial or tumor cell motility during the onset of angiogenesis or tumor cell invasion. SSTNIGF1R, a peptide mimetic of the docking site in Sdc1, competitively disrupts the ternary receptor complex on tumor cells and activated endothelial cells, prevents integrin activation and removes the block to ASK-1 activation, inducing tumor cell death and blocking tumor-induced angiogenesis. Neither activity can be rescued by IGF-1 when the receptor complex is disrupted by SSTNIGF1R, emphasizing the singular role played by the syndecan in this IGF-1R signaling mechanism. Work is currently underway examining the integrin activation mechanism, and SSTNIGF1R-mediated inhibition of tumor growth in mouse models of breast and head & neck cancer, and against human patient-derived head & neck cancer xenografts.

 

Relevant publications

Funding

  • NIH R01-CA212413 Syndecan-1 (CD138) and its synstatins: targeting invasion, survival and angiogenesis in myeloma
  • NIH 1 P50-DE026787 Project 3: Targeting Head and Neck Cancer with Synstatin Therapeutics

Regulation of VLA-4 activation and VEGFR2-mediated polarized cell invasion of myeloma, melanoma, T-lymphoma and vascular endothelial cells.

A juxtamembrane site in the extracellular domain of human Sdc1 (amino acids 210-236) is responsible for engaging very late antigen-4 (VLA-4, also known as the a4b1 integrin) and vascular endothelial growth factor receptor-2 (VEGFR2), respectively. Synstatin peptide mimetics of the VLA-4 or VEGFR2 binding motifs (SSTNVLA-4 or SSTNVEGFR2), prevent VLA-4 or VEGFR2 capture by the syndecan and disrupt invasion signaling that relies on the co-capture of both receptors. VLA-4 undergoes rapid activation when engaged to ligand, involving a conformation change and clustering (avidity modulation) to increase binding affinity. On cells expressing Sdc1 and VLA-4 (e.g., myeloma cells, vascular endothelial cells, melanoma, Jurkat-T cells and others), this activation is blocked by preventing VLA-4 docking with Sdc1 using SSTNVLA-4 or mutating the VLA-4 binding motif in the syndecan. The mechanism underlying the syndecan-dependent integrin activation mechanism is currently under investigation. In addition to carrying out cell-matrix adhesion, VLA-4 activated by this mechanism becomes localized to the leading edge of the cells where it is highly involved in cell invasion when the cells express the heparan-sulfate-degrading enzyme heparanase. Heparanase is a known tumor promoter and enhancer of leukocyte recruitment during inflammation. Trimming of the HS chains on Sdc1 exposes its core protein to cleavage by matrix-metalloproteinase-9 (MMP-9), releasing the syndecan ectodomain. The VLA-4 and VEGFR2 docking motif in the shed syndecan ectodomain couples VEGFR2 to the clustered integrin, causing VEGF-independent activation of VEGFR2. VEGFR2 signaling leads to activation of protein kinase-A (PKA) engaged with the VEGFR2 cytoplasmic domain and phosphorylation of the a4-integrin cytoplasmic domain on serine 988. This leads to Rac1 activation, causing lamellipodium formation, establishment of a leading edge, and polarized invasion of tumor cells and vascular endothelial cells necessary for tumor cell metastasis and angiogenesis. Either prevention of integrin activation by SSTNVLA-4­ or VEGFR2-coupling to Sdc4 by SSTNVEGFR2 serves to block these processes. Work is currently under way examining these processes and the efficacy of the SSTNs in mouse models of multiple myeloma and immune suppression.

Relevant publications

 

Funding

  • NIH R01-CA212413 Syndecan-1 (CD138) and its synstatins: targeting invasion, survival and angiogenesis in myeloma

Coupling of EGFR to integrins by Sdc4 regulates tumor cell proliferation, survival and invasion.

A juxtamembrane site in human Sdc4 (amino acids 87-131) captures the epidermal growth factor receptor (EGFR) and the a3b1 integrin. In addition, the C-terminus of the syndecan engages the cytoplasmic C-terminus of the b4 integrin, which comprises the a6b4 integrin. Several tyrosines in the b4 cytoplasmic “signaling domain” become phosphorylated, and the receptor complex as a whole supports the proliferation, survival and invasion of tumor cells (breast carcinoma, head & neck squamous cell carcinoma) and endothelial cells engaged in pathological angiogenesis. Ongoing work suggests that the receptor complex acts to suppress stress signaling and to promote cell cycle progression in the tumor cells, evidenced by the findings that an inhibitory synstatin mimetic of the docking site in Sdc4 (SSTNEGFR) causes rapid activation of stress MAPKs (p38MAPK and JNK), and rapid G1 and S-phase cell cycle arrest specifically in tumor cells. Ongoing work is examining the mechanism of cell cycle arrest in breast and head & neck cancer, and the mechanism of EGFR activation and signaling when coupled to the syndecan.

 

Relevant publications

Funding

  • NIH R01-CA163662 Role of Syndecans in HER2 and TN breast cancer
  • NIH 1 P50-DE026787 Project 3: Targeting Head and Neck Cancer with Synstatin Therapeutics

Lab Members

Deanna Beauvais, PhDDeanna Beauvais

Associate Scientist

BS, Mechanical Engineering and Biochemistry, United States Military Academy (USMA), West Point

PhD, Molecular & Cellular Pharmacology, University of Wisconsin–Madison

 

Scott Nelson, PhDScott Nelson

Assistant Scientist

BA Biology, 1985, Illinois Wesleyan University

PhD Biochemistry, 1990, University of Minnesota

 

Noah StuevenNoah Stueven

Associate Research Specialist

BS Biotechnology & Chemistry, 2017, University of Wisconsin–River Falls

 

 

Kristin Short, BSKristin Short

Associate Research Specialist & Laboratory Manager

BS Human Biology, 2015, University of Wisconsin–Green Bay

MS Cell and Molecular Biology, 2017, University of Wisconsin–La Crosse

 

Oisun Jung, PhDOisun Jung

Graduate Student

MS Cell and Molecular Biology, 2011, Seoul National University

  • The Hidden Conundrum of Phosphoinositide Signaling in Cancer. Trends Cancer
    Thapa N, Tan X, Choi S, Lambert PF, Rapraeger AC, Anderson RA
    2016 Jul; 2 (7): 378-390
    • More

      Phosphoinositide 3-kinase (PI3K) generation of PI(3,4,5)P3 from PI(4,5)P2 and the subsequent activation of Akt and its downstream signaling cascades (e.g. mTORC1) dominates the landscape of phosphoinositide signaling axis in cancer research. However, PI(4,5)P2 is breaking its boundary as merely a substrate for PI3K and phospholipase C (PLC), and is now an established lipid messenger pivotal for different cellular events in cancer. Here, we review the phosphoinositide signaling axis in cancer, giving due weight to PI(4,5)P2 and its generating enzymes, the phosphatidylinositol phosphate (PIP) kinases (PIPKs). We highlighted how PI(4,5)P2 and PIP kinases serve as a proximal node in phosphoinositide signaling axis and how its interaction with cytoskeletal proteins regulates migratory and invasive nexus of metastasizing tumor cells.

      View details for PubMedID 27819060
  • Heparanase regulation of cancer, autophagy and inflammation: new mechanisms and targets for therapy. FEBS J
    Sanderson RD, Elkin M, Rapraeger AC, Ilan N, Vlodavsky I
    2017 Jan; 284 (1): 42-55
    • More

      Because of its impact on multiple biological pathways, heparanase has emerged as a major regulator of cancer, inflammation and other disease processes. Heparanase accomplishes this by degrading heparan sulfate which regulates the abundance and location of heparin-binding growth factors thereby influencing multiple signaling pathways that control gene expression, syndecan shedding and cell behavior. In addition, heparanase can act via nonenzymatic mechanisms that directly activate signaling at the cell surface. Clinical trials testing heparanase inhibitors as anticancer therapeutics are showing early signs of efficacy in patients further emphasizing the biological importance of this enzyme. This review focuses on recent developments in the field of heparanase regulation of cancer and inflammation, including the impact of heparanase on exosomes and autophagy, and novel mechanisms whereby heparanase regulates tumor metastasis, angiogenesis and chemoresistance. In addition, the ongoing development of heparanase inhibitors and their potential for treating cancer and inflammation are discussed.

      View details for PubMedID 27758044
  • Syndecan-1 (CD138) Suppresses Apoptosis in Multiple Myeloma by Activating IGF1 Receptor: Prevention by SynstatinIGF1R Inhibits Tumor Growth. Cancer Res
    Beauvais DM, Jung O, Yang Y, Sanderson RD, Rapraeger AC
    2016 Sep 01; 76 (17): 4981-93
    • More

      Syndecan-1 (Sdc1/CD138) expression is linked to disease severity in multiple myeloma, although the causal basis for this link remains unclear. Here we report that capture of the IGF1 receptor (IGF1R) by Sdc1 suppresses ASK1-dependent apoptosis in multiple myeloma cells. Sdc1 binds two different fractions of IGF1R, one that is constitutively active and a second that is activated by IGF1 ligand. Notably, IGF1R kinase activity in both fractions is blocked by synstatinIGF1R (SSTNIGF1R), a peptide that inhibits IGF1R capture by Sdc1, as well as by a truncated peptide (SSTNIGF1R-T) that appears to be specific for multiple myeloma cells. Mechanistically, we show that ASK1 is bound to active IGF1R and inhibited by Tyr and Ser83/Ser966 phosphorylation. When IGF1R engagement with Sdc1 is blocked by SSTNIGF1R, ASK1 becomes activated, and initiates JNK- and caspase-3-mediated apoptosis. In pharmacologic tests, we find SSTNIGF1R is highly stable in human plasma and displays a half-life of 27 hours in mice, wherein it significantly reduces both the size and neovascularization of CAG myeloma tumor xenografts. Taken together, our results offer a preclinical proof of concept and mechanistic rationale for the exploration of SSTNIGF1R as an experimental therapeutic to dually attack multiple myeloma tumor cell survival and tumor angiogenesis. Cancer Res; 76(17); 4981-93. ©2016 AACR.

      View details for PubMedID 27364558
  • Heparanase-induced shedding of syndecan-1/CD138 in myeloma and endothelial cells activates VEGFR2 and an invasive phenotype: prevention by novel synstatins. Oncogenesis
    Jung O, Trapp-Stamborski V, Purushothaman A, Jin H, Wang H, Sanderson RD, Rapraeger AC
    2016 Feb 29; 5: e202
    • More

      Multiple myeloma arises when malignant plasma cells invade and form multiple tumors in the bone marrow. High levels of heparanase (HPSE) correlate with poor prognosis in myeloma patients. A likely target of the enzyme is the heparan sulfate (HS) proteoglycan syndecan-1 (Sdc1, CD138), which is highly expressed on myeloma cells and contributes to poor prognosis in this disease. We find that HPSE promotes an invasive phenotype mediated by the very late antigen-4 (VLA-4, or α4β1 integrin) in myeloma cells plated on either fibronectin (FN) or vascular endothelial cell adhesion molecule-1 (VCAM-1), ligands that are prevalent in the bone marrow. The phenotype depends on vascular endothelial cell growth factor receptor-2 (VEGFR2), which is aberrantly expressed in myeloma, and is characterized by a highly protrusive lamellipodium and cell invasion. HPSE-mediated trimming of the HS on Sdc1 and subsequent matrix metalloproteinase-9-mediated shedding of the syndecan exposes a juxtamembrane site in Sdc1 that binds VEGFR2 and VLA-4, thereby coupling VEGFR2 to the integrin. Shed Sdc1 can be mimicked by recombinant Sdc1 ectodomain or by a peptide based on its binding motif, which causes VLA-4 to re-orient from the lagging edge (uropod) to the leading edge of migrating cells, couple with and activate VEGFR2. Peptides (called 'synstatins') containing only the VLA-4 or VEGFR2 binding sites competitively inhibit invasion, as they block coupling of the receptors. This mechanism is also utilized by vascular endothelial cells, in which it is also activated by HPSE, during endothelial cell tube formation. Collectively, our findings reveal for the first time the mechanism through which HPSE modulates Sdc1 function to promote both tumor cell invasion and angiogenesis, thereby driving multiple myeloma progression. The inhibitory synstatins, or inhibitors of HPSE enzyme activity, are likely to show promise as therapeutics against myeloma extravasation and spread.

      View details for PubMedID 26926788
  • Stress-Induced EGFR Trafficking: Mechanisms, Functions, and Therapeutic Implications. Trends Cell Biol
    Tan X, Lambert PF, Rapraeger AC, Anderson RA
    2016 May; 26 (5): 352-66
    • More

      Epidermal growth factor receptor (EGFR) has fundamental roles in normal physiology and cancer, making it a rational target for cancer therapy. Surprisingly, however, inhibitors that target canonical, ligand-stimulated EGFR signaling have proven to be largely ineffective in treating many EGFR-dependent cancers. Recent evidence indicates that both intrinsic and therapy-induced cellular stress triggers robust, noncanonical pathways of ligand-independent EGFR trafficking and signaling, which provides cancer cells with a survival advantage and resistance to therapeutics. Here, we review the mechanistic regulation of noncanonical EGFR trafficking and signaling, and the pathological and therapeutic stresses that activate it. We also discuss the implications of this pathway in clinical treatment of EGFR-overexpressing cancers.

      View details for PubMedID 26827089
  • Syndecan-1 and Syndecan-4 Capture Epidermal Growth Factor Receptor Family Members and the α3β1 Integrin Via Binding Sites in Their Ectodomains: NOVEL SYNSTATINS PREVENT KINASE CAPTURE AND INHIBIT α6β4-INTEGRIN-DEPENDENT EPITHELIAL CELL MOTILITY. J Biol Chem
    Wang H, Jin H, Rapraeger AC
    2015 Oct 23; 290 (43): 26103-13
    • More

      The α6β4 integrin is known to associate with receptor tyrosine kinases when engaged in epithelial wound healing and in carcinoma invasion and survival. Prior work has shown that HER2 associates with α6β4 integrin and syndecan-1 (Sdc1), in which Sdc1 engages the cytoplasmic domain of the β4 integrin subunit allowing HER2-dependent motility and carcinoma cell survival. In contrast, EGFR associates with Sdc4 and the α6β4 integrin, and EGFR-dependent motility depends on cytoplasmic engagement of β4 integrin with Sdc4. However, how HER2 and EGFR assimilate into a complex with the syndecans and integrin, and why kinase capture is syndecan-specific has remained unknown. In the present study, we demonstrate that HER2 is captured via a site, comprised of amino acids 210-240, in the extracellular domain of human Sdc1, and EGFR is captured via an extracellular site comprised of amino acids 87-131 in human Sdc4. Binding assays using purified recombinant proteins demonstrate that the interaction between the EGFR family members and the syndecans is direct. The α3β1 integrin, which is responsible for the motility of the cells, is captured at these sites as well. Peptides based on the interaction motifs in Sdc1 and Sdc4, called synstatins (SSTN210-240 and SSTN87-131) competitively displace the receptor tyrosine kinase and α3β1 integrin from the syndecan with an IC50 of 100-300 nm. The syndecans remain anchored to the α6β4 integrin via its cytoplasmic domain, but the activation of cell motility is disrupted. These novel SSTN peptides are potential therapeutics for carcinomas that depend on these HER2- and EGFR-coupled mechanisms for their invasion and survival.

      View details for PubMedID 26350464
  • Cytoplasmic domain interactions of syndecan-1 and syndecan-4 with α6β4 integrin mediate human epidermal growth factor receptor (HER1 and HER2)-dependent motility and survival. J Biol Chem
    Wang H, Jin H, Beauvais DM, Rapraeger AC
    2014 Oct 31; 289 (44): 30318-32
    • More

      Epithelial cells are highly dependent during wound healing and tumorigenesis on the α6β4 integrin and its association with receptor tyrosine kinases. Previous work showed that phosphorylation of the β4 subunit upon matrix engagement depends on the matrix receptor syndecan (Sdc)-1 engaging the cytoplasmic domain of the β4 integrin and coupling of the integrin to human epidermal growth factor receptor-2 (HER2). In this study, HER2-dependent migration activated by matrix engagement is compared with migration stimulated by EGF. We find that whereas HER2-dependent migration depends on Sdc1, EGF-dependent migration depends on a complex consisting of human epidermal growth factor receptor-1 (HER1, commonly known as EGFR), α6β4, and Sdc4. The two syndecans recognize distinct sites at the extreme C terminus of the β4 integrin cytoplasmic domain. The binding motif in Sdc1 is QEEXYX, composed in part by its syndecan-specific variable (V) region and in part by the second conserved (C2) region that it shares with other syndecans. A cell-penetrating peptide containing this sequence competes for HER2-dependent epithelial migration and carcinoma survival, although it is without effect on the EGFR-stimulated mechanism. β4 mutants bearing mutations specific for Sdc1 and Sdc4 recognition act as dominant negative mutants to block cell spreading or cell migration that depends on HER2 or EGFR, respectively. The interaction of the α6β4 integrin with the syndecans appears critical for it to be utilized as a signaling platform; migration depends on α3β1 integrin binding to laminin 332 (LN332; also known as laminin 5), whereas antibodies that block α6β4 binding are without effect. These findings indicate that specific syndecan family members are likely to have key roles in α6β4 integrin activation by receptor tyrosine kinases.

      View details for PubMedID 25202019
  • Synstatin: a selective inhibitor of the syndecan-1-coupled IGF1R-αvβ3 integrin complex in tumorigenesis and angiogenesis. FEBS J
    Rapraeger AC
    2013 May; 280 (10): 2207-15
    • More

      The syndecans are a family of heparan sulfate-decorated cell-surface proteoglycans: matrix receptors with roles in cell adhesion and growth factor signaling. Their heparan sulfate chains recognize 'heparin-binding' motifs that are ubiquitously present in the extracellular matrix, providing the means for syndecans to constitutively bind and cluster to sites of cell-matrix adhesion. Emerging evidence suggests that specialized docking sites in the syndecan extracellular domains may serve to localize other receptors to these sites as well, including integrins and growth factor receptor tyrosine kinases. A prototype of this mechanism is capture of the αvβ3 integrin and insulin-like growth factor 1 receptor (IGF1R) by syndecan-1 (Sdc1), forming a ternary receptor complex in which signaling downstream of IGF1R activates the integrin. This Sdc1-coupled ternary receptor complex is especially prevalent on tumor cells and activated endothelial cells undergoing angiogenesis, reflecting the up-regulated expression of αvβ3 integrin in such cells. As such, much effort has focused on developing therapeutic agents that target this integrin in various cancers. Along these lines, the site in the Sdc1 ectodomain that is responsible for capture and activation of the αvβ3 or αvβ5 integrins by IGF1R can be mimicked by a short peptide called 'synstatin', which competitively displaces the integrin and IGF1R kinase from the syndecan and inactivates the complex. This review summarizes our current knowledge of the Sdc1-coupled ternary receptor complex and the efficacy of synstatin as an emerging therapeutic agent to target this signaling mechanism.

      View details for PubMedID 23375101
  • Vascular endothelial-cadherin stimulates syndecan-1-coupled insulin-like growth factor-1 receptor and cross-talk between αVβ3 integrin and vascular endothelial growth factor receptor 2 at the onset of endothelial cell dissemination during angiogenesis. FEBS J
    Rapraeger AC, Ell BJ, Roy M, Li X, Morrison OR, Thomas GM, Beauvais DM
    2013 May; 280 (10): 2194-206
    • More

      Vascular endothelial growth factor (VEGF)-stimulated angiogenesis depends on a cross-talk mechanism involving VEGF receptor 2 (VEGFR2), vascular endothelial (VE)-cadherin and the αVβ3 integrin. Because we have shown that αVβ3 integrin activation is dependent on its incorporation, along with the insulin-like growth factor-1 receptor (IGF1R) kinase, into a ternary receptor complex organized by the matrix receptor syndecan-1 (Sdc1), we questioned the role of this core complex in VEGF-stimulated angiogenesis. We find that the Sdc1-coupled ternary receptor complex is required for VEGF signalling and for stimulation of vascular endothelial cell migration by vascular endothelial cadherin (VE-cadherin) engagement. VE-cadherin binding to Fc/VE-cadherin extracellular domain chimera activates Sdc1-coupled IGF1R and αvβ3 integrin; this depends on VEGFR2 and c-Src activated by the cadherin. Blocking homotypic VE-cadherin engagement disrupts VEGF-stimulated cell migration, which is restored by clustering the cadherin in the absence of cell-cell adhesion. This cadherin-dependent stimulation requires VEGFR2 and IGF1R and is blocked by synstatin (SSTN)(92-119), a peptide that competitively disrupts the Sdc1-coupled ternary complex and prevents the αVβ3 integrin activation required for VEGFR2 activation. VEGFR2-stimulated angiogenesis in the mouse aortic ring explant assay is disrupted by SSTN, although only early in the process, suggesting that IGF1R coupling to Sdc1 and αVβ3 integrin comprises a core activation mechanism activated by VE-cadherin that is necessary for VEGFR2 and integrin activation in the initial stages of endothelial cell dissemination during angiogenesis.

      View details for PubMedID 23331867
  • Transmembrane and extracellular domains of syndecan-1 have distinct functions in regulating lung epithelial migration and adhesion. J Biol Chem
    Altemeier WA, Schlesinger SY, Buell CA, Brauer R, Rapraeger AC, Parks WC, Chen P
    2012 Oct 12; 287 (42): 34927-35
    • More

      Syndecan-1 is a cell surface proteoglycan that can organize co-receptors into a multimeric complex to transduce intracellular signals. The syndecan-1 core protein has multiple domains that confer distinct cell- and tissue-specific functions. Indeed, the extracellular, transmembrane, and cytoplasmic domains have all been found to regulate specific cellular processes. Our previous work demonstrated that syndecan-1 controls lung epithelial migration and adhesion. Here, we identified the necessary domains of the syndecan-1 core protein that modulate its function in lung epithelial repair. We found that the syndecan-1 transmembrane domain has a regulatory function in controlling focal adhesion disassembly, which in turn controls cell migration speed. In contrast, the extracellular domain facilitates cell adhesion through affinity modulation of α(2)β(1) integrin. These findings highlight the fact that syndecan-1 is a multidimensional cell surface receptor that has several regulatory domains to control various biological processes. In particular, the lung epithelium requires the syndecan-1 transmembrane domain to govern cell migration and is independent from its ability to control cell adhesion via the extracellular domain.

      View details for PubMedID 22936802
  • Syndecan-1 couples the insulin-like growth factor-1 receptor to inside-out integrin activation. J Cell Sci
    Beauvais DM, Rapraeger AC
    2010 Nov 01; 123 (Pt 21): 3796-807
    • More

      Syndecan-1 (Sdc1) engages and activates the αvβ3 (and/or αvβ5) integrin when clustered in human carcinoma and endothelial cells. Although the engagement is extracellular, the activation mechanism is cytoplasmic. This talin-dependent, inside-out signaling pathway is activated downstream of the insulin-like growth factor-1 receptor (IGF1R), whose kinase activity is triggered by Sdc1 clustering. In vitro binding assays using purified receptors suggest that association of the Sdc1 ectodomain with the integrin provides a 'docking face' for IGF1R. IGF1R docking and activation of the associated integrin is blocked by synstatin (SSTN(92-119)), a peptide derived from the integrin engagement site in Sdc1. IGF1R colocalizes with αvβ3 integrin and Sdc1 in focal contacts, but fails to associate with or activate the integrin in cells either lacking Sdc1 or expressing Sdc1(Δ67-121), a mutant that is unable to form the Sdc1-integrin-IGF1R ternary complex. Integrin activation is also blocked by IGF1R inhibitors or by silencing IGF1R or talin expression with small-interfering RNAs (siRNAs). In both cases, expression of the constitutively active talin F23 head domain rescues integrin activation. We recently reported that SSTN(92-119) blocks angiogenesis and impairs tumor growth in mice, therefore this Sdc1-mediated integrin regulatory mechanism might be a crucial regulator of disease processes known to rely on these integrins, including tumor cell metastasis and tumor-induced angiogenesis.

      View details for PubMedID 20971705
  • Focus on molecules: syndecan-1. Exp Eye Res
    Zhang Y, McKown RL, Raab RW, Rapraeger AC, Laurie GW
    2011 Oct; 93 (4): 329-30
  • Interaction of syndecan and alpha6beta4 integrin cytoplasmic domains: regulation of ErbB2-mediated integrin activation. J Biol Chem
    Wang H, Leavitt L, Ramaswamy R, Rapraeger AC
    2010 Apr 30; 285 (18): 13569-79
    • More

      The alpha6beta4 integrin is a laminin 332 (LN332) receptor central to the formation of hemidesmosomes in epithelial layers. However, the integrin becomes phosphorylated by keratinocytes responding to epidermal growth factor in skin wounds or by squamous cell carcinomas that overexpress/hyperactivate the tyrosine kinase ErbB2, epidermal growth factor receptor, or c-Met. We show here that the beta4-dependent signaling in A431 human squamous carcinoma cells is dependent on the syndecan family of matrix receptors. Yeast two-hybrid analysis identifies an interaction within the distal third (amino acids 1473-1752) of the beta4 cytoplasmic domain and the conserved C2 region of the syndecan cytoplasmic domain. Via its C2 region, Sdc1 forms a complex with the alpha6beta4 integrin along with the receptor tyrosine kinase ErbB2 and the cytoplasmic kinase Fyn in A431 cells. Engagement of LN332 or clustering of the alpha6beta4 integrin with integrin-specific antibodies causes phosphorylation of ErbB2, Fyn, and the beta4 subunit as well as activation of phosphatidylinositol 3-kinase and Akt and their assimilation into this complex. This leads to phosphatidylinositol 3-kinase-dependent cell spreading and Akt-dependent protection from apoptosis. This is disrupted by RNA interference silencing of Sdc1 but can be rescued by mouse Sdc1 or Sdc4 but not by syndecan mutants lacking their C-terminal C2 region. This disruption does not prevent the phosphorylation of ErbB2 or Fyn but blocks the Fyn-mediated phosphorylation of the beta4 tail. We propose that syndecans engage the distal region of the beta4 cytoplasmic domain and bring it to the plasma membrane, where it can be acted upon by Src family kinases.

      View details for PubMedID 20181947
  • Heparanase-enhanced shedding of syndecan-1 by myeloma cells promotes endothelial invasion and angiogenesis. Blood
    Purushothaman A, Uyama T, Kobayashi F, Yamada S, Sugahara K, Rapraeger AC, Sanderson RD
    2010 Mar 25; 115 (12): 2449-57
    • More

      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.

      View details for PubMedID 20097882
  • Syndecan-1 regulates alphavbeta3 and alphavbeta5 integrin activation during angiogenesis and is blocked by synstatin, a novel peptide inhibitor. J Exp Med
    Beauvais DM, Ell BJ, McWhorter AR, Rapraeger AC
    2009 Mar 16; 206 (3): 691-705
    • More

      Syndecan-1 (Sdc1) is a matrix receptor shown to associate via its extracellular domain with the alpha(v)beta(3) and alpha(v)beta(5) 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 Sdc1's interaction with these integrins. Because the alpha(v)beta(3) and alpha(v)beta(5) 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.

      View details for PubMedID 19255147
  • Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin. J Cell Biol
    Ma P, Beck SL, Raab RW, McKown RL, Coffman GL, Utani A, Chirico WJ, Rapraeger AC, Laurie GW
    2006 Sep 25; 174 (7): 1097-106
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      Cell surface heparan sulfate (HS) proteoglycans are carbohydrate-rich regulators of cell migratory, mitogenic, secretory, and inflammatory activity that bind and present soluble heparin-binding growth factors (e.g., fibroblast growth factor, Wnt, Hh, transforming growth factor beta, amphiregulin, and hepatocyte growth factor) to their respective signaling receptors. We demonstrate that the deglycanated core protein of syndecan-1 (SDC1) and not HS chains nor SDC2 or -4, appears to target the epithelial selective prosecretory mitogen lacritin. An important and novel step in this mechanism is that binding necessitates prior partial or complete removal of HS chains by endogenous heparanase. This limits lacritin activity to sites where heparanase appears to predominate, such as sites of exocrine cell migration, secretion, renewal, and inflammation. Binding is mutually specified by lacritin's C-terminal mitogenic domain and SDC1's N terminus. Heparanase modification of the latter transforms a widely expressed HS proteoglycan into a highly selective surface-binding protein. This novel example of cell specification through extracellular modification of an HS proteoglycan has broad implications in development, homeostasis, and disease.

      View details for PubMedID 16982797
  • Heparan sulfate-related oligosaccharides in ternary complex formation with fibroblast growth factors 1 and 2 and their receptors. J Biol Chem
    Jastrebova N, Vanwildemeersch M, Rapraeger AC, Giménez-Gallego G, Lindahl U, Spillmann D
    2006 Sep 15; 281 (37): 26884-92
    • More

      Biosynthesis of heparan sulfate (HS) is strictly regulated to yield products with cell/tissue-specific composition. Interactions between HS and a variety of proteins, including growth factors and morphogens, are essential for embryonic development and for homeostasis in the adult. Fibroblast growth factors (FGFs) and their various receptors (FRs) form ternary complexes with HS, as required for receptor signaling. Libraries of HS-related, radiolabeled oligosaccharides were generated by chemo-enzymatic modification of heparin and tested for affinity to immobilized FR ectodomains in the presence of FGF1 or FGF2. Experiments were designed to enable assessment of N-sulfated 8- and 10-mers with defined numbers of iduronic acid 2-O-sulfate and glucosamine 6-O-sulfate groups. FGF1 and FGF2 were found to require similar oligosaccharides in complex formation with FR1c-3c, FGF2 affording somewhat more efficient oligosaccharide recruitment than FGF1. FR4, contrary to FR1c-3c, bound oligosaccharides at physiological ionic conditions even in the absence of FGFs, and this interaction was further promoted by FGF1 but not by FGF2. In all systems studied, the stability of FGF-oligosaccharide-FR complexes correlated with the overall level of saccharide O-sulfation rather than on the precise distribution of sulfate groups.

      View details for PubMedID 16807244
  • Syndecan-1 regulates alphavbeta5 integrin activity in B82L fibroblasts. J Cell Sci
    McQuade KJ, Beauvais DM, Burbach BJ, Rapraeger AC
    2006 Jun 15; 119 (Pt 12): 2445-56
    • More

      B82L mouse fibroblasts respond to fibronectin or vitronectin via a syndecan-1-mediated activation of the alphavbeta5 integrin. Cells attached to syndecan-1-specific antibody display only filopodial extension. However, the syndecan-anchored cells extend lamellipodia when the antibody-substratum is supplemented with serum, or low concentrations of adsorbed vitronectin or fibronectin, that are not sufficient to activate the integrin when plated alone. Integrin activation is blocked by treatment with (Arg-Gly-Asp)-containing peptides and function-blocking antibodies that target alphav integrins, as well as by siRNA-mediated silencing of beta5 integrin expression. In addition, alphavbeta5-mediated cell attachment and spreading on high concentrations of vitronectin is blocked by competition with recombinant syndecan-1 ectodomain core protein and by downregulation of mouse syndecan-1 expression by mouse-specific siRNA. Taking advantage of the species-specificity of the siRNA, rescue experiments in which human syndecan-1 constructs are expressed trace the activation site to the syndecan-1 ectodomain. Moreover, both full-length mouse and human syndecan-1 co-immunoprecipitate with the beta5 integrin subunit, but fail to do so if the syndecan is displaced by competition with soluble, recombinant syndecan-1 ectodomain. These results suggest that the ectodomain of the syndecan-1 core protein contains an active site that assembles into a complex with the alphavbeta5 integrin and regulates alphavbeta5 integrin activity.

      View details for PubMedID 16720645
  • HSulf-1 and HSulf-2 are potent inhibitors of myeloma tumor growth in vivo. J Biol Chem
    Dai Y, Yang Y, MacLeod V, Yue X, Rapraeger AC, Shriver Z, Venkataraman G, Sasisekharan R, Sanderson RD
    2005 Dec 02; 280 (48): 40066-73
    • More

      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.

      View details for PubMedID 16192265
  • Regulation of fibroblast growth factor-2 activity by human ovarian cancer tumor endothelium. Clin Cancer Res
    Whitworth MK, Backen AC, Clamp AR, Wilson G, McVey R, Friedl A, Rapraeger AC, David G, McGown A, Slade RJ, Gallagher JT, Jayson GC
    2005 Jun 15; 11 (12): 4282-8
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      Fibroblast growth factor-2 (FGF-2) is a potent angiogenic cytokine that is dependent on heparan sulfate for its biological activity. We have investigated the relationship among heparan sulfate, FGF-2, and the signal-transducing receptors in human, advanced-stage, serous ovarian adenocarcinoma. Using a unique molecular probe, FR1c-Ap, which consisted of a soluble FGF receptor 1 isoform IIIc covalently linked to an alkaline phosphatase moiety, the distribution of heparan sulfate that had the ability to support the formation of a heparan sulfate/FGF-2/FGFR1 isoform IIIc alkaline phosphatase heparan sulfate construct complex was determined. This may be taken as a surrogate marker for the distribution of biologically active heparan sulfate and was distributed predominantly in endothelial cells and stroma but was absent from adenocarcinoma cells. In situ hybridization revealed the expression of FGFR1 mRNA in the endothelium and reverse transcription-PCR confirmed the presence of FGFR1 isoform IIIc but not isoform IIIb. The presence of FGF-2 around tumor endothelium was detected through immunohistochemistry. Double-staining techniques showed that heparan sulfate was found predominantly at the basal aspect of the endothelium and suggested that syndecan-3 might function as one of the proteoglycans involved in FGF-2 signaling in the endothelium. The data suggest that the entire extracellular signaling apparatus, consisting of FGF-2, biologically active heparan sulfate, and FGFRs capable of responding to FGF-2, is present in ovarian cancer endothelium, thereby highlighting the cytokine and its cognate receptor as potential targets for the antiangiogenic treatment of this disease.

      View details for PubMedID 15958608
  • The syndecan-1 ectodomain regulates alphavbeta3 integrin activity in human mammary carcinoma cells. J Cell Biol
    Beauvais DM, Burbach BJ, Rapraeger AC
    2004 Oct 11; 167 (1): 171-81
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      The alpha(v)beta(3) 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 alpha(v)beta(3) 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 alpha(v)beta(3) requires the S1 ectodomain (ED), as ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances alpha(v)beta(3) 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 alpha(v)beta(3)-dependent cell spreading and migration. Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated alpha(v)beta(3) integrins.

      View details for PubMedID 15479743
  • Syndecan-1 ectodomain regulates matrix-dependent signaling in human breast carcinoma cells. Exp Cell Res
    Burbach BJ, Ji Y, Rapraeger AC
    2004 Oct 15; 300 (1): 234-47
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      Syndecan-1 was overexpressed in T47D, MCF-7, or Hs578t human breast carcinoma cell lines, mimicking overexpression observed in carcinomas in vivo. Overexpression of syndecan-1, or its ectodomain alone fused to a glycosylphosphatidylinositol anchor (GPI-mS1ED), promotes cell rounding in 2D culture. Deletions within the syndecan-1 ectodomain (S1ED) implicate an active site within the core protein between the glycosaminoglycan attachment region and the transmembrane domain. Polyclonal antibodies directed against the ectodomain, or treatment with the tyrosine kinase inhibitor genistein, block activity and revert GPI-mS1ED overexpressing cells to a normal morphology. Extracellular matrix (ECM)-dependent signaling appears to be targeted, as GPI-mS1ED cells attach and spread similarly to control cells in response to E-cadherin engagement, but fail to spread on integrin-dependent ligands. However, integrin-dependent cell attachment, and integrin activation and subsequent FAK phosphorylation are unaffected, suggesting that the syndecan regulates the integration of signaling following matrix adhesion. In 3D culture, where syndecan-1 may have a more critical role in cell behavior, the disrupted signaling leads to poorly cohesive, invasive colonies. Thus, altered matrix-dependent signaling due to increased levels of cell surface syndecan-1 may lead to epithelial cell invasion during early stages of tumorigenesis.

      View details for PubMedID 15383330
  • Cell-surface heparan sulfate proteoglycans potentiate chordin antagonism of bone morphogenetic protein signaling and are necessary for cellular uptake of chordin. J Biol Chem
    Jasuja R, Allen BL, Pappano WN, Rapraeger AC, Greenspan DS
    2004 Dec 03; 279 (49): 51289-97
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      Signaling by bone morphogenetic proteins (BMPs) plays a central role in early embryonic patterning, organogenesis, and homeostasis in a broad range of species. Chordin, an extracellular antagonist of BMP signaling, is thought to readily diffuse in tissues, thus forming gradients of BMP inhibition that result in reciprocal gradients of BMP signaling. The latter determine cell fates along the embryonic dorsoventral axis. The secreted protein Twisted Gastrulation (TSG) is thought to help shape BMP signaling gradients by acting as a cofactor that enhances Chordin inhibition of BMP signaling. Here, we demonstrate that mammalian Chordin binds heparin with an affinity similar to that of factors known to functionally interact with heparan sulfate proteoglycans (HSPGs) in tissues. We further demonstrate that Chordin binding in mouse embryonic tissues was dependent upon its interaction with cell-surface HSPGs and that Chordin bound to cell-surface HSPGs (e.g. syndecans), but not to basement membranes containing the HSPG perlecan. Surprisingly, mammalian TSG did not bind heparin unless prebound to Chordin and/or BMP-4, although Drosophila TSG has been reported to bind heparin on its own. Results are also presented that indicate that Chordin-HSPG interactions strongly potentiate the antagonism of BMP signaling by Chordin and are necessary for the retention and uptake of Chordin by cells. These data and others regarding Chordin diffusion have implications for the paradigm of how Chordin is thought to regulate BMP signaling in the extracellular space and how gradients of BMP signaling are formed.

      View details for PubMedID 15381701
  • Essential and separable roles for Syndecan-3 and Syndecan-4 in skeletal muscle development and regeneration. Genes Dev
    Cornelison DD, Wilcox-Adelman SA, Goetinck PF, Rauvala H, Rapraeger AC, Olwin BB
    2004 Sep 15; 18 (18): 2231-6
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      Syndecan-3 and syndecan-4 function as coreceptors for tyrosine kinases and in cell adhesion. Syndecan-3(-/-) mice exhibit a novel form of muscular dystrophy characterized by impaired locomotion, fibrosis, and hyperplasia of myonuclei and satellite cells. Explanted syndecan-3(-/-) satellite cells mislocalize MyoD, differentiate aberrantly, and exhibit a general increase in overall tyrosine phosphorylation. Following induced regeneration, the hyperplastic phenotype is recapitulated. While there are fewer apparent defects in syndecan-4(-/-) muscle, explanted satellite cells are deficient in activation, proliferation, MyoD expression, myotube fusion, and differentiation. Further, syndecan-4(-/-) satellite cells fail to reconstitute damaged muscle, suggesting a unique requirement for syndecan-4 in satellite cell function.

      View details for PubMedID 15371336
  • A polymer scaffold for protein oligomerization. J Am Chem Soc
    Griffith BR, Allen BL, Rapraeger AC, Kiessling LL
    2004 Feb 18; 126 (6): 1608-9
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      We report the design and synthesis of well-defined polymers for the noncovalent oligomerization of proteins. The reported scaffolds, which were generated by atom-transfer radical polymerization (ATRP), take advantage of the well-characterized interaction between a Ni2+ complex and an oligohistidine sequence (His tag). Thus, our polymers are designed to facilitate the oligomerization of any protein possessing a His tag. We demonstrate that they can oligomerize fibroblast growth factor-8b (FGF-8b) and promote FGF-8b-mediated cell proliferation in the absence of heparin.

      View details for PubMedID 14871072
  • Syndecans in tumor cell adhesion and signaling. Reprod Biol Endocrinol
    Beauvais DM, Rapraeger AC
    2004 Jan 07; 2: 3
    • More

      Anchorage of cells to "heparin"--binding domains that are prevalent in extracellular matrix (ECM) components is thought to occur primarily through the syndecans, a four-member family of transmembrane heparan sulfate proteoglycans that communicate environmental cues from the ECM to the cytoskeleton and the signaling apparatus of the cell. Known activities of the syndecans trace to their highly conserved cytoplasmic domains and to their heparan sulfate chains, which can serve to regulate the signaling of growth factors and morphogens. However, several emerging studies point to critical roles for the syndecans' extracellular protein domains in tumor cell behavior to include cell adhesion and invasion. Although the mechanisms of these activities remain largely unknown, one possibility involves "co-receptor" interactions with integrins that may regulate integrin function and the cell adhesion-signaling phenotype. Thus, alterations in syndecan expression, leading to either overexpression or loss of expression, both of which take place in tumor cells, may have dramatic effects on tumor cell invasion.

      View details for PubMedID 14711376
  • Spatial and temporal expression of heparan sulfate in mouse development regulates FGF and FGF receptor assembly. J Cell Biol
    Allen BL, Rapraeger AC
    2003 Nov 10; 163 (3): 637-48
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      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.

      View details for PubMedID 14610064
  • Fibroblast growth factor receptor-1 mediates the inhibition of endothelial cell proliferation and the promotion of skeletal myoblast differentiation by SPARC: a role for protein kinase A. J Cell Biochem
    Motamed K, Blake DJ, Angello JC, Allen BL, Rapraeger AC, Hauschka SD, Sage EH
    2003 Oct 01; 90 (2): 408-23
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      The role of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine) in modulation of vascular cell proliferation is believed to be mediated, in part, by its ability to regulate the activity of certain growth factors through direct binding. In this study, we demonstrate that SPARC does not bind to basic fibroblast growth factor (bFGF/FGF-2) or interfere with complex formation between FGF-2 and its high-affinity FGF receptor-1 (FGFR1), yet both native SPARC and a peptide derived from the C-terminal high-affinity Ca(2+)-binding region of protein significantly inhibit ligand-induced autophosphorylation of FGFR1 (>80%), activation of mitogen-activated protein kinases (MAPKs) (>75%), and DNA synthesis in human microvascular endothelial cells (HMVEC) stimulated by FGF-2 (>80%). We also report that in the presence of FGF-2, a factor which otherwise stimulates myoblast proliferation and the repression of terminal differentiation, both native SPARC and the Ca(2+)-binding SPARC peptide significantly promote (>60%) the differentiation of the MM14 murine myoblast cell line that expresses FGFR1 almost exclusively. Moreover, using heparan sulfate proteoglycan (HSPG)-deficient myeloid cells and porcine aortic endothelial cells (PAECs) expressing chimeric FGFR1, we show that antagonism of FGFR1-mediated DNA synthesis and MAPK activation by SPARC does not require the presence of cell-surface, low-affinity FGF-2 receptors, but can be mediated by an intracellular mechanism that is independent of an interaction with the extracellular ligand-binding domain of FGFR1. We also report that the inhibitory effect of SPARC on DNA synthesis and MAPK activation in endothelial cells is mediated in part (>50%) by activation of protein kinase A (PKA), a known regulator of Raf-MAPK pathway. SPARC thus modulates the mitogenic effect of FGF-2 downstream from FGFR1 by selective regulation of the MAPK signaling cascade.

      View details for PubMedID 14505356
  • Syndecan-1 transmembrane and extracellular domains have unique and distinct roles in cell spreading. J Biol Chem
    McQuade KJ, Rapraeger AC
    2003 Nov 21; 278 (47): 46607-15
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      Raji cells expressing syndecan-1 (Raji-S1) adhere and spread when plated on heparan sulfate-binding extracellular matrix ligands or monoclonal antibody 281.2, an antibody directed against the syndecan-1 extracellular domain. Cells plated on monoclonal antibody 281.2 initially extend a broad lamellipodium, a response accompanied by membrane ruffling at the cell margin. Membrane ruffling then becomes polarized, leading to an elongated cell morphology. Previous work demonstrated that the syndecan-1 cytoplasmic domain is not required for these activities, suggesting important roles for the syndecan-1 transmembrane and/or extracellular domains in the assembly of a signaling complex necessary for spreading. Work described here demonstrates that truncation of the syndecan-1 extracellular domain does not affect the initial lamellipodial extension in the Raji-S1 cells but does inhibit the active membrane ruffling that is necessary for cell polarization. Replacement of the entire syndecan-1 transmembrane domain with leucine residues completely blocks the cell spreading. These data demonstrate that the syndecan-1 transmembrane and extracellular domains have important but distinct roles in Raji-S1 cell spreading; the extracellular domain mediates an interaction that is necessary for dynamic cytoskeletal rearrangements whereas an interaction of the transmembrane domain is required for the initial spreading response.

      View details for PubMedID 12975379
  • Syndecan-1 accumulates in lysosomes of poorly differentiated breast carcinoma cells. Matrix Biol
    Burbach BJ, Friedl A, Mundhenke C, Rapraeger AC
    2003 Apr; 22 (2): 163-77
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      Expression patterns of syndecan-1, the cell surface heparan sulfate proteoglycan (HSPG) predominant on epithelial cells, were analyzed in tissue samples from 30 infiltrating human breast carcinomas and in 9 human breast carcinoma cell lines. Immunohistochemical staining demonstrates that while a subset of the breast carcinomas lose syndecan-1, this proteoglycan is expressed or overexpressed in a majority of the cases. Interestingly, cells in poor grade tumors contain intracellular syndecan-1, an observation that has not been previously described and was thus further investigated. Examination of cultured breast carcinoma cell lines indicates that they also display the phenotype of the syndecan-1 positive tumors and thereby provide a model system for analysis of intracellular syndecan-1. All cell lines examined express syndecan-1, and poorly differentiated lines such as BT549 cells internalize the proteoglycan from the cell surface where it accumulates as intact HSPG in intracellular vesicles. Colocalization studies using fluorescent markers identify these to be lysosomes. This finding is unexpected, as the accepted mechanism for degradation of syndecan HSPG following endocytosis is fragmentation of the protein core and glycosaminoglycan chains in endosomes, followed by delivery of the fragments to lysosomes. Lysosomal inactivation using ammonium chloride demonstrates that well-differentiated lines such as T47D and MCF-7 cells, which maintain the majority of syndecan-1 on their cell surfaces, also target intact constitutively endocytosed syndecan-1 to lysosomes. Taken together, these results suggest that mammary epithelial cells utilize a previously uncharacterized mechanism for syndecan-1 catabolism. In this pathway the proteoglycan remains intact as it passes through the endosomal system, prior to arriving at its site of intracellular degradation in lysosomes.

      View details for PubMedID 12782143
  • Syndecan-1-mediated cell spreading requires signaling by alphavbeta3 integrins in human breast carcinoma cells. Exp Cell Res
    Beauvais DM, Rapraeger AC
    2003 Jun 10; 286 (2): 219-32
    • More

      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 Mn(2+), suggesting that activation of a beta(1) or beta(3) integrin partner is required. Surprisingly, pretreatment of cells with a function-activating beta(1) antibody does not induce spreading, whereas function-blocking beta(1) integrin antibodies do, suggesting involvement of a beta(1)-to-beta(3) integrin cross-talk. Indeed, blockade of beta(1) integrin activation induces alpha(v)beta(3) 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 alpha(v)beta(3) integrins; signaling via this complex is negatively regulated by beta(1) integrins.

      View details for PubMedID 12749851
  • Heparan sulfate-growth factor interactions. Methods Cell Biol
    Rapraeger AC
    2002; 69: 83-109
  • Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration. Dev Biol
    Cornelison DD, Filla MS, Stanley HM, Rapraeger AC, Olwin BB
    2001 Nov 01; 239 (1): 79-94
    • More

      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.

      View details for PubMedID 11784020
  • Role of heparan sulfate as a tissue-specific regulator of FGF-4 and FGF receptor recognition. J Cell Biol
    Allen BL, Filla MS, Rapraeger AC
    2001 Nov 26; 155 (5): 845-58
    • More

      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.

      View details for PubMedID 11724824
  • Tissue-specific binding by FGF and FGF receptors to endogenous heparan sulfates. Methods Mol Biol
    Friedl A, Filla M, Rapraeger AC
    2001; 171: 535-46
  • Molecular interactions of syndecans during development. Semin Cell Dev Biol
    Rapraeger AC
    2001 Apr; 12 (2): 107-16
    • More

      The syndecans, cell surface heparan sulfate proteoglycans (HSPGs), bind numerous ligands via their HS glycosaminoglycan chains. The response to this binding is flavored by the identity of the core protein that bears the HS chains. Each of the syndecan core proteins has a short cytoplasmic domain that binds cytosolic regulatory factors. The syndecans also contain highly conserved transmembrane domain and extracellular domains for which important activities are slowly emerging. These protein domains, which will be the focus of this review, localize the syndecan to sites at the cell surface during development where they collaborate with other receptors to regulate signaling and cytoskeletal organization.

      View details for PubMedID 11292376
  • Syndecan-1 signals independently of beta1 integrins during Raji cell spreading. Exp Cell Res
    Lebakken CS, McQuade KJ, Rapraeger AC
    2000 Sep 15; 259 (2): 315-25
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      Syndecan-1-expressing Raji lymphoid cells (Raji-S1 cells) bind and spread rapidly when attaching to matrix ligands that contain heparan sulfate-binding domains. However, these ligands also contain binding sites for integrins, which are widely known to signal, raising the question of whether the proteoglycan core protein participates in generation of the signal for spreading. To address this question, the spreading of the Raji-S1 cells is examined on ligands specific for either beta1 integrins, known to be present on the Raji cells, or the syndecan-1 core protein. The cells adhere and spread on invasin, a ligand that activates beta1 integrins, the IIICS fragment of fibronectin, which is a specific ligand for the alpha4beta1 integrin, or mAb281.2, an antibody specific for the syndecan-1 core protein. The signaling resulting from adhesion to the syndecan-specific antibody appears integrin independent as (i) the morphology of the cells spreading on the antibody is distinct from spreading initiated by the integrins alone; (ii) spreading on the syndecan or integrin ligands is affected differently by the kinase inhibitors tyrphostin 25, genistein, and staurosporine; and (iii) spreading on the syndecan-specific antibody is not disrupted by blocking beta1 integrin activation with mAb13, a beta1 inhibitory antibody. These data demonstrate that ligation of syndecan-1 initiates intracellular signaling and suggest that this signaling occurs when cells expressing syndecan-1 adhere to matrix ligands containing heparan sulfate-binding domains.

      View details for PubMedID 10964499
  • Syndecan-regulated receptor signaling. J Cell Biol
    Rapraeger AC
    2000 May 29; 149 (5): 995-8
  • Differential ability of heparan sulfate proteoglycans to assemble the fibroblast growth factor receptor complex in situ. FASEB J
    Chang Z, Meyer K, Rapraeger AC, Friedl A
    2000 Jan; 14 (1): 137-44
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      Fibroblast growth factors (FGFs) require heparan sulfate proteoglycans (HSPGs) as cofactors for signaling. The heparan sulfate chains (HS) mediate stable high affinity binding of FGFs to their receptor tyrosine kinases (FR) and may specifically regulate FGF activity. A novel in situ binding assay was developed to examine the ability of HSPGs to promote FGF/FR binding using a soluble FR fusion construct (FR1-AP). This fusion protein probe forms a dimer in solution, simulating the dimerization or oligomerization that is thought to occur at the cell surface physiologically. In frozen sections of human skin, FGF-2 binds to keratinocytes and basement membranes of epidermis and dermal blood vessels. In contrast, in skin preincubated with FGF-2, FR1-AP binds avidly to FGF-2 immobilized on keratinocyte cell surfaces, but fails to bind to basement membranes at the dermo-epidermal junction or dermal microvessels despite the fact that these structures bind large amounts of FGF-2. Apparently, basement membrane and cell surface HSPGs differ in their ability to mediate the assembly of a FGF/FR signaling complex presumably due to structural differences of the heparan sulfate chains.

      View details for PubMedID 10627288
  • Tyrosine phosphorylation of syndecan-1 and -4 cytoplasmic domains in adherent B82 fibroblasts. J Biol Chem
    Ott VL, Rapraeger AC
    1998 Dec 25; 273 (52): 35291-8
    • More

      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.

      View details for PubMedID 9857070
  • Molecular interactions of the syndecan core proteins. Curr Opin Cell Biol
    Rapraeger AC, Ott VL
    1998 Oct; 10 (5): 620-8
    • More

      The syndecan family of cell-surface heparan sulfate proteoglycans participate in multiple cell behaviors ranging from growth factor signaling to cell adhesion. Participation in these activities is dependent on specific binding interactions of their heparan sulfate chains and molecular interactions of their core proteins with cytoskeletal and signaling molecules. The highly conserved features of the core proteins have long suggested important functions, which are only now beginning to be understood. Recent advances point to important roles for the extracellular, transmembrane and cytoplasmic domains of the syndecan core proteins in the assembly of these proteoglycans into an intracellular cytoskeletal and signaling apparatus. The proteins display interactions that may be common among the different family members, as well as interactions that provide signaling capabilities that are specific to individual members.

      View details for PubMedID 9818173
  • Characterization of the high affinity cell-binding domain in the cell surface proteoglycan syndecan-4. J Biol Chem
    McFall AJ, Rapraeger AC
    1998 Oct 23; 273 (43): 28270-6
    • More

      The syndecan family of cell surface proteoglycans regulates cell adhesion via their glycosaminoglycan chains and discrete domains of their core proteins. Core protein domains that are variable between syndecan family members may regulate syndecan-specific associations, thereby endowing individual syndecans with unique functions. A syndecan-4-specific domain has been identified in the extracellular syndecan-4 protein. This region mediates cell adhesion when provided as an artificial substratum and is localized within amino acids 56-109 of the recombinant extracellular protein domain of mouse syndecan-4 (mS4ED) (McFall, A. J., and Rapraeger, A. C. (1997) J. Biol. Chem. 272, 12901-12904). To characterize its interaction with the cell surface, radiolabeled ligand binding studies were performed. A single high affinity interaction, with a dissociation constant of 2 x 10(-9) M, was observed between mS4ED and both human and mouse cells. Both chicken S4ED and mS4ED compete for this interaction, although they are only 34% identical within the cell-binding domain sequence. The extracellular protein domains of syndecan-1, -2, and -3, however, fail to compete. The interaction is also observed with native syndecan-4 shed from cell surfaces. Interestingly, the extracellular protein domain of syndecan-1 also mediates cell adhesion, suggesting a similar but discrete interaction for this family member.

      View details for PubMedID 9774449
  • The cell surface proteoglycan syndecan-1 mediates fibroblast growth factor-2 binding and activity. J Cell Physiol
    Filla MS, Dam P, Rapraeger AC
    1998 Mar; 174 (3): 310-21
    • More

      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.

      View details for PubMedID 9462693
  • Identification of an adhesion site within the syndecan-4 extracellular protein domain. J Biol Chem
    McFall AJ, Rapraeger AC
    1997 May 16; 272 (20): 12901-4
    • More

      The syndecan family of cell surface proteoglycans regulates cell adhesion and growth factor signaling by binding components of the extracellular matrix and growth factors. To date, all known ligand interactions are via the covalently attached glycosaminoglycan chains. To assay for potential extracellular interactions via the core proteins directly, the recombinant extracellular domain of syndecan-4 (S4ED), one of the four syndecan family members, was tested as a substratum for the attachment of mammalian cells. Human foreskin fibroblasts bind to mouse S4ED, and both mouse and chicken S4ED can block this binding, with 50% inhibition observed between 0.1 and 1 x 10(-7) M. The extracellular domain of another syndecan family member, syndecan-1, fails to compete for cell binding to mouse S4ED. Amino acids 56-109 of the 120-amino acid mouse S4ED compete fully, suggesting that the cell binding domain is within this region. The ability of syndecan-4 to interact with molecules at the cell surface via its core protein as well as its glycosaminoglycan chains may uniquely regulate the formation of cell surface signaling complexes following engagement of this proteoglycan with its extracellular ligands.

      View details for PubMedID 9148893
  • Differential binding of fibroblast growth factor-2 and -7 to basement membrane heparan sulfate: comparison of normal and abnormal human tissues. Am J Pathol
    Friedl A, Chang Z, Tierney A, Rapraeger AC
    1997 Apr; 150 (4): 1443-55
    • More

      Fibroblast growth factors (FGFs) play multiple roles during development and in adult tissues as paracrine regulators of growth and differentiation. FGFs signal through transmembrane receptor tyrosine kinases, but heparan sulfate is also required for signaling by members of the FGF family. In addition, heparan sulfate may be involved in determining tissue distribution of FGFs. Using biotinylated FGF-2 and FGF-7 (KGF) as probes, we have identified specific interactions between FGFs and heparan sulfates in human tissues. Both FGF species bind to tissue mast cells and to epithelial cell membranes. Binding to basement membrane heparan sulfate is tissue source dependent and specific. Although FGF-2 strongly binds to basement membrane heparan sulfate in skin and most other tissue sites examined, FGF-7 fails to bind to basement membrane heparan sulfate in most locations. However, in subendothelial matrix in blood vessels and in the basement membrane of a papillary renal cell carcinoma, strong FGF-7 binding is seen. In summary, distinct and specific affinities of heparan sulfates for different FGFs were identified that may affect growth factor activation and local distribution. Heparan sulfate may have a gatekeeper function to either restrict or permit diffusion of heparin-binding growth factors across the basement membrane.

      View details for PubMedID 9094999
  • Pervanadate activation of intracellular kinases leads to tyrosine phosphorylation and shedding of syndecan-1. Biochem J
    Reiland J, Ott VL, Lebakken CS, Yeaman C, McCarthy J, Rapraeger AC
    1996 Oct 01; 319 ( Pt 1): 39-47
    • More

      Syndecan-1 is a transmembrane haparan sulphate proteoglycan that binds extracellular matrices and growth factors, making it a candidate to act between these regulatory molecules and intracellular signalling pathways. It has a highly conserved transmembrane/cytoplasmic domain that contains four conserved tyrosines. One of these is in a consensus sequence for tyrosine kinase phosphorylation. As an initial step to investigating whether or not phosphorylation of these tyrosines is part of a signal-transduction pathway, we have monitored the tyrosine phosphorylation of syndecan-1 by cytoplasmic tyrosine kinases in intact cells. Tyrosine phosphorylation of syndecan-1 is observed when NMuMG cells are treated with sodium orthovanadate or pervanadate, which have been shown to activate intracellular tyrosine kinases. Initial studies with sodium orthovanadate demonstrate a slow accumulation of phosphotyrosine on syndecan-1 over the course of several hours. Pervanadate, a more effective inhibitor of phosphatases, allows detection of phosphotyrosine on syndecan-1 within 5 min, with peak phosphorylation seen by 15 min. Concurrently, in a second process activated by pervanadate, syndecan-1 ectodomain is cleaved and released into the culture medium. Two phosphorylated fragments of syndecan-1 of apparent sizes 6 and 8 kDa remain with the cell after shedding of the ectodomain. The 8 kDa size class appears to be a highly phosphorylated form of the 6 kDa product, as it disappears if samples are dephosphorylated. These fragments contain the C-terminus of syndecan-1 and also retain at least a portion of the transmembrane domain, suggesting that they are produced by a cell surface cleavage event. Thus pervanadate treatment of cells results in two effects of syndecan-1: (i) phosphorylation of one or more of its tyrosines via the action of a cytoplasmic kinase(s) and (ii) cleavage and release of the ectodomain into the medium, producing a C-terminal fragment containing the transmembrane/cytoplasmic domain.

      View details for PubMedID 8870647
  • Two hierarchies of FGF-2 signaling in heparin: mitogenic stimulation and high-affinity binding/receptor transphosphorylation. Biochemistry
    Krufka A, Guimond S, Rapraeger AC
    1996 Aug 27; 35 (34): 11131-41
    • More

      FGF-2 activates multiple signaling pathways by a cell surface signaling complex assembled with FGF, its receptor tyrosine kinase, and heparan sulfate proteoglycan. Heparan sulfate binds to a site on the receptor and at least one site on the growth factor. Several models propose an important role for heparan sulfate not only in facilitating FGF-2 binding to its receptor tyrosine kinase but also in promoting signaling via formation of receptor dimers. Such dimers are capable of transphosphorylation of the cytoplasmic domain of the receptor, leading to the generation of phosphotyrosines that are important initiators of intracellular signaling pathways. To explore the participation of heparan sulfates in the formation of a signaling complex that activates these pathways, the binding and activity of FGF-2 on Swiss 3T3 fibroblasts and F32 lymphoid cells is examined with either native or modified forms of heparin. As shown previously, fibroblasts treated with chlorate, which inhibits the sulfation of heparan sulfate and its subsequent binding to FGF-2, display a dramatically reduced response to picomolar concentrations of FGF-2, but binding to receptors and a mitogenic response is restored by heparin. However, the restoration of high-affinity binding is seen only at an optimal concentration of heparin. Excess heparin competes for binding sites within the signaling complex such that high-affinity binding and receptor transphosphorylation are reduced. Despite this, mitogenic signaling is not diminished. A similar result is observed using heparin fragments that promote mitogenesis but not high-affinity binding. These results suggest that the high-affinity signaling complex that is necessary for stable receptor transphosphorylation differs from the signaling complex sufficient for triggering mitogenesis. We speculate that heparan sulfate in vivo participates in two hierarchies of receptor activation. In one, heparan sulfate participates in FGF-2 binding to its receptor tyrosine kinase and activation of mitogenic signaling, perhaps through monomeric receptors or the transient formation of receptor dimers. In the second hierarchy, heparan sulfate participates in the stabilization of a signaling complex that is likely to be comprised of receptor multimers that carry out effective receptor transphosphorylation. A further description of this mechanism may lead to an understanding of how heparan sulfate or its homologues can regulate specific signaling pathways within the cell.

      View details for PubMedID 8780517
  • Syndecan-1 mediates cell spreading in transfected human lymphoblastoid (Raji) cells. J Cell Biol
    Lebakken CS, Rapraeger AC
    1996 Mar; 132 (6): 1209-21
    • More

      Syndecan-1 is a cell surface proteoglycan containing a highly conserved transmembrane and cytoplasmic domain, and an extracellular domain bearing heparan sulfate glycosaminoglycans. Through these domains, syndecan-1 is proposed to have roles in growth factor action, extracellular matrix adhesion, and cytoskeletal organization that controls cell morphology. To study the role of syndecan-1 in cell adhesion and cytoskeleton reorganization, mouse syndecan-1 cDNA was transfected into human Raji cells, a lymphoblastoid cell line that grows as suspended cells and exhibits little or no endogenous cell surface heparan sulfate. High expressing transfectants (Raji-Sl cells) bind to and spread on immobilized thrombospondin or fibronectin, which are ligands for the heparan sulfate chains of the proteoglycan. This binding and spreading as not dependent on the cytoplasmic domain of the core protein, is mutants expressing core proteins with cytoplasmic deletions maintain the ability to spread. The spreading is mediated through engagement of the syndecan-1 core protein, as the Raji-S 1 cells also bind to and spread on immobilized mAb 281.2, an antibody specific for the ectodomain of the syndecan-1 core protein. Spreading on the antibody is independent of the heparan sulfate glycosaminoglycan chains and can be inhibited by competition with soluble mAb 281.2. The spreading can be inhibited by treatment with cytochalasin D or colchicine. These data suggest that the core protein of syndecan-1 mediates spreading through the formation of a multimolecular signaling complex at the cell surface that signals cytoskeleton reorganization. This complex may form via intramembrane or extracellular interactions with the syndecan core protein.

      View details for PubMedID 8601596
  • Elevated levels of syndecan-1 expression confer potent serum-dependent growth in human 293T cells. Cancer Res
    Numa F, Hirabayashi K, Tsunaga N, Kato H, O'Rourke K, Shao H, Stechmann-Lebakken C, Varani J, Rapraeger A, Dixit VM
    1995 Oct 15; 55 (20): 4676-80
    • More

      Syndecan-1 is the best studied integral membrane proteoglycan and functions to modulate epithelial cell attachment and physiology. Extracellularly, syndecan-1 binds both growth factors and extracellular matrix components, and intracellularly, its cytoplasmic portion interacts with cytoskeletal components. To investigate the possible role of syndecan-1 in epithelial cell transformation that is characterized by alteration in extracellular matrix interactions and cytoskeleton architecture, we established stable transfectants of syndecan-1 in a highly transformed human renal epithelial line expressing two viral oncogenes, adenovirus E1a and SV40 large T antigen (293T cell line). Expression of syndecan-1 core protein and appropriate posttranslational attachment of glycosaminoglycan chains was confirmed by enzymatic digestion and Western blot analysis. Overexpresser cells grew at a significantly faster rate than the vector-transfected control cells in serum-rich media but showed a proliferative disadvantage in serum-reduced media. In addition to this serum dependency, syndecan-1 overexpression caused a partial reversal of the transformed phenotype with the expressing clones becoming more anchorage dependent and less motile than the vector-transfected counterparts. Surprisingly, the overexpressers were more tumorigenic when injected s.c. into nude mice. These results indicate that syndecan-1 expression plays a role in the control of cell proliferation and suggest that serum-dependent growth may be the more reflective of tumorigenicity in nude mice.

      View details for PubMedID 7553648
  • In the clutches of proteoglycans: how does heparan sulfate regulate FGF binding? Chem Biol
    Rapraeger AC
    1995 Oct; 2 (10): 645-9
    • More

      Fibroblast growth factors and their receptors bind to heparan sulfate glycosaminoglycans. This is thought to promote ligand-receptor binding and enhance signaling by promoting receptor multimerization. Synthetic mimetics designed to occupy these binding sites may provide the means to understand and to regulate FGF signaling.

      View details for PubMedID 9383470
  • Role of FGFs in skeletal muscle and limb development. Mol Reprod Dev
    Olwin BB, Arthur K, Hannon K, Hein P, McFall A, Riley B, Szebenyi G, Zhou Z, Zuber ME, Rapraeger AC
    1994 Sep; 39 (1): 90-100; discussion 100-1
    • More

      Fibroblast growth factors (FGFs) are a family of nine proteins that bind to three distinct types of cell surface molecules: (i) FGF receptor tyrosine kinases (FGFR-1 through FGFR-4); (ii) a cysteine-rich FGF receptor (CFR); and (iii) heparan sulfate proteoglycans (HSPGs). Signaling by FGFs requires participation of at least two of these receptors: the FGFRs and HSPGs form a signaling complex. The length and sulfation pattern of the heparan sulfate chain determines both the activity of the signaling complex and, in part, the ligand specificity for FGFR-1. Thus, the heparan sulfate proteoglycans are likely to play an essential role in signaling. We have recently identified a role for FGF in limb bud development in vivo. In the chick limb bud, ectopic expression of the 18 kDa form of FGF-2 or FGF-2 fused to an artificial signal peptide at its amino terminus causes skeletal duplications. These data, and the observations that FGF-2 is localized to the subjacent mesoderm and the apical ectodermal ridge in the early developing limb, suggest that FGF-2 plays an important role in limb outgrowth. We propose that FGF-2 is an apical ectodermal ridge-derived factor that participates in limb outgrowth and patterning.

      View details for PubMedID 7999366
  • Regulation by heparan sulfate in fibroblast growth factor signaling. Methods Enzymol
    Rapraeger AC, Guimond S, Krufka A, Olwin BB
    1994; 245: 219-40
    • More

      The integral role of heparan sulfate proteoglycans in FGF signaling provides a potential means of regulating FGF activity. This regulation may be used by the cell, where the modification of heparan sulfate glycosaminoglycans during their synthesis in the Golgi can produce cell type- and potentially ligand-specific sulfation sequences. The description of these sequences will not only provide information on how this regulation is achieved, perhaps lending insight into other heparan sulfate-ligand interactions, but may also discern sulfated mimetics that can be used to disrupt or alter FGF signaling. These mimetics may be useful in the treatment disrupt or alter FGF signaling. These mimetics may be useful in the treatment of disease, or in understanding how FGF signaling via discrete pathways within the cell leads to specific cellular responses, such as activation of mitogenic signaling pathways, calcium fluxes, and cellular differentiation.

      View details for PubMedID 7760735
  • Activating and inhibitory heparin sequences for FGF-2 (basic FGF). Distinct requirements for FGF-1, FGF-2, and FGF-4. J Biol Chem
    Guimond S, Maccarana M, Olwin BB, Lindahl U, Rapraeger AC
    1993 Nov 15; 268 (32): 23906-14
    • More

      Chlorate-treated Swiss 3T3 fibroblasts, with impaired synthesis of heparan sulfate proteoglycan, were used as target cells in assessing the ability of exogenous heparin-derived saccharides to promote the mitogenic activity of basic fibroblast growth factor 2 (FGF-2). Full-size native heparin (carrying iduronosyl 2-O-sulfate and glucosaminyl 6-O-sulfate groups), as well as a dodecasaccharide fraction isolated after limited deaminative cleavage of heparin, were efficient promoters, whereas the corresponding decasaccharides, or smaller oligosaccharides, were inactive. Neither selectively 2-O-desulfated nor preferentially 6-O-desulfated heparin were active. However, the latter derivative competed with native heparin for binding to FGF-2 and thus blocked the ability of native heparin to promote the mitogenic activity of FGF-2. The 6-O-desulfated heparin also prevented the ability of FGF-2 to suppress myogenic differentiation in MM14 mouse myoblasts. The binding region for FGF-2 has been identified as a pentasaccharide sequence containing a single essential O-sulfate group, at C2 of iduronic acid (1). It is proposed that the dodecasaccharide sequence required to promote receptor signaling by FGF-2 encompasses this pentasaccharide region, which binds the growth factor, and a site interacting with the receptor that contains essential 2-O- and 6-O-sulfate groups. Similar studies involving the related growth factors, FGF-1 and FGF-4, revealed differential effects of saccharides. The mitogenic effect induced by FGF-1 thus was not blocked by either the 2-O- or the 6-O-desulfated heparins. However, both of these derivatives, at high concentrations, promote mitogenic activity of FGF-4. It is concluded that specific saccharide sequences within heparan sulfate glycosaminoglycan chains favor the signaling by distinct members of the FGF family.

      View details for PubMedID 7693696
  • Membrane-anchored proteoglycans of mouse macrophages: P388D1 cells express a syndecan-4-like heparan sulfate proteoglycan and a distinct chondroitin sulfate form. J Cell Physiol
    Yeaman C, Rapraeger AC
    1993 Nov; 157 (2): 413-25
    • More

      Proteoglycan accumulation by thioglycollate-elicited mouse peritoneal macrophages and a panel of murine monocyte-macrophage cell lines has been examined to determine whether these cells express plasma membrane-anchored heparan sulfate proteoglycans. Initially, cells were screened for heparan sulfate and chondroitin sulfate glycosaminoglycans after metabolic labeling with radiosulfate. Chondroitin sulfate is secreted to a variable extent by every cell type examined. In contrast, heparan sulfate is all but absent from immature pre-monocytes and is associated predominantly with the cell layer of mature macrophage-like cells. In the P388D1 cell line, the cell-associated chondroitin sulfate is largely present as a plasma membrane-anchored proteoglycan containing a 55 kD core protein moiety, which appears to be unique. In contrast, the cell-associated heparan sulfate is composed of a proteoglycan fraction and protein-free glycosaminoglycan chains, which accumulate intracellularly. A fraction of the heparan sulfate proteoglycan contains a lipophilic domain and can be released from cells following mild treatment with trypsin, suggesting that it is anchored in the plasma membrane. Isolation of this proteoglycan indicates that it is likely syndecan-4: it is expressed as a heparan sulfate proteoglycan at the cell surface, it is cleaved from the plasma membrane by low concentrations of trypsin, and it consists of a single 37 kD core protein moiety that co-migrates with syndecan-4 isolated from NMuMG mouse mammary epithelial cells. Northern analysis reveals that a panel of macrophage-like cell lines accumulate similar amounts of syndecan-4 mRNA, demonstrating that this proteoglycan is expressed by a variety of mature macrophage-like cells. Syndecan-1 mRNA is present only in a subset of these cells, suggesting that the expression of this heparan sulfate proteoglycan may be more highly regulated by these cells.

      View details for PubMedID 8227171
  • The coordinated regulation of heparan sulfate, syndecans and cell behavior. Curr Opin Cell Biol
    Rapraeger AC
    1993 Oct; 5 (5): 844-53
    • More

      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.

      View details for PubMedID 8240828
  • Post-transcriptional regulation of syndecan-1 expression by cAMP in peritoneal macrophages. J Cell Biol
    Yeaman C, Rapraeger AC
    1993 Aug; 122 (4): 941-50
    • More

      Syndecan-1 is a cell surface heparan sulfate proteoglycan that is proposed to serve in cell-cell adhesion, cell-matrix anchorage, and growth factor signaling. Its expression is temporally and spatially regulated during epithelial-mesenchymal interactions in many developing tissues. In some cases, this regulation appears to be achieved at the level of transcription. However, induction of syndecan-1 expression in the embryonic kidney mesenchyme is suggested to occur at the level of mRNA translation (Vainio, S., M. Jalkanen, M. Bernfield, and L. Saxén. 1992. Dev. Biol. 152:221-232). To identify a system in which the regulatory mechanisms controlling syndecan-1 expression can be studied, cells of the monocyte-macrophage lineage, which regulate the expression of many cell surface receptors, were screened for syndecan-1 expression. The syndecan-1 gene is active in blood monocytes as well as resident and thioglycollate-elicited mouse peritoneal macrophages, but expression of the proteoglycan is regulated at two levels. First, elicited macrophages accumulate nine-fold more syndecan-1 mRNA than do resident macrophages or circulating blood monocytes. Another member of the syndecan family of proteoglycans, syndecan-4, shows a distinct pattern of expression, suggesting that this regulation is specific for syndecan-1. Second, utilization of the mRNA for syndecan-1 production encounters a post-transcriptional block in the elicited macrophages that can be overcome by triggering agents such as E-type prostaglandins or dibutyryl cAMP, which raise intracellular cAMP levels. Dibutyryl cAMP does not induce syndecan-1 expression in resident peritoneal macrophages, which lack a pool of stored mRNA. This suggests that this agent promotes the post-transcriptional utilization of stored syndecan-1 mRNA. The induced proteoglycan appears at the cell surface as a integral of 100-kD heparan sulfate-rich isoform of syndecan-1. This suggests that a cAMP-dependent post-transcriptional control mechanism may be present in a variety of tissues when syndecan-1 expression is regulated.

      View details for PubMedID 8394371
  • Heparan sulfate proteoglycan and FGF receptor target basic FGF to different intracellular destinations. J Cell Sci
    Reiland J, Rapraeger AC
    1993 Aug; 105 ( Pt 4): 1085-93
    • More

      Basic FGF is a prototype of a family of heparin binding growth factors that regulate a variety of cellular responses including cell growth, morphogenesis and differentiation. At least two families of receptors bind bFGF and could mediate its response: (1) tyrosine kinase-containing FGF receptors, designated FGFR-1 to FGFR-4, and (2) heparan sulfate proteoglycans that bind bFGF through their heparan sulfate chains. Both are known to undergo internalization and thus bFGF bound to the different receptors may be internalized via more than one pathway. It is not known whether the intracellular fate of bFGF differs depending upon which receptor binds it at the cell surface. To investigate the respective roles of these receptors in the intracellular targeting of bFGF, we utilized NMuMG cells that bind and internalize bFGF through their heparan sulfate proteoglycans, but do not express detectable levels of FGFRs nor respond to bFGF. Basic FGF conjugated to saporin (bFGF-saporin) was used as a probe to study targeting of bFGF by the different receptors. Saporin is a cytotoxin that has no effect on cells if added exogenously. However, it kills cells if it gains access to the cytoplasm. The NMuMG cells internalize bFGF-saporin but are not killed. Transfecting these cells with FGFR-1 results in bFGF-responsive cells, which bind and internalize bFGF through FGFR-1, and are killed. Removing the heparan sulfate from these cells eliminates killing by bFGF-saporin.(ABSTRACT TRUNCATED AT 250 WORDS)

      View details for PubMedID 8227197
  • Repression of myogenic differentiation by aFGF, bFGF, and K-FGF is dependent on cellular heparan sulfate. J Cell Biol
    Olwin BB, Rapraeger A
    1992 Aug; 118 (3): 631-9
    • More

      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.

      View details for PubMedID 1379245
  • Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation. Science
    Rapraeger AC, Krufka A, Olwin BB
    1991 Jun 21; 252 (5013): 1705-8
    • More

      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.

      View details for PubMedID 1646484
  • Transforming growth factor (type beta) promotes the addition of chondroitin sulfate chains to the cell surface proteoglycan (syndecan) of mouse mammary epithelia. J Cell Biol
    Rapraeger A
    1989 Nov; 109 (5): 2509-18
    • More

      Cultured monolayers of NMuMG mouse mammary epithelial cells have augmented amounts of cell surface chondroitin sulfate glycosaminoglycan (GAG) when cultured in transforming growth factor-beta (TGF-beta), presumably because of increased synthesis on their cell surface proteoglycan (named syndecan), previously shown to contain chondroitin sulfate and heparan sulfate GAG. This increase occurs throughout the monolayer as shown using soluble thrombospondin as a binding probe. However, comparison of staining intensity of the GAG chains and syndecan core protein suggests variability among cells in the attachment of GAG chains to the core protein. Characterization of purified syndecan confirms the enhanced addition of chondroitin sulfate in TGF-beta: (a) radiosulfate incorporation into chondroitin sulfate is increased 6.2-fold in this proteoglycan fraction and heparan sulfate is increased 1.8-fold, despite no apparent increase in amount of core protein per cell, and (b) the size and density of the proteoglycan are increased, but reduced by removal of chondroitin sulfate. This is shown in part by treatment of the cells with 0.5 mM xyloside that blocks the chondroitin sulfate addition without affecting heparan sulfate. Higher xyloside concentrations block heparan sulfate as well and syndecan appears at the cell surface as core protein without GAG chains. The enhanced amount of GAG on syndecan is partly attributed to an increase in chain length. Whereas this accounts for the additional heparan sulfate synthesis, it is insufficient to explain the total increase in chondroitin sulfate; an approximately threefold increase in chondroitin sulfate chain addition occurs as well, confirmed by assessing chondroitin sulfate ABC lyase (ABCase)-generated chondroitin sulfate linkage stubs on the core protein. One of the effects of TGF-beta during embryonic tissue interactions is likely to be the enhanced synthesis of chondroitin sulfate chains on this cell surface proteoglycan.

      View details for PubMedID 2509487
  • A quantitative solid-phase assay for identifying radiolabeled glycosaminoglycans in crude cell extracts. Anal Biochem
    Rapraeger A, Yeaman C
    1989 Jun; 179 (2): 361-5
    • More

      Extraction of radiosulfate-labeled cell layers in denaturing urea and nonionic detergent allows the quantitative binding of GAG-containing materials from up to 96 discrete samples to a single cationic nylon blot. Free sulfate and/or sulfated lipids fail to bind. Washing the blot with differential salt concentrations discriminates between native proteoglycans and free glycosaminoglycan chains or fragments. In addition, chondroitin sulfates and heparan sulfate are identified either by prior digestion with chondroitin ABC or AC lyase, as generated disaccharides fail to bind to the blot, or by treatment of the entire blot with nitrous acid following binding. Similarly, heparan sulfate can be identified on chromatograms or Western transfers from polyacrylamide gel electrophoresis by autoradiography before and after treatment of the blot with nitrous acid.

      View details for PubMedID 2505639
  • Heparan sulfate-mediated binding of epithelial cell surface proteoglycan to thrombospondin. J Biol Chem
    Sun X, Mosher DF, Rapraeger A
    1989 Feb 15; 264 (5): 2885-9
    • More

      Purified NMuMG mouse mammary epithelial cell surface proteoglycan (PG), a membrane-intercalated core protein bearing both heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) chains, binds to a thrombospondin (TSP) affinity column and is eluted by a salt gradient. Double immunofluorescence microscopy demonstrates extensive co-localization of bound exogenous TSP and cells bearing exposed cell surface PG at their apical surface. The binding, as assayed by both methods, is heparitinase-sensitive, but not chondroitinase-sensitive. Alkali-released heparan sulfate chains bind to a TSP affinity column, similarly to native PG, whereas the chrondroitin sulfate chains do not. Core protein does not bind to TSP. These results indicate that NMuMG cells bind TSP via their surface PG and that the binding is mediated by the heparan sulfate chains.

      View details for PubMedID 2521631
  • Altered structure of the hybrid cell surface proteoglycan of mammary epithelial cells in response to transforming growth factor-beta. J Cell Biol
    Rasmussen S, Rapraeger A
    1988 Nov; 107 (5): 1959-67
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      Transforming growth factor beta (TGF-beta) is a polypeptide growth factor that affects the accumulation of extracellular matrix by many cell types. We have examined the ability of mouse mammary epithelial (NMuMG) cells to respond to TGF-beta and assessed the effect of the growth factor on the expression of their cell surface heparan sulfate/chondroitin sulfate hybrid proteoglycan. NMuMG cells respond maximally to 3 ng/ml TGF-beta and the response is consistent with occupancy of the type III receptor. However, cells that are polarized, as shown by sequestration of the cell surface PG at their basolateral surfaces, must have the growth factor supplied to that site for maximal response. Immunological quantification of proteoglycan core protein on treated cells suggests that the cells have an unchanging number of this proteoglycan at their cell surface. Nonetheless, metabolic labeling with radiosulfate shows a approximately 2.5-fold increase in 35SO4-glycosaminoglycans in this proteoglycan fraction, defined either by its lipophilic, antigenic, or cell surface properties. Kinetic studies indicate that the enhanced radiolabeling is due to augmented synthesis, rather than slower degradation. Analysis of the glycosaminoglycan composition of the proteoglycan shows an increased amount of chondroitin sulfate, suggesting that the increased labeling per cell may be attributed to an augmented synthesis of chondroitin sulfate glycosaminoglycan on the core protein that also bears heparan sulfate, thus altering the proportions of these two glycosaminoglycans on this hybrid proteoglycan. We conclude that TGF-beta may affect NMuMG cell behavior by altering the structure and thus the activity of this proteoglycan.

      View details for PubMedID 3141430
  • Mouse mammary epithelial cells produce basement membrane and cell surface heparan sulfate proteoglycans containing distinct core proteins. J Cell Biol
    Jalkanen M, Rapraeger A, Bernfield M
    1988 Mar; 106 (3): 953-62
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      Cultured mouse mammary (NMuMG) cells produce heparan sulfate-rich proteoglycans that are found at the cell surface, in the culture medium, and beneath the monolayer. The cell surface proteoglycan consists of a lipophilic membrane-associated domain and an extracellular domain, or ectodomain, that contains both heparan and chondroitin sulfate chains. During culture, the cells release into the medium a soluble proteoglycan that is indistinguishable from the ectodomain released from the cells by trypsin treatment. This medium ectodomain was isolated, purified, and used as an antigen to prepare an affinity-purified serum antibody from rabbits. The antibody recognizes polypeptide determinants on the core protein of the ectodomain of the cell surface proteoglycan. The reactivity of this antibody was compared with that of a serum antibody (BM-1) directed against the low density basement membrane proteoglycan of the Englebarth-Holm-Swarm tumor (Hassell, J. R., W. C. Leyshon, S. R. Ledbetter, B. Tyree, S. Suzuki, M. Kato, K. Kimata, and H. Kleinman. 1985. J. Biol. Chem. 250:8098-8105). The BM-1 antibody recognized a large, low density heparan sulfate-rich proteoglycan in the cells and in the basal extracellular materials beneath the monolayer where it accumulated in patchy deposits. The affinity-purified anti-ectodomain antibody recognized the cell surface proteoglycan on the cells, where it is seen on apical cell surfaces in subconfluent cultures and in fine filamentous arrays at the basal cell surface in confluent cultures, but detected no proteoglycan in the basal extracellular materials beneath the monolayer. The amino acid composition of the purified medium ectodomain was substantially different from that reported for the basement membrane proteoglycan. Thus, NMuMG cells produce at least two heparan sulfate-rich proteoglycans that contain distinct core proteins, a cell surface proteoglycan, and a basement membrane proteoglycan. In newborn mouse skin, these proteoglycans localize to distinct sites; the basement membrane proteoglycan is seen solely at the dermal-epidermal boundary and the cell surface proteoglycan is seen solely at the surfaces of keratinocytes in the basal, spinous, and granular cell layers. These results suggest that although heparan sulfate-rich proteoglycans may have similar glycosaminoglycan chains, they are sorted by the epithelial cells to different sites on the basis of differences in their core proteins.

      View details for PubMedID 2964452
  • Cell surface proteoglycan of mouse mammary epithelial cells is shed by cleavage of its matrix-binding ectodomain from its membrane-associated domain. J Cell Biol
    Jalkanen M, Rapraeger A, Saunders S, Bernfield M
    1987 Dec; 105 (6 Pt 2): 3087-96
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      The cell surface proteoglycan on normal murine mammary gland (NMuMG) epithelial cells consists of a lipophilic domain, presumably intercalated into the plasma membrane, and an ectodomain that binds via its glycosaminoglycan chains to matrix components, is released intact by proteases and is detected by monoclonal antibody 281-2. The antibody 281-2 also detects a proteoglycan in the culture medium conditioned by NMuMG cells. This immunoactive proteoglycan was purified to homogeneity using DEAE-cellulose chromatography, isopycnic centrifugation, and 281-2 affinity chromatography. Comparison of the immunoreactive medium proteoglycan with the trypsin-released ectodomain revealed that these proteoglycans are indistinguishable by several criteria as both: (a) contain heparan sulfate and chondroitin sulfate chains; and (b) are similar in hydrodynamic size and buoyant density; (c) have the same size core protein (Mr approximately 53 kD); (d) are nonlipophilic as studied by liposomal intercalation and transfer to silicone-treated paper. Kinetic studies of the release of proteoglycan from the surface of suspended NMuMG cells are interpreted to indicate that the immunoreactive medium proteoglycan is derived directly from the cell surface proteoglycan. Suspension of the cells both augments the release and inhibits the replacement of cell surface proteoglycan. These results indicate that the cell surface proteoglycan of NMuMG cells can be shed by cleavage of its matrix-binding ectodomain from its membrane-associated domain, providing a mechanism by which the epithelial cells can loosen their proteoglycan-mediated attachment to the matrix.

      View details for PubMedID 3320062
  • Cell surface proteoglycan associates with the cytoskeleton at the basolateral cell surface of mouse mammary epithelial cells. J Cell Biol
    Rapraeger A, Jalkanen M, Bernfield M
    1986 Dec; 103 (6 Pt 2): 2683-96
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      The cell surface proteoglycan on normal murine mammary gland mouse mammary epithelial cells consists of an ectodomain bearing heparan and chondroitin sulfate chains and a lipophilic domain that is presumed to be intercalated into the plasma membrane. Because the ectodomain binds to matrix components produced by stromal cells with specificity and high affinity, we have proposed that the cell surface proteoglycan is a matrix receptor that binds epithelial cells to their underlying basement membrane. We now show that the proteoglycan surrounds cells grown in subconfluent or newly confluent monolayers, but becomes restricted to the basolateral surface of cells that have been confluent for a week or more; Triton X-100 extraction distinguishes three fractions of cell surface proteoglycan: a fraction released by detergent and presumed to be free in the membrane, a fraction bound via a salt-labile linkage, and a nonextractable fraction; the latter two fractions co-localize with actin filament bundles at the basal cell surface; and when proteoglycans at the apical cell surface are cross-linked by antibodies, they initially assimilate into detergent-resistant, immobile clusters that are subsequently aggregated by the cytoskeleton. These findings suggest that the proteoglycan, initially present on the entire surface and free in the plane of the membrane, becomes sequestered at the basolateral cell surface and bound to the actin-rich cytoskeleton as the cells become polarized in vitro. Binding of matrix components may cross-link proteoglycans at the basal cell surface and cause them to associate with the actin cytoskeleton, providing a mechanism by which the cell surface proteoglycan acts as a matrix receptor to stabilize the morphology of epithelial sheets.

      View details for PubMedID 3025223
  • The cell surface proteoglycan from mouse mammary epithelial cells bears chondroitin sulfate and heparan sulfate glycosaminoglycans. J Biol Chem
    Rapraeger A, Jalkanen M, Endo E, Koda J, Bernfield M
    1985 Sep 15; 260 (20): 11046-52
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      The cell surface proteoglycan fraction isolated by mild trypsin treatment of NMuMG mouse mammary epithelial cells contains largely heparan sulfate, but also 15-24% chondroitin sulfate glycosaminoglycans. We conclude that this fraction contains a unique hybrid proteoglycan bearing both heparan sulfate and chondroitin sulfate glycosaminoglycans because (i) the proteoglycan behaves as a single species by sizing, ion exchange and collagen affinity chromatography, and by isopycnic centrifugation, even in the presence of 8 M urea or 4 M guanidine hydrochloride, (ii) the behavior of the chondroitin sulfate in these separation techniques is affected by heparan sulfate-specific probes and vice versa, and (iii) proteoglycan core protein bearing both heparan sulfate and chondroitin sulfate is recognized by a single monoclonal antibody. Removal of both types of glycosaminoglycan reduces the proteoglycan to a core protein of approximately 53 kDa. The proteoglycan fraction is heterogeneous in size, largely due to a variable number and/or length of the glycosaminoglycan chains. We estimate that one or two chondroitin sulfate chains (modal Mr of 17,000) exist on the proteoglycan for every four heparan sulfate chains (modal Mr of 36,000). Synthesis of these chains is reportedly initiated on an identical trisaccharide that links the chains to the same amino acid residues on the core protein. Therefore, some regulatory information, perhaps residing in the amino acid sequence of the core protein, must determine the type of chain synthesized at any given linkage site. Post-translational addition of these glycosaminoglycans to the protein may provide information affecting its ultimate localization. It is likely that the protein is directed to specific sites on the cell surface because of the ability of the glycosaminoglycans to recognize and bind extracellular components.

      View details for PubMedID 3161889
  • Heparan sulfate proteoglycans from mouse mammary epithelial cells: localization on the cell surface with a monoclonal antibody. J Cell Biol
    Jalkanen M, Nguyen H, Rapraeger A, Kurn N, Bernfield M
    1985 Sep; 101 (3): 976-84
    • More

      Mouse mammary epithelial cells, of the normal murine mammary gland (NMuMG) cell line, bear a heparan sulfate-rich proteoglycan (HSPG) on their surfaces. A hybridoma (281-2) secreting a monoclonal antibody that recognizes this HSPG was produced by fusion of SP-2/0 myeloma cells with spleen cells from rats immunized with NMuMG cells. The 281-2 monoclonal antibody is directed against the core protein of the cell surface HSPG, as demonstrated by (a) recognition of the isolated proteoglycan but not its glycosaminoglycan chains, (b) co-localization of 281-2-specific antigen and radioactive cell surface HSPG on gradient polyacrylamide gel electrophoresis and on isopycnic centrifugation, and (c) abolition of immunofluorescent staining of the NMuMG cell surface by the intact, but not the protease-digested ectodomain of the cell surface HSPG. The antibody is specific for cell surface HSPG and does not recognize the HSPG that accumulates extracellularly beneath the basal cell surface. Therefore, the 281-2 antibody may be used to isolate the cell surface HSPG and to explore its distribution in tissues.

      View details for PubMedID 3161899
  • Heparan sulfate proteoglycans from mouse mammary epithelial cells. Cell surface proteoglycan as a receptor for interstitial collagens. J Biol Chem
    Koda JE, Rapraeger A, Bernfield M
    1985 Jul 05; 260 (13): 8157-62
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      A heparan sulfate-rich proteoglycan is on the surface of NMuMG mouse mammary epithelial cells apparently intercalated into their plasma membranes. Mild treatment of the cells with trypsin releases the GAG-bearing region (ectodomain) of this molecule as a discrete proteoglycan which is readily purified. At physiological pH and ionic strength, the ectodomain binds collagen types I, III, and V but not types II, IV, or denatured type I. The proteoglycan binds to a single class of high affinity saturable sites on type I collagen fibrils, sites which are selective for heparin-like glycosaminoglycans. The binding of NMuMG cells to type I collagen duplicates that of their cell surface proteoglycan; cells bind to native but not denatured collagen, and binding is inhibited by heparin but not by other glycosaminoglycans. These binding properties suggest that cell surface heparan sulfate proteoglycans could act as receptors for interstitial collagens and mediate changes in cell behavior induced by collagenous matrices.

      View details for PubMedID 3159726
  • Cell surface proteoglycan of mammary epithelial cells. Protease releases a heparan sulfate-rich ectodomain from a putative membrane-anchored domain. J Biol Chem
    Rapraeger A, Bernfield M
    1985 Apr 10; 260 (7): 4103-9
    • More

      Heparan sulfate-rich proteoglycan is present on the surface of NMuMG mouse mammary epithelial cells. All of this cell surface fraction is lipophilic, assessed by intercalation into lipid vesicles, and requires proteolytic cleavage to be released from the cell surface. No proteoglycan is competitively displaced by heparin. The cell surface lipophilic proteoglycan constitutes 52-55% of the total cellular proteoglycan while the remaining proteoglycan is apparently intracellular, comprising a nonlipophilic fraction (35%) and a small (10-13%) lipophilic fraction. Trypsin or chymotrypsin cleaves a labile site between the region of the cell surface proteoglycan bearing the glycosaminoglycan chains and the cell-associated portion of the core protein, producing a proteoglycan that is nonlipophilic, has an increased bouyant density, and is smaller than the parent molecule. We refer to this proteoglycan as the ectodomain of the cell surface proteoglycan. The correlation between its cell surface location and lipophilic properties suggests that a hydrophobic domain of its core protein may anchor this proteoglycan in the plasma membrane. In vivo, the proteoglycan may be cleaved from this putative anchor, generating nonlipophilic proteoglycan present as a matrix component, or it may remain a membrane component, anchoring the cell directly to the extracellular matrix.

      View details for PubMedID 3156852
  • Remodelling of the basement membrane: morphogenesis and maturation. Ciba Found Symp
    Bernfield M, Banerjee SD, Koda JE, Rapraeger AC
    1984; 108: 179-96
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      We have analysed the reciprocal interactions between mouse embryo submandibular epithelium and mesenchyme which result in branching morphogenesis of the epithelium. The interactions modify the composition and metabolism of the basal and reticular laminae which comprise the basement membrane lying between these tissues. The mesenchyme remodels the basement membrane by depositing a type I collagen-rich matrix on the basal lamina and by producing a neutral hyaluronidase, which degrades hyaluronate and chondroitin sulphate, components of this basal lamina. By analogy with mouse mammary epithelial cells, the submandibular epithelial cells have a heparan sulphate-rich proteoglycan on their cell surfaces which is anchored to the cells. The extracellular domain of this integral membrane proteoglycan binds to interstitial collagen. Interfering with the collagen-proteoglycan interaction appears to reduce the morphological stability of the cells. Together with other processes, including epithelial cell proliferation, this remodelling leads to branching epithelial morphogenesis. Basement membrane remodelling may be a general process for regulating cell behaviour during development and is one of the mechanisms of morphogenetic tissue interaction. Remodelling may also cause maturation of basement membranes from a dynamic state of high turnover in the embryo to their persistence and stability in the adult organism.

      View details for PubMedID 6569828
  • Heparan sulfate proteoglycans from mouse mammary epithelial cells. A putative membrane proteoglycan associates quantitatively with lipid vesicles. J Biol Chem
    Rapraeger AC, Bernfield M
    1983 Mar 25; 258 (6): 3632-6
    • More

      Mouse mammary epithelial (NMuMG) cells produce both cellular and extracellular heparan sulfate-rich proteoglycans. A cellular proteoglycan, but no extracellular proteoglycans, associates quantitatively and vectorially with lipid vesicles, as assessed by column chromatography and centrifugation. This lipophilic cellular proteoglycan is extracted as an aggregate when cells are treated with 4 M guanidine HCl, but is extracted as a single component in the presence of detergent, suggesting that it aggregates with cellular lipid. An association with lipid is confirmed by intercalation of the proteoglycan into the bilayer of lipid vesicles. Formation of lipid vesicles in the presence of the proteoglycan causes the proteoglycan to have the chromatographic and sedimentation behavior of the vesicles while destruction of the vesicles with detergent nullifies this effect. The proteoglycan is intercalated nullifies this effect. The proteoglycan is intercalated into the vesicles with its glycosaminoglycan-containing domain exposed to the exterior since mild trypsin treatment quantitatively removes this portion of the proteoglycan from the vesicle. After cleavage from the vesicle, the released proteoglycan chromatographs with an apparent molecular size similar to that of the whole proteoglycan, but no longer aggregates with lipid. Thus, trypsin removes a lipophilic domain which is responsible for its interaction with lipid and presumably anchors the proteoglycan in cellular membranes.

      View details for PubMedID 6219993
  • The appearance of an extracellular arylsulfatase during morphogenesis of the sea urchin Strongylocentrotus purpuratus. Dev Biol
    Rapraeger AC, Epel D
    1981 Dec; 88 (2): 269-78

Contact Information

Alan Rapraeger, PhD

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Madison, WI 53705
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