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Jorge Filmus - One of the best experts on this subject based on the ideXlab platform.
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Glypican 3 binds to frizzled and plays a direct role in the stimulation of canonical wnt signaling
Journal of Cell Science, 2014Co-Authors: Mariana I Capurro, Wen Shi, Tonya Martin, Jorge FilmusAbstract:Glypican-3 (GPC3) is a proteoglycan that is bound to the cell surface. It is expressed by most hepatocellular carcinomas (HCCs) but not by normal hepatocytes. GPC3 stimulates HCC growth by promoting canonical Wnt signaling. Because Glypicans interact with Wnts, it has been proposed that these proteoglycans stimulate signaling by increasing the amount of Wnt at the cell membrane, thus facilitating the interaction of this growth factor with its signaling receptor, Frizzled. However, in this study, we demonstrate that GPC3 plays a more direct role in the stimulation of Wnt signaling. Specifically, we show that, in addition to interacting with Wnt, GPC3 and Frizzled interact directly through the glycosaminoglycan chains of GPC3, indicating that this Glypican stimulates the formation of signaling complexes between Wnt and Frizzled. Consistent with this, we show that the binding of Wnt at the cell membrane triggers the endocytosis of a complex that includes Wnt, Frizzled and GPC3. Additional support for our model is provided by the finding that Glypican-6 (GPC6) inhibits canonical Wnt signaling, despite the fact that it binds to Wnt at the cell membrane.
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Glypican 3 a marker and a therapeutic target in hepatocellular carcinoma
FEBS Journal, 2013Co-Authors: Jorge Filmus, Mariana CapurroAbstract:Glypican-3 (GPC3) is a member of the Glypican family. Glypicans are proteoglycans that are attached to the cell surface by a glycosyl-phosphatidylinositol anchor. They regulate the signaling activity of several growth factors, including Wnts. This regulation is based on the ability of Glypicans to stimulate or inhibit the interaction of these growth factors with their respective signaling receptors. It has been clearly established that whereas GPC3 is expressed by most hepatocellular carcinomas (HCCs), this Glypican is not detected in normal and cirrhotic liver, or in benign hepatic lesions. Consequently, immunostaining of liver biopsies for GPC3 is currently being used by clinical pathologists to confirm HCC diagnosis when the malignant nature of the lesion is difficult to establish. In addition to being a marker of HCC, GPC3 plays a role in the progression of the disease. GPC3 promotes the growth of HCC by stimulating canonical Wnt signaling. It has been proposed that this stimulation is based on the ability of GPC3 to increase the binding of Wnt to its signaling receptor, Frizzled. Two therapeutic approaches for HCC that target GPC3 are currently being tested in phase II clinical trials. One of them is based on the use of a humanized GPC3 monoclonal antibody that inhibits the in vivo growth of HCC xenografts by inducing antibody-dependent cellular cytotoxicity. The second approach employs a vaccine that consists of two GPC3-derived peptides that induce cytotoxic T lymphocytes against these peptides. Targeting of GPC3 might offer a new tool for the treatment of HCC.
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Abstract 3191: Glypican-5 stimulates rhabdomyosarcoma cell proliferation by activating hedgehog signaling
Cellular and Molecular Biology, 2010Co-Authors: Wen Shi, Mariana Capurro, Jorge FilmusAbstract:Glypican-5 is one of the six members of the Glypican family. It has been previously reported that Glypican-5 stimulates the proliferation of rhabdomyosarcoma cells. In this study we show that this stimulatory activity of Glypican-5 is due to its ability to promote Hedgehog signaling. We have previously shown that Glypican-3, another member of the Glypican family, inhibits Hedgehog signaling by competing with Patched for Hedgehog binding. Furthermore, we showed that Glypican-3 binds to Hedgehog through its core protein but not to Patched. Here we demonstrate that Glypican-5 can increase the binding of Sonic Hedgehog to Patched. We also show that Glypican-5 can bind to both Hedgehog and Patched through its glycosaminoglycan chains. Interestingly, we found that the heparan sulfate chains of Glypican-5 display a significantly higher degree of sulfation than those from Glypican-3. In addition, we show that Glypican-5 interacts with Hedgehog with a lower affinity than Glypican-3. Based on these results, we propose that Glypican-5 stimulates Hedgehog signaling by facilitating/stabilizing the interaction between Hedgehog and Patched. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3191.
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Glypican 3 inhibits hedgehog signaling during development by competing with patched for hedgehog binding
Developmental Cell, 2008Co-Authors: Mariana I Capurro, Wen Shi, Angela Jia, Jorge FilmusAbstract:Loss-of-function mutations in Glypican-3 (GPC3), one of the six mammalian Glypicans, causes the Simpson-Golabi-Behmel overgrowth syndrome (SGBS), and GPC3 null mice display developmental overgrowth. Because the Hedgehog signaling pathway positively regulates body size, we hypothesized that GPC3 acts as an inhibitor of Hedgehog activity during development. Here, we show that GPC3 null embryos display increased Hedgehog signaling and that GPC3 inhibits Hedgehog activity in cultured mouse embryonic fibroblasts. In addition, we report that GPC3 interacts with high affinity with Hedgehog but not with its receptor, Patched, and that GPC3 competes with Patched for Hedgehog binding. Furthermore, GPC3 induces Hedgehog endocytosis and degradation. Surprisingly, the heparan sulfate chains of GPC3 are not required for its interaction with Hedgehog. We conclude that GPC3 acts as a negative regulator of Hedgehog signaling during mammalian development and that the overgrowth observed in SGBS patients is, at least in part, the consequence of hyperactivation of the Hedgehog signaling pathway.
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Soluble Glypican 3 inhibits the growth of hepatocellular carcinoma
Cancer Research, 2008Co-Authors: Sandra I. Zittermann, Wen Shi, Jorge FilmusAbstract:1516 Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver, and the third most common cause of cancer-related mortality. Currently, there are not effective treatments for HCCs that cannot be removed by surgery. Work from our laboratory and from other groups has demonstrated that Glypican-3 (GPC3) is expressed by most HCCs (~ 75 %), while it is undetectable in hepatocytes from normal liver and benign liver disease. GPC3 is a member of the Glypican family. Glypicans are heparan sulfate proteoglycans that are bound to the exocytoplasmic surface of the plasma membrane through a glycosyl-phosphatidylinositol anchor. Experimental evidence accumulated during the last few years indicates that Glypicans regulate the activity of several signaling pathways, including those triggered by Wnts, Hedgehogs, BMPs and FGFs. This regulatory activity is based on the ability of Glypicans to facilitate or inhibit the interaction of these ligands withtheir signaling receptors. Our laboratory has previously shown that GPC3 promotes the in vitro and in vivo growth of HCC cells by stimulating theWnt signaling pathway. Preliminary evidence suggests that GPC3 facilitates the interaction of Wnt with its receptor Frizzled. Because the growth stimulatory activity of GPC3 in HCC cells requires the attachment of GPC3 to the cell membrane, we havehypothesized that a mutated GPC3 lacking the GPI anchoring domain (soluble GPC3, sGPC3) will inhibit HCC growth. To investigate this hypothesis, Huh7, Li7 and Huh6 HCC cell lines were transduced with alentivirus containingsGPC3 or viruscontrol. We observed that sGPC3-expressing cells have a lower proliferation rate and form fewer colonies in agar than the controls. In addition, sGPC3 significantly inhibited the in vivo growth of HCC cells. Our results suggest that sGPC3 could be used as a therapeutic tool for HCC.
Guido David - One of the best experts on this subject based on the ideXlab platform.
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Induction of a CXCL8 binding site on endothelial syndecan‐3 in rheumatoid synovium
Arthritis & Rheumatism, 2005Co-Authors: Angela M. Patterson, Guido David, Lucy Gardner, Jennifer Shaw, Emilie Loreau, Luc Aguilar, Brian A. Ashton, Jim MiddletonAbstract:Objective To identify and characterize which endothelial heparan sulfate proteoglycans (HSPGs) bind the chemokine CXCL8 (interleukin-8) in human rheumatoid arthritis (RA) and nonrheumatoid synovia. Method CXCL8 binding to endothelial HSPGs in RA and nonrheumatoid synovia was determined by heparinase treatment followed by an in situ binding assay and autoradiography. Endothelial HSPGs were characterized by immunohistochemical analysis and quantitative reverse transcriptase–polymerase chain reaction (RT-PCR). Phosphatidyinositol-specific phospholipase C (PI-PLC) and antibodies to HSPGs were used in in situ binding experiments to identify which HSPGs bound CXCL8. Results The expression of heparan sulfate on microvascular endothelial cells was demonstrated in RA and nonrheumatoid synovia. Using antibodies to syndecan-1–4 and Glypican-1, -3, and -4, the selective expression of syndecan-3 by endothelial cells was detected in RA and nonrheumatoid synovia. In addition, RT-PCR showed the presence of syndecan-3 messenger RNA in endothelial cells extracted from RA and nonrheumatoid synovia. 125I-CXCL8 bound to venular endothelial cells; treatment with heparinases I and III significantly reduced this binding in RA but not nonrheumatoid synovia. 125I-CXCL8 binding was not reduced after treatment with PI-PLC, which cleaves glycosyl phosphatidylinositol linkages, suggesting that CXCL8 did not bind to Glypicans. Treatment of synovia with a syndecan-3 antibody reduced CXCL8 binding to RA but not nonrheumatoid endothelial cells; however, no reduction in binding was observed with syndecan-2 or Glypican-4 antibodies. Conclusion Our results show the selective induction of a CXCL8 binding site on endothelial syndecan-3 in RA synovium. This site may be involved in leukocyte trafficking into RA synovial tissue.
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induction of a cxcl8 binding site on endothelial syndecan 3 in rheumatoid synovium
Arthritis & Rheumatism, 2005Co-Authors: Angela M. Patterson, Guido David, Lucy Gardner, Jennifer Shaw, Emilie Loreau, Luc Aguilar, Brian A. Ashton, Jim MiddletonAbstract:Objective To identify and characterize which endothelial heparan sulfate proteoglycans (HSPGs) bind the chemokine CXCL8 (interleukin-8) in human rheumatoid arthritis (RA) and nonrheumatoid synovia. Method CXCL8 binding to endothelial HSPGs in RA and nonrheumatoid synovia was determined by heparinase treatment followed by an in situ binding assay and autoradiography. Endothelial HSPGs were characterized by immunohistochemical analysis and quantitative reverse transcriptase–polymerase chain reaction (RT-PCR). Phosphatidyinositol-specific phospholipase C (PI-PLC) and antibodies to HSPGs were used in in situ binding experiments to identify which HSPGs bound CXCL8. Results The expression of heparan sulfate on microvascular endothelial cells was demonstrated in RA and nonrheumatoid synovia. Using antibodies to syndecan-1–4 and Glypican-1, -3, and -4, the selective expression of syndecan-3 by endothelial cells was detected in RA and nonrheumatoid synovia. In addition, RT-PCR showed the presence of syndecan-3 messenger RNA in endothelial cells extracted from RA and nonrheumatoid synovia. 125I-CXCL8 bound to venular endothelial cells; treatment with heparinases I and III significantly reduced this binding in RA but not nonrheumatoid synovia. 125I-CXCL8 binding was not reduced after treatment with PI-PLC, which cleaves glycosyl phosphatidylinositol linkages, suggesting that CXCL8 did not bind to Glypicans. Treatment of synovia with a syndecan-3 antibody reduced CXCL8 binding to RA but not nonrheumatoid endothelial cells; however, no reduction in binding was observed with syndecan-2 or Glypican-4 antibodies. Conclusion Our results show the selective induction of a CXCL8 binding site on endothelial syndecan-3 in RA synovium. This site may be involved in leukocyte trafficking into RA synovial tissue.
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Mapping of the rat Glypican genes.
Cytogenetics and cell genetics, 2001Co-Authors: Claude Szpirer, Mark Veugelers, Josiane Szpirer, Michèle Riviere, P. Van Vooren, Guido DavidAbstract:The Glypicans compose a family of glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycans that play a role in the control of cell division and growth regulation. So far, six members (GPC1-6) of this family are known in vertebrates. The rat Glypican gene 3 (Gpc3) was previously assigned to chromosome Xq36 (Shen et al., 1997). Using standard and radiation cell hybrids, we localized the five other rat Glypican genes.
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Glypican-6, a New Member of the Glypican Family of Cell Surface Heparan Sulfate Proteoglycans
Journal of Biological Chemistry, 1999Co-Authors: Mark Veugelers, Joris Vermeesch, Helga Ceulemans, Anne-marie Bruystens, Christien Coomans, Joachim Dürr, Peter Marynen, Guido DavidAbstract:Abstract The Glypicans compose a family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans. Mutations in dally, a gene encoding aDrosophila Glypican, and in GPC3, the gene for human Glypican-3, implicate Glypicans in the control of cell growth and division. So far, five members of the Glypican family have been identified in vertebrates. By sequencing expressed sequence tag clones and products of rapid amplifications of cDNA ends, we identified a sixth member of the Glypican family. The Glypican-6 mRNA encodes a protein of 555 amino acids that is most homologous to Glypican-4 (identity of 63%). Expression of this protein in Namalwa cells shows a core protein of ∼60 kDa that is substituted with heparan sulfate only. GPC6, the gene encoding human Glypican-6, contains nine exons. Like GPC5, the gene encoding Glypican-5,GPC6 maps to chromosome 13q32. Clustering of theGPC5/GPC6 genes on chromosome 13q32 is strongly reminiscent of the clustering of the GPC3/GPC4 genes on chromosome Xq26 and suggests GPCs arose from a series of gene and genome duplications. Based on similarities in sequence and gene organization, Glypican-1, Glypican-2, Glypican-4, and Glypican-6 appear to define a subfamily of Glypicans, differing from the subfamily comprising so far Glypican-3 and Glypican-5. Northern blottings indicate that Glypican-6 mRNA is widespread, with prominent expressions in human fetal kidney and adult ovary. In situ hybridization studies localize Glypican-6 to mesenchymal tissues in the developing mouse embryo. High expressions occur in smooth muscle cells lining the aorta and other major blood vessels and in mesenchymal cells of the intestine, kidney, lung, tooth, and gonad. Growth factor signaling in these tissues might in part be regulated by the presence of Glypican-6 on the cell surface.
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Glypican-1 Is a VEGF165 Binding Proteoglycan That Acts as an Extracellular Chaperone for VEGF165
The Journal of biological chemistry, 1999Co-Authors: Stela Gengrinovitch, Guido David, Bluma Berman, Larry Witte, Gera Neufeld, Dina RonAbstract:Abstract Glypican-1 is a member of a family of glycosylphosphatidylinositol anchored cell surface heparan sulfate proteoglycans implicated in the control of cellular growth and differentiation. The 165-amino acid form of vascular endothelial growth factor (VEGF165) is a mitogen for endothelial cells and a potent angiogenic factor in vivo. Heparin binds to VEGF165 and enhances its binding to VEGF receptors. However, native HSPGs that bind VEGF165 and modulate its receptor binding have not been identified. Among the Glypicans, Glypican-1 is the only member that is expressed in the vascular system. We have therefore examined whether Glypican-1 can interact with VEGF165. Glypican-1 from rat myoblasts binds specifically to VEGF165 but not to VEGF121. The binding has an apparent dissociation constant of 3 × 10−10 m. The binding of Glypican-1 to VEGF165 is mediated by the heparan sulfate chains of Glypican-1, because heparinase treatment abolishes this interaction. Only an excess of heparin or heparan sulfates but not other types of glycosaminoglycans inhibited this interaction. VEGF165 interacts specifically not only with rat myoblast Glypican-1 but also with human endothelial cell-derived Glypican-1. The binding of125I-VEGF165 to heparinase-treated human vascular endothelial cells is reduced following heparinase treatment, and addition of Glypican-1 restores the binding. Glypican-1 also potentiates the binding of 125I-VEGF165 to a soluble extracellular domain of the VEGF receptor KDR/flk-1. Furthermore, we show that Glypican-1 acts as an extracellular chaperone that can restore the receptor binding ability of VEGF165, which has been damaged by oxidation. Taken together, these results suggest that Glypican-1 may play an important role in the control of angiogenesis by regulating the activity of VEGF165, a regulation that may be critical under conditions such as wound repair, in which oxidizing agents that can impair the activity of VEGF are produced, and in situations were the concentrations of active VEGF are limiting.
Arthur D. Lander - One of the best experts on this subject based on the ideXlab platform.
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Glypican 1 is overexpressed in human breast cancer and modulates the mitogenic effects of multiple heparin binding growth factors in breast cancer cells
Cancer Research, 2001Co-Authors: Kei Matsuda, Arthur D. Lander, Haruhisa Maruyama, Fang Guo, Jorg Kleeff, Jun Itakura, Yoshiro Matsumoto, Murray KorcAbstract:Glypicans are a family of glycosylphosphatidylinositol-anchored cell surface heparan sulfate proteoglycans implicated in the control of cellular growth and differentiation. Here we show that Glypican-1 is strongly expressed in human breast cancers, whereas expression of Glypican-1 is low in normal breast tissues. In contrast, the expression of Glypican-3 and -4 is only slightly increased in breast cancers by comparison with normal breast tissues, and Glypican-2 and -5 are below the level of detection by Northern blotting in both normal and cancer samples. Treatment of MDA-MB-231 and MDA-MB-468 breast cancer cells with phosphoinositide-specific phospholipase-C abrogated the mitogenic response to two heparin-binding growth factors, heparin-binding epidermal growth factor-like growth factor and fibroblast growth factor 2. Stable transfection of these cells with a Glypican-1 antisense construct markedly decreased Glypican-1 protein levels and the mitogenic response to the same heparin-binding growth factors, as well as that to heregulin alpha, heregulin beta, and hepatocyte growth factor. Syndecan-1 was also expressed at high levels in both breast cancer tissues and breast cancer cells when compared with normal breast tissues. There was a good correlation between Glypican-1 and syndecan-1 expression in the tumors. However, clones expressing the Glypican-1 antisense construct did not exhibit decreased syndecan-1 levels, indicating that loss of responsiveness to heparin-binding growth factors in these clones was not due to altered syndecan-1 expression. Furthermore, 8 of 10 tumors with stage 2 or 3 disease exhibited high levels of Glypican-1 by Northern blot analysis. In contrast, low levels of Glypican-1 mRNA were evident in 1 of 10 tumors with stage 2 or 3 disease and in 9 of 10 tumors with stage 1 disease. Taken together, these data suggest that Glypican-1 may play a pivotal role in the ability of breast cancer cells to exhibit a mitogenic response to multiple heparin-binding growth factors and may contribute to disease progression in this malignancy.
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Mechanisms Underlying Preferential Assembly of Heparan Sulfate on Glypican-1
The Journal of biological chemistry, 2000Co-Authors: Robert L. Chen, Arthur D. LanderAbstract:Glypicans are major cell surface heparan sulfate proteoglycans, the structures of which are characterized by the presence of a cysteine-rich globular domain, a short glycosaminoglycan (GAG) attachment region, and a glycosylphosphatidylinositol membrane anchor. Despite strong evolutionary conservation of the globular domains of Glypicans, no function has yet been attributed to them. By using a novel quantitative approach for assessing proteoglycan glycosylation, we show here that removal of the globular domain from rat Glypican-1 converts the proteoglycan from one that bears approximately 90% heparan sulfate (HS) to one that bears approximately 90% chondroitin sulfate. Mutational analysis shows that sequences at least 70 amino acids away from the Glypican-1 GAG attachment site are required for preferential HS assembly, although more nearby sequences also play a role. The effects of the Glypican-1 globular domain on HS assembly could also be demonstrated by fusing this domain to sequences representing the GAG attachment sites of other proteoglycans or, surprisingly, simply by expressing the isolated globular domain in cells and analyzing effects either on an exogenously expressed Glypican-1 GAG attachment domain or on endogenous proteoglycans. Quantitative analysis of the effect of the globular domain on GAG addition to proteoglycan core proteins suggested that preferential HS assembly is achieved, at least in part, through the inhibition of chondroitin sulfate assembly. These data identify the Glypican-1 globular domain as a structural motif that potently influences GAG class determination and suggest that an important role of Glypican globular domains is to ensure a high level of HS substitution of these proteoglycans.
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stable transfection of a Glypican 1 antisense construct decreases tumorigenicity in panc 1 pancreatic carcinoma cells
Pancreas, 1999Co-Authors: Jorg Kleeff, Arthur D. Lander, Asli Kumbasar, Helmut Friess, Stefan Wildi, Murray KorcAbstract:Glypican-1 belongs to a family of glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycans (HSPGs) that affect cell growth, invasion, and adhesion. Cell-surface HSPGs are believed to act as co-receptors for heparin-binding mitogenic growth factors. It was reported that Glypican-1 is strongly expressed in human pancreatic cancer, and that it may play an essential role in regulating growth-factor responsiveness in pancreatic carcinoma cells. In this study we investigated the effects of decreased Glypican-1 expression in PANC-1 pancreatic cancer cells. To this end, PANC-1 cells were stable transfected with a full-length Glypican-1 antisense construct. The Glypican- antisense transfected clones displayed markedly reduced Glypican- protein levels and a marked attenuation of the mitogenic responses to heparin-binding growth factors that are commonly overexpressed in pancreatic cancer: fibroblast growth factor-2 (FGF2), heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), and hepatocyte growth factor (HGF). In addition, Glypican-1 antisense-expressing PANC-1 cells exhibited a significantly reduced ability to form tumors in nude mice in comparison with parental and sham-transfected PANC-1 cells. These data suggest that Glypican-1 plays an important role in the responses of pancreatic cancer cells to heparin-binding growth factors, and documents for the first time that its expression may enhance tumorigenic potential in vivo.
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heparan sulfate proteoglycans as adhesive and anti invasive molecules syndecans and Glypican have distinct functions
Journal of Biological Chemistry, 1998Co-Authors: Wei Liu, Arthur D. Lander, E D Litwack, Michelle J Stanley, J K Langford, Ralph D SandersonAbstract:ARH-77 cells do not adhere to type I collagen and readily invade into collagen gels, but following expression of the transmembrane heparan sulfate proteoglycan syndecan-1, they bind collagen and fail to invade. We now show that cells transfected with syndecan-2 or syndecan-4 also bind collagen and are non-invasive. In contrast, cells transfected with the glycosylphosphatidylinositol-anchored proteoglycan Glypican-1 do not bind to collagen and remain invasive, even though Glypican- and syndecan-expressing cells have similar surface levels of heparan sulfate, and their proteoglycans have similar affinities for collagen. Analysis of cells expressing syndecan-1-Glypican-1 chimeric proteoglycans reveals that inhibition of invasion requires the extracellular domain of syndecan but not its transmembrane or cytoplasmic domain. Surprisingly, cells bearing a chimera composed of the Glypican extracellular domain fused to the syndecan transmembrane and cytoplasmic domains bind to collagen but remain invasive, implying that adhesion to collagen is not by itself sufficient to inhibit invasion. Apparently, the extracellular domain of syndecan-1, presumably by interacting with cell-surface signal transducing molecules, directly regulates complex cell behaviors such as motility and invasiveness. These results also show for the first time that syndecans and Glypicans can have distinct functions, even when expressed by the same cell type.
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Expression of the heparan sulfate proteoglycan Glypican‐1 in the developing rodent
Developmental dynamics : an official publication of the American Association of Anatomists, 1998Co-Authors: Ernest D. Litwack, Stephenie Paine-saunders, Jonathan K. Ivins, Asli Kumbasar, Christopher S. Stipp, Arthur D. LanderAbstract:The Glypicans are a family of glycosylphosphatidylinositol (GPI)-anchored proteoglycans that, by virtue of their cell-surface localization and possession of heparan sulfate chains, may regulate the responses of cells to numerous heparin-binding growth factors, cell adhesion molecules, and extracellular matrix components. Mutations in one Glypican cause a syndrome of human birth defects, suggesting important roles for these proteoglycans in development. Glypican-1, the first-discovered member of this family, was originally found in cultured fibroblasts, and later shown to be a major proteoglycan of the mature and developing brain. Here we examine the pattern of Glypican-1 mRNA and protein expression more widely in the developing rodent, concentrating on late embryonic and early postnatal stages. High levels of Glypican-1 expression were found throughout the brain and skeletal system. In the brain, Glypican-1 mRNA was widely, and sometimes only transiently, expressed by zones of neurons and neuroepithelia. Glypican-1 protein localized strongly to axons and, in the adult, to synaptic terminal fields as well. In the developing skeletal system, Glypican-1 was found in the periosteum and bony trabeculae in a pattern consistent with expression by osteoblasts, as well as in the bone marrow. Glypican-1 was also observed in skeletal and smooth muscle, epidermis, and in the developing tubules and glomeruli of the kidney. Little or no expression was observed in the developing heart, lung, liver, dermis, or vascular endothelium at the stages examined. The tissue-, cell type-, and in some cases stage-specific expression of Glypican-1 revealed in this study are likely to provide insight into the functions of this proteoglycan in development.
Douglas C Mcfarland - One of the best experts on this subject based on the ideXlab platform.
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Modulation of turkey myogenic satellite cell differentiation through the shedding of Glypican-1.
Comparative biochemistry and physiology. Part A Molecular & integrative physiology, 2012Co-Authors: Sandra G. Velleman, Yan Song, Jonghyun Shin, Douglas C McfarlandAbstract:Glypican-1 is a cell membrane heparan sulfate proteoglycan. It is composed of a core protein with covalently attached glycosaminoglycan, and N-linked glycosylated (N-glycosylated) chains, and is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) linkage. Glypican-1 plays a key role in the growth and development of muscle by regulating fibroblast growth factor 2 (FGF2). The GPI anchor of Glypican-1 can be cleaved, resulting in Glypican-1 being secreted or shed into the extracellular matrix environment. The objective of the current study was to investigate the role of Glypican-1 shedding and the glycosaminoglycan and N-glycosylated chains in regulating the differentiation of turkey myogenic satellite cells. A Glypican-1 construct without the GPI anchor was cloned into the mammalian expression vector pCMS-EGFP, and Glypican-1 without the GPI anchor and glycosaminoglycan and N-glycosylated chains were also cloned. These constructs were co-transfected into turkey myogenic satellite cells with a small interference RNA targeting the GPI anchor of endogenous Glypican-1. The soluble Glypican-1 mutants were not detected in the satellite cells but in the cell medium, suggesting the secretion of the soluble Glypican-1 mutants. Soluble Glypican-1 increased satellite cell differentiation and enhanced myotube formation in the presence of exogenous FGF2. The increase in differentiation was supported by the elevated expression of myogenin. In conclusion, the shedding of Glypican-1 from the satellite cell surface acts as a positive regulator of satellite cell differentiation and sequesters FGF2, permitting further differentiation.
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the effect of fibroblast growth factor 2 on the in vitro expression of syndecan 4 and Glypican 1 in turkey satellite cells
Poultry Science, 2008Co-Authors: S G Velleman, X Li, Douglas C McfarlandAbstract:The membrane-associated heparan sul- fate proteoglycan families, consisting of the syndecans and Glypicans, are low-affinity receptors for fibroblast growth factor 2 (FGF2) that are essential in regulat- ing the cellular response to FGF2. Fibroblast growth factor 2 is a potent stimulator of skeletal muscle cell proliferation and a strong inhibitor of differentiation. The regulation of the expression of the syndecans and Glypicans will likely play a role in modulating the ef- fects of FGF2 on cellular growth properties. In the present study, the effect of FGF2 on the expression of syndecan-4 and Glypican-1 was measured by real-time PCR during turkey myogenic satellite cell prolifera- tion and differentiation in vitro. Both syndecan-4 and Glypican-1 transcription were influenced by the addi- tion of exogenous FGF2. Syndecan-4 mRNA expression was reduced only during proliferation, whereas glypi- can-1 expression was reduced during both proliferation and differentiation. These results suggest that FGF2 growth factor signaling is, in part, regulated by an autoregulatory loop involving FGF2 regulation of syn- decan-4 and Glypican-1 expression and will affect the growth of skeletal muscle by modulating the prolifera- tion and differentiation of satellite cells.
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effect of syndecan 1 syndecan 4 and Glypican 1 on turkey muscle satellite cell proliferation differentiation and responsiveness to fibroblast growth factor 2
Poultry Science, 2007Co-Authors: S G Velleman, Douglas C McfarlandAbstract:ABSTRACT The membrane-associated heparan sulfate proteoglycan families consisting of the syndecans and Glypicans are low-affinity receptors for fibroblast growth factor 2 (FGF2). Fibroblast growth factor 2 is a potent stimulator of skeletal muscle cell proliferation and a strong inhibitor of differentiation. Because syndecan-1, syndecan-4, and Glypican-1 potentially play unique, but pivotal, roles in muscle cell proliferation and differentiation, these proteoglycans were examined for their effect on muscle cell proliferation and differentiation and FGF2 responsiveness. In the present study, turkey Randombred Control 2 line myogenic satellite cells were transfected with expression vector constructions of syndecan-1, syndecan-4, or Glypican-1 to assay their role during muscle development and the effect on FGF2 responsiveness. During proliferation, only syndecan-1 increased proliferation. Both syndecan-4 and Glypican-1 decreased proliferation at 72 h but generally did not affect the proliferation process. There was no interaction between the transfected gene and cell proliferation response to FGF2. Glypican-1 increased differentiation early in the process (24 h), and at later times differentiation was decreased by Glypican-1. Both syndecan-1 and syndecan-4 overexpression decreased differentiation. During differentiation, except for Glypican-1 at 48 h of differentiation, there was no interaction between gene treatment and FGF2 responsiveness. This result indicates that FGF2 responsiveness was not affected by the overexpression of syndecan-1, syndecan-4, and Glypican-1 during differentiation. These data demonstrate that syndecan-1, syndecan-4, or Glypican-1 differentially affect the processes of turkey muscle cell proliferation and differentiation, and can regulate these developmental stages in an FGF2-independent manner.
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Developmental regulated expression of syndecan-1 and Glypican in pectoralis major muscle in turkeys with different growth rates.
Development growth & differentiation, 2004Co-Authors: Xiaosong Liu, Douglas C Mcfarland, Karl E. Nestor, Sandra G. VellemanAbstract:Heparan sulfate proteoglycans, syndecan-1 and Glypican, are low-affinity receptors for fibroblast growth factor 2 (FGF2). Since FGF2 stimulates skeletal muscle cell proliferation but inhibits differentiation, differences in syndecan-1 and Glypican expression might affect muscle development and growth by changing the intensity of FGF2 signaling. In the present study, the pectoralis major muscle from 14 to 24-day-old-embryos, and from 1 to 16-week-old birds from a turkey line (F) selected for increased 16-week bodyweight and its genetic control line (RBC2), were used to address how syndecan-1 and Glypican are expressed during skeletal muscle formation. The expression of syndecan-1 and Glypican was measured by semiquantitative reverse transcription polymerase chain reaction. For males, the F-line embryos expressed more syndecan-1 (days 14 and 16) and Glypican (days 14 and 18) than the RBC2 line. Similar line differences for males were observed during posthatch development. The male embryos from both lines expressed more syndecan-1 at days 18 through 22 and more Glypican at days 20 and 22 than the corresponding females. The temporal and spatial distribution of syndecan-1 and Glypican was detected by in situ hybridization. Syndecan-1 was identified in all muscle fibers at all embryonic stages studied, whereas the Glypican was detected from embryonic day 18. The data from the current study provided new information about the expression of syndecan-1 and Glypican as it relates to skeletal muscle growth properties.
Jim Middleton - One of the best experts on this subject based on the ideXlab platform.
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Induction of a CXCL8 binding site on endothelial syndecan‐3 in rheumatoid synovium
Arthritis & Rheumatism, 2005Co-Authors: Angela M. Patterson, Guido David, Lucy Gardner, Jennifer Shaw, Emilie Loreau, Luc Aguilar, Brian A. Ashton, Jim MiddletonAbstract:Objective To identify and characterize which endothelial heparan sulfate proteoglycans (HSPGs) bind the chemokine CXCL8 (interleukin-8) in human rheumatoid arthritis (RA) and nonrheumatoid synovia. Method CXCL8 binding to endothelial HSPGs in RA and nonrheumatoid synovia was determined by heparinase treatment followed by an in situ binding assay and autoradiography. Endothelial HSPGs were characterized by immunohistochemical analysis and quantitative reverse transcriptase–polymerase chain reaction (RT-PCR). Phosphatidyinositol-specific phospholipase C (PI-PLC) and antibodies to HSPGs were used in in situ binding experiments to identify which HSPGs bound CXCL8. Results The expression of heparan sulfate on microvascular endothelial cells was demonstrated in RA and nonrheumatoid synovia. Using antibodies to syndecan-1–4 and Glypican-1, -3, and -4, the selective expression of syndecan-3 by endothelial cells was detected in RA and nonrheumatoid synovia. In addition, RT-PCR showed the presence of syndecan-3 messenger RNA in endothelial cells extracted from RA and nonrheumatoid synovia. 125I-CXCL8 bound to venular endothelial cells; treatment with heparinases I and III significantly reduced this binding in RA but not nonrheumatoid synovia. 125I-CXCL8 binding was not reduced after treatment with PI-PLC, which cleaves glycosyl phosphatidylinositol linkages, suggesting that CXCL8 did not bind to Glypicans. Treatment of synovia with a syndecan-3 antibody reduced CXCL8 binding to RA but not nonrheumatoid endothelial cells; however, no reduction in binding was observed with syndecan-2 or Glypican-4 antibodies. Conclusion Our results show the selective induction of a CXCL8 binding site on endothelial syndecan-3 in RA synovium. This site may be involved in leukocyte trafficking into RA synovial tissue.
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induction of a cxcl8 binding site on endothelial syndecan 3 in rheumatoid synovium
Arthritis & Rheumatism, 2005Co-Authors: Angela M. Patterson, Guido David, Lucy Gardner, Jennifer Shaw, Emilie Loreau, Luc Aguilar, Brian A. Ashton, Jim MiddletonAbstract:Objective To identify and characterize which endothelial heparan sulfate proteoglycans (HSPGs) bind the chemokine CXCL8 (interleukin-8) in human rheumatoid arthritis (RA) and nonrheumatoid synovia. Method CXCL8 binding to endothelial HSPGs in RA and nonrheumatoid synovia was determined by heparinase treatment followed by an in situ binding assay and autoradiography. Endothelial HSPGs were characterized by immunohistochemical analysis and quantitative reverse transcriptase–polymerase chain reaction (RT-PCR). Phosphatidyinositol-specific phospholipase C (PI-PLC) and antibodies to HSPGs were used in in situ binding experiments to identify which HSPGs bound CXCL8. Results The expression of heparan sulfate on microvascular endothelial cells was demonstrated in RA and nonrheumatoid synovia. Using antibodies to syndecan-1–4 and Glypican-1, -3, and -4, the selective expression of syndecan-3 by endothelial cells was detected in RA and nonrheumatoid synovia. In addition, RT-PCR showed the presence of syndecan-3 messenger RNA in endothelial cells extracted from RA and nonrheumatoid synovia. 125I-CXCL8 bound to venular endothelial cells; treatment with heparinases I and III significantly reduced this binding in RA but not nonrheumatoid synovia. 125I-CXCL8 binding was not reduced after treatment with PI-PLC, which cleaves glycosyl phosphatidylinositol linkages, suggesting that CXCL8 did not bind to Glypicans. Treatment of synovia with a syndecan-3 antibody reduced CXCL8 binding to RA but not nonrheumatoid endothelial cells; however, no reduction in binding was observed with syndecan-2 or Glypican-4 antibodies. Conclusion Our results show the selective induction of a CXCL8 binding site on endothelial syndecan-3 in RA synovium. This site may be involved in leukocyte trafficking into RA synovial tissue.