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Abstract: Well-differentiated hepatocellular carcinoma (HCC) may be difficult to distinguish from a benign lesion. Glypican 3 (GPC-3) is an oncofetal protein, which has been demonstrated to be up-regulated in HCC. The aim of this study is to evaluate the diagnostic role of combined GPC-3 and CD34 immunoassaying in the distinction between HCC and benign hepatic mimickers. This study was performed on 100 cases of formalin-fixed, paraffin-embedded cases of hepatic focal lesions obtained from the files of pathology laboratory of our university from 2009 to 2012. The following groups were studied: group A (n = 60) (hepatocellular malignant lesions) and group B (n = 40) (Hepatocellular nonmalignant lesions). All cases were stained with GPC-3 and CD34. Sensitivity, specificity, and positive and negative predictive values were calculated for both antibodies. Glypican 3 and complete CD34 staining pattern expression in group A was significantly higher than in group B. The results of costaining showed that, in HCCs, almost all the GPC-3–positive cases had a complete CD34 staining pattern, whereas in the 40 hepatocellular nonmalignant lesions, none stained up with the 2 markers. Therefore, although the sensitivity declined (82%), the specificity and positive predictive value (PPV) of costaining reached 100% and were greater than that observed for single staining with GPC-3 (specificity, 92.5%; PPV, 94.3%) or CD34 (specificity, 97.5%; PPV, 98.3%). Our data demonstrate that GPC-3 and CD34 costaining has better diagnostic value for differentiating HCC from nonmalignant hepatocellular lesions than does single staining. [Copyright &y& Elsevier]

Objective To investigate the effects of silencing glypican - 3 ( GPC - 3 ) gene transcription by shRNA on the proliferation and apoptosis of hepatoma cells. Methods GPC - 3 - shRNA was inserted into pGPU6/GFP/Neo vector, ad HepG2 cells were transfected with the vector. The mRNA and protein expression of GPC - 3 was measured by fluorescence quantitative PCR ad Western blot ; the proliferation of HepG2 cefls was evaluated by MTT assay ; the cefl cycle ad apoptosis of HepG2 cells were analyzed by flow cytometry, Annexin - V - P E / 7 - AAD staining, ad apoptotic DNA ladder kit- Results After the HepG2 cefls were transfected with GPC - 3 - shRNA, the GPC - 3 mRNA silencing rate was up to 89- 3%, in accordance with the down - regulation of GPC - 3 protein ; the proliferation inhibition rate of HepG2 cefls was as high as 71 - 1 % ; the HepG2 cefls were arrested in G1 phase, and the apoptosii rate of HepG2 cefls reached 65. 6% - Conclusion Silencing GPC - 3 gene transcription by shRNA can significatly inhibit the proliferation ad promote the apoptosis of hepatoma ceHs- [ABSTRACT FROM AUTHOR]

Regulated intercellular signaling is critical for the normal development and maintenance of multicellular organisms. Glypicans have been shown to regulate signaling by TGF-βs, hedgehogs and Wnts in several cellular contexts. Glypicans comprise a conserved family of heparan sulfated, glycosylphosphatidylinositol (GPI)-linked extracellular proteins. The structural complexity of glypicans may underlie their functional complexity. In a recent study, 31 we built on previous findings that one of the two C. elegans glypicans, LON-2, specifically inhibits signaling by the TGF-β superfamily member DBL-1. We tested the functional requirements of LON-2 protein core components and post-translational modifications for LON-2 activity. We provide the first evidence that two parts of a glypican can independently regulate TGF-β superfamily signaling in vivo: the N-terminal furin protease product and a C-terminal region containing heparan sulfate attachment sites. Furthermore, we show a protein-protein interaction motif is crucial for LON-2 activity in the N-terminal protein core, suggesting that LON-2 acts by serving as a scaffold for DBL-1 and an RGDbinding protein. In addition, we demonstrate specificity of glypican function by showing C. elegans GPN-1 does not functionally substitute for LON-2. This work reveals a molecular foundation for understanding the complexity and specificity of glypican function. [ABSTRACT FROM AUTHOR]

Abstract: From birth, the vault of the skull grows at a prodigious rate, driven by the activity of osteoblastic cells at the fibrous joints (sutures) that separate the bony calvarial plates. One in 2500 children is born with a medical condition known as craniosynostosis because of premature bony fusion of the calvarial plates and a cessation of bone growth at the sutures. Bone morphogenetic proteins (BMPs) are potent growth factors that promote bone formation. Previously, we found that Glypican-1 (GPC1) and Glypican-3 (GPC3) are expressed in cranial sutures and are decreased during premature suture fusion in children. Although glypicans are known to regulate BMP signalling, a mechanistic link between GPC1, GPC3 and BMPs and osteogenesis has not yet been investigated. We now report that human primary suture mesenchymal cells coexpress GPC1 and GPC3 on the cell surface and release them into the media. We show that they inhibit BMP2, BMP4 and BMP7 activities, which both physically interact with BMP2 and that immunoblockade of endogenous GPC1 and GPC3 potentiates BMP2 activity. In contrast, increased levels of GPC1 and GPC3 as a result of overexpression or the addition of recombinant protein, inhibit BMP2 signalling and BMP2-mediated osteogenesis. We demonstrate that BMP signalling in suture mesenchymal cells is mediated by both SMAD-dependent and SMAD-independent pathways and that GPC1 and GPC3 inhibit both pathways. GPC3 inhibition of BMP2 activity is independent of attachment of the glypican on the cell surface and post-translational glycanation, and thus appears to be mediated by the core glypican protein. The discovery that GPC1 and GPC3 regulate BMP2-mediated osteogenesis, and that inhibition of endogenous GPC1 and GPC3 potentiates BMP2 responsiveness of human suture mesenchymal cells, indicates how downregulation of glypican expression could lead to the bony suture fusion that characterizes craniosynostosis. [Copyright &y& Elsevier]

Bone formation is remarkable for the convergence in the activity of four major signalling pathways, the bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (HH) and wingless-integrated (WNT) pathways. These pathways cooperate in morphogenetic, proliferative and differentiative processes that underpin the development, growth and repair of skeletal structures. They are regulated by pathway-specific modulators and by another class of molecules, the glypicans. Glypicans are proteoglycans located on the cell surface, where they act as coreceptors to promote or inhibit signalling by ligands of the BMP, FGF, HH and WNT pathways, through protein-protein and protein-carbohydrate interactions. In this review, we discuss glypican structure, expression and function in the context of bone development and growth, with emphasis on the long bone growth plate where five of the six glypicans are expressed in overlapping patterns in the chondrogenic zone. Analyses of gene knockout models and the human conditions of Simpson-Golabi-Behmel syndrome and omodysplasia, which arise from mutations in glypican 3 (GPC3) and GPC6, respectively, highlight both subtle and striking effects of glypicans on bone growth. We draw attention to challenges and areas of opportunity, where the actions of glypicans on BMP, FGF, HH and WNT signalling might be profitably studied to help illuminate the complex interplay of signalling that drives bone growth. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Hedgehog (Hh) family proteins are secreted signaling ligands whose short- and long-range activities transform cellular fates in multiple contexts in organisms ranging from metazoans to humans. In the developing Drosophila wing, extracellular Hh binds to cell-bound glypican heparan sulfate proteoglycans (HSPGs) and the secreted protein Shifted (Shf), a member of Wnt inhibitory factor 1 (WIF1) family. The glypicans and Shf are required for long-range Hh movement and signaling; it has been proposed that Shf promotes long-range Hh signaling by reinforcing binding between Hh and the glypicans, and that much or all of glypican function in Hh signaling requires Shf. However, we will show here that Shf maintains short-range Hh signaling in the wing via a mechanism that does not require the presence of or binding to the Drosophila glypicans Dally and Dally-like protein. Conversely, we demonstrate interactions between Hh and the glypicans that are maintained, and even strengthened, in the absence of Shf. We present evidence that Shf binds to the CDO/BOC family Hh co-receptors Interference hedgehog (Ihog) and Brother of Ihog, suggesting that Shf regulates short-range Hh signaling through interactions with the receptor complex. In support of a functional interaction between Ihog and members of the Shf/WIF1 family, we show that Ihog can increase the Wnt-inhibitory activity of vertebrate WIF1; this result raises the possibility of interactions between WIF1 and vertebrate CDO/BOC family members. [ABSTRACT FROM AUTHOR]

Abstract: Glypicans are multifunctional proteoglycans with regulatory roles in several intercellular signaling pathways. Here, we examine the functional requirements for glypican regulation of bone morphogenetic protein (BMP)-mediated body length in C. elegans. We provide evidence that two parts of C. elegans glypican LON-2 can independently inhibit BMP signaling in vivo: the N-terminal furin protease product and the C-terminal region containing heparan sulfate attachment sequences. While the C-terminal protease product is dispensable for LON-2 minimal core protein activity, it does affect the localization of LON-2. Cleavage of LON-2 into two parts at the conserved furin protease site is not required for LON-2 to inhibit BMP-like signaling. The glycosyl-phosphatidylinositol (GPI) membrane anchor is also not absolutely required for LON-2 activity. Finally, we show that an RGD protein–protein interaction motif in the LON-2 N-terminal domain is necessary for LON-2 core protein activity, suggesting that LON-2 inhibits BMP signaling by acting as a scaffold for BMP and an RGD-binding protein. [Copyright &y& Elsevier]

The membrane lipid-anchored glypicans (Gpcs) [heparan sulfate (HS) proteoglycans (PGs)] are present in both vertebrates and invertebrates and serve as important modulators of growth factors and morphogens during development. Their core proteins are similar and consist of a large N-terminal domain comprising 14 evolutionary conserved cysteines and a C-terminal stalk carrying the HS side chains and the lipid anchor. Cysteines in Gpc-1 can be S-nitrosylated but their positions have not been identified. The recently determined crystal structure of the N-terminal domain of Gpc-1 has revealed that all the evolutionary conserved cysteines form intramolecular disulfide bonds. However, Gpc-1 contains two more, non-conserved cysteines in the C-terminal stalk, located near the HS attachment sites. We show here that the non-conserved cysteines are free thiols as a Gpc-1 core protein containing the C-terminal stalk could be biotinylated by 1-biotinamido-4-(4′-[maleimidomethyl-cyclohexane]-carboxyamido)butane. After S-nitrosylation by using a nitric oxide (NO) donor and copper ions, the Gpc-1 core protein was retained on an affinity matrix substituted with HS oligosaccharides containing N-unsubstituted glucosamines (GlcNH2/NH3+). The protein was displaced with 0.2 M glucosamine but also by 2 mM ascorbate. In the latter case, the HS of the affinity matrix was simultaneously cleaved into fragments containing anhydromannose (anMan). We propose that the S-nitrosocysteine residues interact with closely located GlcNH2/NH3+ in the HS side chains of the Gpc-1 PG. Addition of ascorbate induces a series of reactions that eventually releases HS fragments with reducing terminal anMan, presumably without the formation of free NO. [ABSTRACT FROM AUTHOR]

Proteoglycans have been studied to a limited extent in lymphoid cells. In this study we have investigated the expression of proteoglycans in B-cells, CD4+ T-cells, CD8+ T-cells, natural killer cells, as well as in nine different cell lines established from patients with lymphoid malignancies. Serglycin was the major proteoglycan expressed at mRNA level by the primary lymphocytes. None of the syndecans or glycpicans was detected at mRNA level in the primary lymphocytes, except for syndecan-4 in CD4+ T-cells and CD8+ T-cells. All lymphoid cell lines expressed serglycin mRNA, as well as one or several members of the syndecan and glypican families. Further, increased synthesis of proteoglycans was found in the cell lines compared to the primary lymphocytes, as well as the presence of heparan sulfate on the cell surface of five of the cells lines. Western blot analysis showed a close correlation between serglycin mRNA level and expression of serglycin core protein. Our results show that serglycin is a major proteoglycan in all the normal lymphoid cells and that these cells carry little, or none, proteoglycans on the cell surface. Serglycin was also a major proteoglycan in the malignant lymphoid cells, but these also expressed one or more types of cell surface proteoglycans. Thus, malignant transformation of lymphoid cells may be followed by increased synthesis of proteoglycans and expression of cell surface proteoglycans. [ABSTRACT FROM AUTHOR]

Novel sulphation motifs within the glycosaminoglycan chain structure of chondroitin sulphate (CS) containing proteoglycans (PGs) are associated with sites of growth, differentiation and repair in many biological systems and there is compelling evidence that they function as molecular recognition sites that are involved in the binding, sequestration or presentation of soluble signalling molecules (e.g. morphogens, growth factors and cytokines). Here, using monoclonal antibodies 3B3(−), 4C3 and 7D4, we examine the distribution of native CS sulphation motifs within the developing connective tissues of the human foetal knee joint, both during and after joint cavitation. We show that the CS motifs have broad, overlapping distributions within the differentiating connective tissues before the joint has fully cavitated; however, after cavitation, they all localise very specifically to the presumptive articular cartilage tissue. Comparisons with the labelling patterns of heparan sulphate (HS), HS-PGs (perlecan, syndecan-4 and glypican-6) and FGF-2, molecules with known signalling roles in development, indicate that these also become localised to the future articular cartilage tissue after joint cavitation. Furthermore, they display interesting, overlapping distributions with the CS motifs, reflective of early tissue zonation. The overlapping expression patterns of these molecules at this site suggests they are involved, or co-participate, in early morphogenetic events underlying articular cartilage formation; thus having potential clinical relevance to mechanisms involved in its repair/regeneration. We propose that these CS sulphation motifs are involved in modulating the signalling gradients responsible for the cellular behaviours (proliferation, differentiation, matrix turnover) that shape the zonal tissue architecture present in mature articular cartilage. [ABSTRACT FROM AUTHOR]

Frizzled and Smoothened are homologous seven-transmembrane proteins functioning in the Wnt and Hedgehog signaling pathways, respectively. They harbor an extracellular cysteine-rich domain (FZ-CRD), a mobile evolutionary unit that has been found in a number of other metazoan proteins and Frizzled-like proteins in Dictyostelium. Domains distantly related to FZ-CRDs, in Hedgehog-interacting proteins (HHIPs), folate receptors and riboflavin-binding proteins (FRBPs), and Niemann-Pick Type C1 proteins (NPC1s), referred to as HFN-CRDs, exhibit similar structures and disulfide connectivity patterns compared with FZ-CRDs. We used computational analyses to expand the homologous set of FZ-CRDs and HFN-CRDs, providing a better understanding of their evolution and classification. First, FZ-CRD-containing proteins with various domain compositions were identified in several major eukaryotic lineages including plants and Chromalveolata, revealing a wider phylogenetic distribution of FZ-CRDs than previously recognized. Second, two new and distinct groups of highly divergent FZ-CRDs were found by sensitive similarity searches. One of them is present in the calcium channel component Mid1 in fungi and the uncharacterized FAM155 proteins in metazoans. Members of the other new FZ-CRD group occur in the metazoan-specific RECK ( reversion-inducing- cysteine-rich protein with Kazal motifs) proteins that are putative tumor suppressors acting as inhibitors of matrix metalloproteases. Finally, sequence and three-dimensional structural comparisons helped us uncover a divergent HFN-CRD in glypicans, which are important morphogen-binding heparan sulfate proteoglycans. Such a finding reinforces the evolutionary ties between the Wnt and Hedgehog signaling pathways and underscores the importance of gene duplications in creating essential signaling components in metazoan evolution. [ABSTRACT FROM AUTHOR]

Glypicans are members of the heparan sulfate proteoglycans (HSPGs) and are involved in various growth factor signaling mechanisms. Although HSPGs affect the β-catenin-dependent and -independent pathways of Wnt signaling, how they regulate distinct Wnt pathways is not clear. It has been suggested that the β-catenin-dependent pathway is initiated through receptor endocytosis in lipid raft microdomains and the independent pathway is activated through receptor endocytosis in non-lipid raft microdomains. Here, evidence is presented that glypican-4 (GPC4) is localized to both membrane microdomains and that the localization affects its ability to regulate distinct Wnt pathways. GPC4 bound to Wnt3a and Wnt5a, which activate the β-catenin-dependent and -independent pathways, respectively, and colocalized with Wnts on the cell surface. LRP6, one of Wnt3a coreceptors, was present in lipid raft microdomains, whereas Ror2, one of Wnt5a coreceptors, was localized to non-lipid raft microdomains. Expression of GPC4 enhanced the Wnt3adependent β-catenin pathway and the Wnt5a-dependent β-catenin-independent pathway, and knockdown of GPC4 suppressed both pathways. A GPC4 mutant that was localized to only non-lipid raft microdomains inhibited the β-catenin-dependent pathway but enhanced the β-catenin-independent pathway. These results suggest that GPC4 concentrates Wnt3a and Wnt5a to the vicinity of their specific receptors in different membrane microdomains, thereby regulating distinct Wnt signaling. [ABSTRACT FROM AUTHOR]

Abstract: The objective of this study was to determine the effects of fatty acids on the proliferation, differentiation, and expression of syndecan-4 and glypican-1 in avian myogenic satellite cells (SC). SC derived from the pectoralis major (PM) and biceps femoris (BF) muscles of the turkey and chicken were individually administered 8 different fatty acids in defined medium during proliferation. A parallel set of turkey SC was induced to differentiate. Highest levels of proliferation of turkey PM and BF SC occurred in cultures containing oleate. Linoleate and oleate were equipotent in supporting proliferation of chicken SC. Microscopic examination revealed that inclusion of docosahexaenoate or eicosapentaenoate was toxic towards both PM and BF SC from both species. Linolenate and arachidonate diminished levels of differentiation. Expression of glypican-1 varied between treatments to a greater extent with turkey BF than with PM SC. Expression in chicken PM and BF SC demonstrated a similar pattern in response to treatments. Turkey PM syndecan-4 expression varied between treatments, whereas expression in turkey BF SC was similar between treatments. Expression in chicken SC varied little between treatments. The results demonstrate species and muscle-specific differences in the parameters examined. It is proposed that changes in lipid raft receptor interactions may contribute to these observed differences. [Copyright &y& Elsevier]

Signaling peptides of the extracellular environment regulate cell biological processes underlying embryonic development, tissue homeostasis, and pathophysiology. The heparan sulphate proteoglycans, glypicans, have evolved as essential modulators of key regulatory proteins such as Wnt, Bmp, Fgf, and Shh. By acting on signal spreading and receptor activation, glypicans can control signal read-out and fate in targeted cells. Genetic and embryological studies have highlighted that glypicans act in a temporal and spatially regulated manner to modulate distinct cellular events. However, alterations of glypican function underlie human congenital malformations and cancer. Recent reports are starting to reveal their mechanism of action and how they can ensure tight modulation of cell signaling. [ABSTRACT FROM AUTHOR]

The conformational conversion of the cellular prion protein (PrPC) to the infectious form (PrPSc) is the critical step in the pathogenesis of prion diseases such as Creutzfeldt-Jakob disease in humans and scrapie in sheep. Cholesterol-rich lipid rafts play a key role in the conversion of PrPC to PrPSc and other cellular components have been identified as important cofactors to trigger, enhance, or accelerate prion formation. Amongst these heparan sulphate proteoglycans (HSPGs) and their glycosaminoglycan side-chains have been implicated in prion metabolism. Recently, the cell-surface HSPG glypican-1 was demonstrated to co-localise with PrPC on the cell surface and to promote its association with lipid rafts. Both PrPC and PrPSc co-immunoprecipitated with glypican-1 and the interaction was dependent on the glycosaminoglycan side chains of glypican-1. Critically, depletion of glypican-1 in scrapieinfected N2a cells reduced the formation of PrPSc, indicating that this HSPG is involved in prion formation. Further work is required to understand the molecular and cellular mechanisms underpinning the role of glypican-1 and possibly other members of the glypican family in prion metabolism, and to determine whether glypican-1 facilitates PrPSc formation in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Glypicans are conserved cell surface heparan sulfate proteoglycans expressed in a spatiotemporally regulated manner in many developing tissues including the nervous system. Here, we show that Glypican-1 (GPC1) is expressed by trigeminal placode cells as they ingress and contribute to trigeminal sensory neurons in the chick embryo. Either expression of full-length or truncated GPC1 in vivo causes defects in trigeminal gangliogenesis in a manner that requires heparan sulfate side chains. This leads to either abnormal placodal differentiation or organization, respectively, with near complete loss of the ophthalmic (OpV) trigeminal ganglion in the most severe cases after overexpression of full-length GPC1. Interestingly, modulating GPC1 alters levels of endogenous Wnt signaling activity in the forming trigeminal ganglion, as indicated by Wnt reporter expression. Accordingly, GPC1 overexpression phenocopies Wnt inhibition in causing loss of OpV placodal neurons. Furthermore, increased Wnt activity rescues the effects of GPC1 overexpression. Taken together, these results suggest that appropriate levels of GPC1 are essential for proper regulation of canonical Wnt signaling during differentiation and organization of trigeminal placodal cells into ganglia. [ABSTRACT FROM AUTHOR]

Abstract: Cerebellum controls motor coordination, balance, eye movement, and has been implicated in memory and addiction. As in other parts of the CNS, correct embryonic and postnatal development of the cerebellum is crucial for adequate performance in the adult. Cellular and molecular defects during cerebellar development can lead to severe phenotypes, such as ataxias and tumors. Knowing how the correct development occurs can shed light into the mechanisms of disease. Heparan sulfate proteoglycans are complex molecules present in every higher eukaryotic cells and changes in their level of expression as well as in their structure lead to drastic functional alterations. This work aimed to investigate changes in heparan sulfate proteoglycans expression during cerebellar development that could unveil control mechanisms. Using real time RT-PCR we evaluated the expression of syndecans, glypicans and modifying enzymes by isolated cerebellar granule cell precursors, and studied the influence of soluble glial factors on the expression of those genes. We evaluated the possible involvement of Runx transcription factors in the response of granule cell precursors to glial factors. Our data show for the first time that cerebellar granule cell precursors express members of the Runx family and that the expression of those genes can also be controlled by glial factors. Our results also show that the expression of all genes studied vary during postnatal development and treatment of precursors with glial factors indicate that the expression of heparan sulfate proteoglycan genes as well as genes encoding heparan sulfate modifying enzymes can be modulated by the microenvironment, reflecting the intricate relations between neuron and glia. [Copyright &y& Elsevier]

GPC-1 (glypican-1) is a cell surface heparan sulfate proteoglycan that acts as a co-receptor for heparin-binding growth factors and members of the TGF-β(transforming growth factor beta-1) family. The function of cell-surface proteoglycans in the reparative dentine process has been under investigation. Gpc-1 was detected with similar frequency as tgf-β1 in the cDNA library using mRNA from the odontoblast-like cell-enriched pulp of rat incisors. The aim of this study was to test our hypothesis that gpc-1 may be related to reparative dentine formation. We examined the expression of this gene during the reparative dentine process, as well as the effect of gpc-1 on odontoblast-like cell differentiation using siRNA (small interfering RNA) to down-regulate gpc-1 expression. Immunohistological examination showed that GPC-1 was expressed in pulp cells entrapped by fibrodentine and odontoblast-like cells as well as TGF-β1. The mRNAs for gpc-1, -3 and -4, except for gpc-2, were expressed during odontoblast-like cell differentiation in pulp cells. The relative levels of gpc-1 mRNA were increased prior to the differentiation stages and were decreased during the secretory and maturation stages of pulp cells. Down-regulation of gpc-1 expression resulted in a 3.9-fold increase in tgf-β1 expression in pulp cells and a 0.3- fold decrease in dspp (dentine sialophosphoprotein) expression compared with control. These results suggested that gpc-1 and tgfβ-1 expression are necessary for the onset of differentiation, but should be down-regulated before other molecules are implicated in the formation of reparative dentine. In conclusion, gpc-1 expression in odontoblast-like cells is associated with the early differentiation but not with the formation of reparative dentine. [ABSTRACT FROM AUTHOR]

Background and Aims: The role of glypican-3 (GPC3), a novel serum marker, in differentiating hepatocellular carcinoma (HCC) from non-malignant chronic liver disease and other malignant space-occupying lesions in the liver is largely unknown. The aims of this study were to evaluate its diagnostic role and clinical correlations in patients with HCC. Methods: Six groups were studied which included 40 healthy subjects, 50 patients with chronic hepatitis (CH), 50 patients with liver cirrhosis (LC), 100 patients with HCC, 50 patients with intrahepatic cholangiocarcinoma (ICC) and 50 patients with metastatic carcinoma (MCA). Serum GPC3 levels were measured by using a sandwich enzyme-linked immunosorbent assay method. Results: Fifty-three percent of HCC patients had elevated serum GPC3 levels with values ranging 35.5–7826.6 ng/mL. The serum marker was undetectable in other groups except one patient (2%) with LC and another patient (2%) with MCA. In most cases of HCC, elevated GPC3 values did not correlate with α-fetoprotein (AFP) levels. Detectable GPC3 was significantly correlated with the presence of viral hepatitis markers but was not correlated with tumor size and stage of HCC. Serum GPC3 was superior to AFP in detecting small HCC (56.3% and 31.3%, respectively). A combination of serum GPC3 and AFP yielded an improved sensitivity for detecting small HCC to 75%. Conclusion: Serum GPC3 is highly specific for detecting HCC. The combined use of serum GPC3 and AFP provides a potentially promising tool to better differentiate HCC from benign liver disorders, as well as from other liver cancers. [ABSTRACT FROM AUTHOR]

Heparan sulfate proteoglycans are a remarkably diverse family of glycosaminoglycan-bearing protein cores that include the syndecans, the glypicans, perlecan, agrin, and collagen XVIII. Members of this protein class play key roles during normal processes that occur during development, tissue morphogenesis, and wound healing. As key components of basement membranes in organs and tissues, they also participate in selective filtration of biological fluids, in establishing cellular barriers, and in modulation of angiogenesis. The ability to perform these functions is provided both by the features of the protein cores as well as by the unique properties of heparan sulfate, which is assembled as a polymer of N-acetylglucosamine and glucuronic acid and modified by specific enzymes to generate specialized biologically active structures. This article discusses the structures and functions of this amazing family of proteoglycans and provides a platform for further study of the individual members. [ABSTRACT FROM AUTHOR]

Abstract: Here we demonstrate a synergistic and tumor selective cytotoxic effect by combined treatment with naphthoxylosides, polyamine synthesis inhibitor, and polyamine based nitric oxide (NO) donor, using in vitro human tumor models. We have earlier reported that heparan sulfate priming naphthoxyloside, 2-(6-hydroxynaphthyl)-O-β-d-xylopyranoside, which inhibits growth of human tumor cells in vitro and in vivo models, undergoes NO dependent cleavage and accumulates in the nuclei of tumor cells. Polyamine depletion using α-difluoromethylornithine (DFMO) increases both the number of NO sensitive sites in heparan sulfate and uptake of the polyamine based NO donor, spermineNONOate, thereby enhancing formation of growth-inhibitory NO induced heparan sulfate products with specific cytotoxic effect on tumor cells. We also show that peracetylation of xylosides doubles the antiproliferative effect towards human cancer cells by making these compounds more permeable to the cells. [Copyright &y& Elsevier]

Abstract: The aorta-gonad-mesonephros (AGM) region is involved in the generation and maintenance of the first definitive hematopoietic stem cells (HSCs). A mouse AGM-derived cell line, AGM-S3, was shown to support the development of HSCs. To elucidate the molecular mechanisms regulating early hematopoiesis, we obtained subclones from AGM-S3, one of which was hematopoiesis supportive (S3-A9) and the other one of which was non-supportive (S3-A7), and we analyzed their gene expression profiles by gene chip analysis. In the present study, we found that Glypican-1 (GPC1) was highly expressed in the supportive subclone AGM-S3-A9. Over-expression of GPC1 in non-supportive cells led to the proliferation of progenitor cells in human cord blood when cocultured with the transfected-stromal cells. Thus, GPC1 may have an important role in the establishment of a microenvironment that supports early events in hematopoiesis. [Copyright &y& Elsevier]

Using murine colon adenocarcinoma-derived clones with different metastatic potentials, the cellular localization of matrix metalloporteinase-9 (MMP-9) and its role in the cell motility were examined. Highly metastatic LuM1 clone aggressively invaded into adjacent tissue in vivo, but low metastatic NM11 clone did not. As compared with the NM11 clone, the LuM1 clone expressed and secreted a remarkably large amount of MMP-9, and exhibited higher abilities of cell migration and invasion in vitro, which were suppressed by MMP-2/MMP-9 inhibitor IV. MMP-9, exhibiting high affinity to heparin, was demonstrated to be condensed on tips of cellular podia. Treatment of the cells with heparitinase-I or heparin resulted in release of MMP-9 from the cell surface, which caused concomitant suppression of their motility to a similar level to that with the MMP inhibitor. Immunoprecipitation of a LuM1 cell lysate with an anti-MMP-9 antibody resulted in co-precipitation of phosphatidylinositol-specific phospholipase C-susceptible heparan sulphate proteoglycans having 66 and 64 kDa core proteins. Taken together, the present results demonstrate that secreted MMP-9 associates with glypican-like proteoglycans through their heparan sulphate chains, and plays a crucial role in cell motility of LuM1 cells. [ABSTRACT FROM PUBLISHER]

The glypicans are a family of cell-surface heparan sulfate proteoglycans consisting of a core protein covalently attached with glycosaminoglycans (GAG). Only glypican-1 is expressed in skeletal muscle and in- creases in expression during myoblast differentiation. Previous studies have suggested that glypican-1 influences fibroblast growth factor 2 (FGF2) signaling pathway by its heparan sulfate chains. Fibroblast growth factor 2 is a potent stimulator of muscle cell proliferation and an intense inhibitor of differentiation. To investigate the functional contribution of each GAG chain attachment site, a turkey glypican-1 full length cDNA (1,650 bp, Gen-Bank accession number AY551002) was cloned into the pCMS-EGFP vector and mutated at 2 or all 3 potential GAG attachment sites at Ser433, Ser485, and Ser487 to obtain 1-chain and no-chain mutants, respectively. The unmu- tated glypican-1, 1-chain, and no-chain mutants, and the pCMS-EGFP vector without an insert were transfected into turkey myogenic satellite cells. The transfected cell cultures were assayed for cell proliferation, differentiation, and FGF2 responsiveness. The overexpression of glypican-1 increased FGF2 responsiveness during proliferation compared with the 1-chain, no-chain mutants, and the pCMS-EGFP vector without an insert, but there was no significant interaction between FGF2 and glypican-i. The overexpression of glypican-1 also increased differentiation but did not affect proliferation when compared with the 1-chain, no-chain mutants, and the pCMS-EGFP vector without an insert. To support the overexpression data, glypican-1 expression was reduced using a small interfering RNA against turkey glypican-1. Inhibition of glypican-1 expression decreased myogenic satellite cell proliferation, differentiation, and FGF2 responsiveness during proliferation. These data indicate that glypican- 1 function requires the GAG chain attachment sites for myogenic satellite cell FGF2 responsiveness during proliferation and to affect the process of differentiation. [ABSTRACT FROM AUTHOR]

Abstract: Division abnormally delayed (Dally) is one of two glycosylphosphatidylinositol (GPI)-linked heparan sulfate proteoglycans in Drosophila. Numerous studies have shown that it influences Decapentaplegic (Dpp) and Wingless signaling. It has been generally assumed that Dally affects signaling by directly interacting with these growth factors, primarily through its heparan sulfate (HS) chains. To understand the functional contributions of HS chains and protein core we have (1) assessed the growth factor binding properties of purified Dally using surface plasmon resonance, (2) generated a form of Dally that is not HS modified and evaluated its signaling capacity in vivo. Purified Dally binds directly to FGF2, FGF10, and the functional Dpp homolog BMP4. FGF binding is abolished by preincubation with HS, but BMP4 association is partially HS-resistant, suggesting the Dally protein core contributes to binding. Cell binding and co-immunoprecipitation studies suggest that non-HS-modified Dally retains some ability to bind Dpp or BMP4. Expression of HS-deficient Dally in vivo showed it does not promote signaling as well as wild-type Dally, yet it can rescue several dally mutant phenotypes. These data reveal that heparan sulfate modification of Dally is not required for all in vivo activities and that significant functional capacity resides in the protein core. [Copyright &y& Elsevier]

Abstract: Glypicans are cell surface molecules that influence signaling and gradient formation of secreted morphogens and growth factors. Several distinct functions have been ascribed to glypicans including acting as co-receptors for signaling proteins. Recent data show that glypicans are also necessary for morphogen propagation in the tissue. In the present study, a model describing the interaction of a morphogen with glypicans is formulated, analyzed and compared with measurements of the effect of glypican Dally-like (Dlp) overexpression on Wingless (Wg) morphogen signaling in Drosophila melanogaster wing imaginal discs. The model explains the opposing effect that Dlp overexpression has on Wg signaling in the distal and proximal regions of the disc and makes a number of quantitative predictions for further experiments. In particular, our model suggests that Dlp acts by allowing Wg to diffuse on cell surface while protecting it from loss and degradation, and that Dlp rather than acting as Wg co-receptor competes with receptors for morphogen binding. [Copyright &y& Elsevier]

Heparan sulfate chains have been found to be associated with amyloid deposits in a number of diseases including transmissible spongiform encephalopathies. Diverse lines of evidence have linked proteoglycans and their glycosaminoglycan chains, and especially heparan sulfate, to the metabolism of the prion protein isoforms. Glypicans are a family of glycosylphosphatidylinositol-anchored, heparan sulfate-containing, cell-associated proteoglycans. Cysteines in glypican-1 can become nitrosylated by endogenously produced nitric oxide. When glypican-1 is exposed to a reducing agent, such as ascorbate, nitric oxide is released and autocatalyses deaminative cleavage of heparan sulfate chains. These processes take place while glypican-1 recycles via a non-classical, caveolin-associated pathway. We have previously demonstrated that prion protein provides the Cu2+ ions required to nitrosylate thiol groups in the core protein of glypican-1. By using confocal immunofluorescence microscopy and immunomagnetic techniques, we now show that copper induces co-internalization of prion protein and glypican-1 from the cell surface to perinuclear compartments. We find that prion protein is controlling both the internalization of glypican-1 and its nitric oxide-dependent autoprocessing. Silencing glypican-1 expression has no effect on copper-stimulated prion protein endocytosis, but in cells expressing a prion protein construct lacking the copper binding domain internalization of glypican-1 is much reduced and autoprocessing is abrogated. We also demonstrate that heparan sulfate chains of glypican-1 are poorly degraded in prion null fibroblasts. The addition of either Cu2+ ions, nitric oxide donors, ascorbate or ectopic expression of prion protein restores heparan sulfate degradation. These results indicate that the interaction between glypican-1 and Cu2+-loaded prion protein is required both for co-internalization and glypican-1 self-pruning. [ABSTRACT FROM AUTHOR]

Abstract: The anosmin-1 protein family regulates cell migration, axon guidance, and branching, by mechanisms that are not well understood. We show that the C. elegans anosmin-1 ortholog KAL-1 promotes migrations of ventral neuroblasts prior to epidermal enclosure. KAL-1 does not modulate FGF signaling in neuroblast migration and acts in parallel to other neuroblast migration pathways. Defects in heparan sulfate (HS) synthesis or in specific HS modifications disrupt neuroblast migrations and affect the KAL-1 pathway. KAL-1 binds the cell surface HS proteoglycans syndecan/SDN-1 and glypican/GPN-1. This interaction is mediated via HS side chains and requires specific HS modifications. SDN-1 and GPN-1 are expressed in ventral neuroblasts and have redundant roles in KAL-1-dependent neuroblast migrations. Our findings suggest that KAL-1 interacts with multiple HSPGs to promote cell migration. [Copyright &y& Elsevier]

The heparan sulfate proteoglycan, glypican-1, is a low affinity receptor for fibroblast growth factor 2 (FGF2). Fibroblast growth factor 2 is a potent stimulator of skeletal muscle cell proliferation and an inhibitor of differentiation. Heparan sulfate proteoglycans like glypican-1 are required for FGF2 to transduce an intracellular signal. Understanding the role of glypican-1 in the regulation of FGF2-mediated signaling is important in furthering the understanding of the biological processes involved in muscle development and growth. In the current study, a turkey glypican-1 expression vector construct was transfected into turkey myogenic satellite cells resulting in the overexpression of glypican-1. The proliferation, differentiation, and responsiveness to FGF2 were measured in control and transfected cell cultures. The overexpression of glypican-1 in turkey myogenic satellite cells increased both satellite cell proliferation and FGF2 responsiveness, but decreased the rate of differentiation. The current data support glypican-1 modulation of both proliferation and differentiation through an FGF2-mediated pathway. [ABSTRACT FROM AUTHOR]

Heparan sulfate proteoglycans (HSPG) are key components of the cell membrane and extracellular matrix of skeletal muscle cells. Two major groups of membrane-associated HSPG found in skeletal muscle are syndecans (SYN) and glypicans (GPC), both of which can regulate growth factor activities and, thus, modulate cell proliferation and differentiation. In the current study, the mRNA expression of a group of membrane-associated HSPG (SYN 2 through 4 and GPC 1) was investigated in embryonic pectoralis major muscle [embryonic days (ED) 14, 16, 18, 20, 22, 24] and myogenic satellite cells isolated from males of a turkey genetic line selected for increased 16-wk BW (F line) and an unselected randombred control (RBC2 line) from which the F line was developed. The mRNA expression was measured by a real-time quantitative PCR approach. The SYN 2 and SYN 4 expression exhibited a similar pattern during embryonic p. major muscle development, which remained constant from ED 14 to ED 22 and declined sharply from ED 22 to ED 24 to a very low level. In contrast, the SYN 3 and GPC 1 expression showed a continuous decline from ED 14 to ED 24. The F line had higher SYN 2 (ED 14, 18, 20, 22), SYN 3 (ED 22), and SYN 4 (ED 22) expression than the RBC2 line. In myogenic satellite cells, initiating differentiation resulted in a decrease in SYN 2 expression and an increase in GPC 1 expression. Both SYN 3 and SYN 4 expression stayed almost constant through both the proliferation and differentiation stages. The proliferating satellite cells from the F line displayed higher SYN 4 expression than those from the RBC2 line. Collectively, the results from the current study suggest that membrane-associated HSPG are differentially expressed in both embryonic p. major muscle tissue and satellite cells isolated from F-line and RBC2-line male turkeys, implying their distinct roles in myogenesis and differing influence on muscle growth properties. [ABSTRACT FROM AUTHOR]

Abstract: Glypicans, a family of heparan sulfate proteoglycans attached to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor, play essential roles in morphogen signaling and distributions. A Drosophila glypican, Dally, regulates the gradient formation of Decapentaplegic (Dpp) in the developing wing. To gain insights into the function of glypicans in morphogen signaling, we examined the activities of two mutant forms of Dally: a transmembrane form (TM-Dally) and a secreted form (Sec-Dally). Misexpression of tm-dally in the wing disc had a similar yet weaker effect in enhancing Dpp signaling compared to that of wild-type dally. In contrast, Sec-Dally shows a weak dominant negative activity on Dpp signal transduction. Furthermore, sec-dally expression led to patterning defects as well as a substantial overgrowth of tissues and animals through the expansion of the action range of Hh. These findings support the recently proposed model that secreted glypicans have opposing and/or distinct effects on morphogen signaling from the membrane-tethered forms. [Copyright &y& Elsevier]

The heparan sulfate proteoglycans, syndecan-1 and glypican-1 (glypican), are low affinity receptors for fibroblast growth factor 2 (FGF2). Because FGF2 stimulates skeletal muscle cell proliferation but inhibits differentiation, changes in FGF2 signaling due to early posthatch feed deprivation may play a significant role in modulating muscle growth. To study the effect of early posthatch feed deprivation in chickens on heparan sulfate proteoglycan relative protein concentration, syndecan-1 expression, and glypican mRNA expression, pectoralis major muscle tissue was isolated from pretreatment d 0 chicis and chicks fed or feed deprived for 3 d, and after d 3 feeding was resumed in the feed-deprived birds until d 7. Heparan sulfate proteoglycan protein concentration was measured by ELISA analysis and was significantly decreased in the feed-deprived birds beginning at d 2 (P <0.05). The expression of syndecan-1 and glypican was measured by semi-quantitative reverse transcription PCR. Syndecan-1 expression was unaffected by feed withdrawal and refeeding (P > 0.05). Glypican mRNA expression was decreased in the muscle tissue from feed-deprived birds at d 3 (P < 0.05), but by d 7, after initiating feeding on d 4, it was significantly elevated compared with in muscle tissue from chicks maintained on feed (P <0.05). The results from the present study demonstrate that the heparan sulfate proteoglycan protein concentration and syndecan-1 and glypican mRNA expressions are differentially affected by early posthatch feed deprivation, which may alter signaling events associated with muscle growth. [ABSTRACT FROM AUTHOR]

Proteoglycans are well-known modulators of intercellular communication and signaling. Remodeling of the proteoglycans in the human uterus occurs throughout pregnancy, and during labor. We therefore hypothesize that heparan sulfate proteoglycans (HSPGs) play an important role in establishing normal labor. In this study HSPGs were characterized and localized in human uterine tissue.Uterine biopsies were obtained from four nonpregnant women, four women during elective cesarean section and four during emergency cesarean section. The biopsies were extracted using 4 mguanidinium hydrochloride (GuHCL). HSPGs were then purified by repeated ion-exchange chromatography on dehydroepiandrosterone (DEAE)-cellulose after digestion with chondroitinase ABC and finally precipitated with Alcian blue. HSPGs were identified by agarose gel electrophoresis and Western blotting. Controlled degradation of the heparan sulfate (HS) side-chains was performed using heparitinase or deglycosylation with trifluoromethanesulfonic acid (TFMS). The resulting core proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and visualized by Coomassie staining. HSPGs were localized in uterine tissue by immunohistochemistry.SDS–PAGE after deglycosylation indicated the presence of multiple distinct core proteins tentatively identified as syndecans 1–4 and glypican 1. Western blots confirmed the presence of these proteoglycans and also perlecan. Immunohistochemistry revealed that the HSPGs were localized mainly in the smooth muscle with few in the extracellular matrix (ECM). Syndecan 3, the dominant proteoglycan, showed the most pronounced changes during pregnancy and labor.For the first time several heparan sulfate proteoglycans have been identified and localized in the human uterus and shown to vary in expression during pregnancy and labor. Syndecan 3 had the most outstanding features in this respect. [ABSTRACT FROM AUTHOR]

We have examined the respective contribution of Heparan Sulfate Proteoglycans (HSPGs) and Frizzled (Fz) proteins in the establishment of the Wingless (Wg) morphogen gradient. From the analysis of mutant clones of sulfateless/N-deacetylase-sulphotransferase in the wing imaginal disc, we find that lack of Heparan Sulfate (HS) causes a dramatic reduction of both extracellular and intracellular Wg in receiving cells. Our studies, together with others [Kirkpatrick, C.A., Dimitroff, B.D., Rawson, J.M., Selleck, S.B., 2004. Spatial regulation of Wingless morphogen distribution and signalling by Dally-like protein. Dev. Cell (in press)], reveals that the Glypican molecule Dally-like Protein (Dlp) is associated with both negative and positive roles in Wg short- and long-range signaling, respectively. In addition, analyses of the two Fz proteins indicate that the Fz and DFz2 receptors, in addition to transducing the signal, modulate the slope of the Wg gradient by regulating the amount of extracellular Wg. Taken together, our analysis illustrates how the coordinated activities of HSPGs and Fz/DFz2 shape the Wg morphogen gradient. [Copyright &y& Elsevier]

The heparan sulfate proteoglycan glypican-1 is essential as a co-receptor for heparin binding growth factors, such as HB-EGF and FGF-2, in pancreatic cancer cells. In the present study, the role of glypican-1 in the regulation of TGF-β signaling was investigated. Colo-357 pancreatic cancer cells were stably transfected with a full-length glypican-1 antisense construct. Cell growth was determined by MTT and soft agar assays. TGF-β1 induced p21 expression and Smad2 phosphorylation were analyzed by immunoblotting. PAI-1 promoter activity was determined by luciferase assays. Down-regulation of glypican-1 expression by stable transfection of a full-length glypican-1 antisense construct resulted in decreased anchorage-dependent and -independent cell growth in Colo-357 pancreatic cancer cells and attenuated TGF-β1 induced cell growth inhibition, Smad2 phosphorylation, and PAI-1 promoter activity. There was, however, no significant difference in TGF-β1 induced p21 expression and Smad2 nuclear translocation. In conclusion, glypican-1 is required for efficient TGF-β1 signaling in pancreatic cancer cells. [Copyright &y& Elsevier]