Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.2.2.7 (heparinase)
1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The myeloid 32D cell line, which grows in suspension and does not express FGF receptors or heparan sulfate proteoglycans, was transfected with the cDNA encoding FGF receptor-1 (32D-flg cells). When co-cultured with glutaraldehyde-fixed Chinese hamster ovary (CHO) cells, the 32D-flg cells remained in suspension in the absence of FGF-2 but attached to the CHO monolayer in the presence of 10 ng/ml FGF-2. In contrast, 32D cells transfected with the vector alone did not attach to the CHO monolayer in the presence of FGF-2. FGF-2-dependent attachment of 32D-flg cells was prevented by inclusion of 10 micrograms/ml heparin in the incubation medium and was diminished when CHO mutants in glycosaminoglycan synthesis or wild-type CHO cells treated with heparinase were used, indicating that the attachment occurred through FGF-2 interactions with heparan sulfates on the CHO cells. Attachment of 32D-flg cells to wild-type CHO cells was half-maximal at 0.4 ng/ml FGF-2 and was also observed with FGF-1 but not FGF-4. 32D-flg cells also attached to living CHO cells in a FGF-2-dependent manner, but attachment was transient at 37 degrees C. Induction of new proteins was not required for FGF-2-dependent attachment, since attachment occurred when the co-cultures were incubated at 4 degrees C and when the 32D-flg cells were preincubated with cycloheximide. FGF-2-dependent attachment of 32D-flg cells was also observed with Balb/C 3T3, NIH 3T3, and bovine capillary endothelial cells. We conclude that attachment is due to FGF-2 binding simultaneously to receptors on the 32D-flg cells and heparan sulfates on the CHO monolayers; thus, the FGF-2 acts as a bridge between receptor-expressing cells and heparan sulfate-bearing cells. In addition, induction of DNA synthesis in 32D-flg cells in response to FGF-2 was potentiated by the CHO-associated heparan sulfates to the same extent as by soluble heparin, indicating that this interaction has functional significance.
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PMID:Fibroblast growth factor-2 can mediate cell attachment by linking receptors and heparan sulfate proteoglycans on neighboring cells. 759 23

The MCF-7 breast cancer cells exhibit remarkable growth enhancement in response to basic fibroblast growth factor (FGF-2) stimulation in a dose dependent manner. To investigate the involvement of proteoglycans on control of FGF-2 induced proliferation, polysaccharide chains were degraded by specific enzymes. Our results showed that MCF-7 cells were unsensitive to FGF-2 after enzymatic degradation of heparin sulfate proteoglycans (HSPG) by heparinase. After metabolic inhibition of sulphation by sodium chloride, radiolabelled proteoglycans were purified and quantified by ion exchange chromatography. Sodium chlorate treatment reduced by 70% sulfation of proteoglycans. This decrease of sulphation totally inhibited FGF-2-mediated proliferation. The sulphated glycosaminoglycans which were critical in FGF-2-induced proliferation were strictly HSPG, as an addition of heparin in cell culture medium can restore FGF-2 mitogenic activity. In contrast, other glycosaminoglycans (chondroitin sulfate/hyaluronic acid) did not show any effect. These results provide clear evidence for the critical role of HSPG in FGF-2-induced proliferation on MCF-7 breast cancer cells.
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PMID:[Involvement of sulfated proteoglycans in the control of proliferation of MCF-7 breast cancer cells]. 865 6

Heparan sulfate (HS) proteoglycans play a key role in cell proliferation induced by basic fibroblast growth factor (FGF-2) and other heparin-binding growth factors. To modulate the involvement of HS, we have used a synthetic, nonsulfated polyanionic aromatic compound (RG-13577) that mimics functional features of heparin/HS. FGF-2-stimulated proliferation of vascular endothelial cells was markedly inhibited in the presence of 5-10 microg/ml compound RG-13577 (poly-4-hydroxyphenoxy acetic acid; Mr approximately 5 kD). Direct interaction between RG-13577 and FGF-2 was demonstrated by the ability of the former to compete with heparin on binding to FGF-2. RG-13577 inhibited FGF-2 binding to soluble- and cell surface-FGF receptor 1 (FGFR1). Unlike heparin, RG-13577 alone failed to mediate dimerization of FGF-2. Moreover, it abrogated heparin-mediated dimerization of FGF-2 and FGFR1, as well as FGF-2 mitogenic activity in HS-deficient F32 lymphoid cells. The antiproliferative effect of compound RG-13577 was associated with abrogation of FGF-2-induced tyrosine phosphorylation of FGFR1 and of cytoplasmic proteins involved in FGF-2 signal transduction, such as p90 and mitogen-activated protein kinase. A more effective inhibition of tyrosine phosphorylation was obtained after removal of the cell surface HS by heparinase. In contrast, tyrosine phosphorylation of an approximately 200-kD protein was stimulated by RG-13577, but not by heparin or FGF-2. RG-13577 prevented microvessel outgrowth from rat aortic rings embedded in a collagen gel. Development of nontoxic polyanionic compounds may provide an effective strategy to inhibit FGF-2-induced cell proliferation associated with angiogenesis, arteriosclerosis, and restenosis.
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PMID:Modulation of fibroblast growth factor-2 receptor binding, dimerization, signaling, and angiogenic activity by a synthetic heparin-mimicking polyanionic compound. 912

Using an ELISA approach, we demonstrate that recombinant human IL-12 (rhIL-12) binds strongly to an immobilized heparin-BSA complex. This binding is completely displaceable with soluble heparin, IC50 approximately 0.1 microg/ml, corresponding to approximately 10 nM. By interpolation with our previous findings, this indicates an affinity for heparin greater than that of antithrombin III and comparable with that of FGF-2, two high-affinity heparin-binding proteins. Recombinant murine IL-12 also binds strongly to heparin. The binding of rhIL-12 to heparin shows specificity because chondroitin sulfates A and C fail to compete, whereas chondroitin B inhibits weakly. A highly sulfated heparan sulfate is a strong competitor, whereas other heparan sulfates show weak or no activity. Small heparin fragments inhibit binding, although activity decreases with size. An octasaccharide pool derived by cleavage of heparin with nitrous acid is a significantly stronger inhibitor than its heparinase I-derived counterpart, further indicating structural specificity in the interaction between rhIL-12 and heparin. The binding of recombinant p40 to heparin appears indistinguishable from that of the IL-12 heterodimer, implying that the heparin binding site is largely if not solely located in this subunit. These results show for the first time that IL-12 is a heparin-binding cytokine, a property common to the other Th1-response-inducing cytokines, IFN-gamma and IL-2. Our findings strongly suggest that IL-12 will tend to be retained close to its sites of secretion in the tissues by binding to heparin-like glycosaminoglycans, thus favoring a paracrine role for IL-12.
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PMID:IL-12 is a heparin-binding cytokine. 991 34

A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the FGF receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation and supports the binding of activating FGF to self-associated FGFR. Here we show that in contrast to heparin, cellular heparan sulfate forms a binary complex with FGFR that discriminates between FGF-1 and FGF-2. FGFR type 4 (FGFR4) in liver parenchymal cells binds only FGF-1, whereas FGFR1 binds FGF-1 and FGF-2 equally. Cell-free complexes of heparin and recombinant FGFR4 bound FGF-1 and FGF-2 equally. However, in contrast to FGFR1, when recombinant FGFR4 was expressed back in epithelial cells by transfection, it failed to bind FGF-2 unless heparan sulfate was depressed by chlorate or heparinase treatment. Isolated heparan sulfate proteoglycan (HSPG) from liver cells in cell-free complexes with FGFR4 restored the specificity for FGF-1 and supported the binding of both FGF-1 and FGF-2 when complexed with FGFR1. In contrast, FGF-2 bound equally well to complexes of both FGFR1 and FGFR4 formed with endothelial cell-derived HSPG, but the endothelial HSPG was deficient for the binding of FGF-1 to both FGFR complexes. These data suggest that a heparan sulfate subunit is a cell type- and FGFR-specific determinant of the selectivity of the FGFR signaling complex for FGF. In a physiological context, the heparan sulfate subunit may limit the redundancy among the current 18 FGF polypeptides for the 4 known FGFR.
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PMID:Specificity for fibroblast growth factors determined by heparan sulfate in a binary complex with the receptor kinase. 1033 1

Human basement membrane heparan sulfate proteoglycan (HSPG) perlecan binds and activates fibroblast growth factor (FGF)-2 through its heparan sulfate (HS) chains. Here we show that perlecans immunopurified from three cellular sources possess different HS structures and subsequently different FGF-2 binding and activating capabilities. Perlecan isolated from human umbilical arterial endothelial cells (HUAEC) and a continuous endothelial cell line (C11 STH) bound similar amounts of FGF-2 either alone or complexed with FGFRalpha1-IIIc or FGFR3alpha-IIIc. Both perlecans stimulated the growth of BaF3 cell lines expressing FGFR1b/c; however, only HUAEC perlecan stimulated those cells expressing FGFR3c, suggesting that the source of perlecan confers FGF and FGFR binding specificity. Despite these differences in FGF-2 activation, the level of 2-O- and 6-O-sulfation was similar for both perlecans. Interestingly, perlecan isolated from a colon carcinoma cell line that was capable of binding FGF-2 was incapable of activating any BaF3 cell line unless the HS was removed from the protein core. The HS chains also exhibited greater bioactivity after digestion with heparinase III. Collectively, these data clearly demonstrate that the bioactivity of HS decorating a single PG is dependent on its cell source and that subtle changes in structure including secondary interactions have a profound effect on biological activity.
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PMID:Not all perlecans are created equal: interactions with fibroblast growth factor (FGF) 2 and FGF receptors. 1184 21

Understanding the process of wound healing will provide valuable insight for the development of new strategies to treat diseases associated with improper regeneration, such as blindness induced by corneal scarring. Heparan sulfate proteoglycans (HSPG) are not normally expressed in the corneal stroma, but their presence at sites of injury suggests their involvement in the wound healing response. Primary cultured corneal stromal fibroblasts constitutively express HSPG and represent an injured phenotype. Recently, nuclear localization of HSPG was shown to increase in corneal stromal fibroblasts plated on fibronectin (FN), an extracellular matrix protein whose appearance in the corneal stroma correlates with injury. One possible role for the nuclear localization of HSPG is to function as a shuttle for the nuclear transport of heparin-binding growth factors, such as basic fibroblast growth factor (FGF-2). Once in the nucleus, these growth factors might directly modulate cellular activities. To investigate this hypothesis, cells were treated with (125)I-labelled FGF-2 under various conditions and fractionated. Our results show that nuclear localization of FGF-2 was increased in cells plated on FN compared to those on collagen type I (CO). Interestingly, FGF-2-stimulated proliferation was increased in cells plated on FN compared to CO and this effect was absent in the presence of heparinase III. Furthermore, pre-treatment with heparinase III decreased nuclear FGF-2, and CHO cells defective in the ability to properly synthesize heparan sulfate chains showed reduced nuclear FGF-2 indicating that the heparan sulfate chains of HSPG are critical for this process. HSPG signaling, particularly through the cytoplasmic tails of syndecans, was investigated as a potential mechanism for the nuclear localization of FGF-2. Treatment with phorbol 12-myristate-13-acetate (PMA), under conditions that caused downregulation of protein kinase Calpha (PKCalpha), decreased nuclear FGF-2. Using pharmacological inhibitors of specific PKC isozymes, we elucidated a potential mode of regulation whereby PKCalpha mediates the nuclear localization of FGF-2 and PKCdelta inhibits it. Our studies suggest a novel mechanism in which FGF-2 translocates to the nucleus in response to injury.
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PMID:Nuclear localization of basic fibroblast growth factor is mediated by heparan sulfate proteoglycans through protein kinase C signaling. 1264 3

We investigated how lipid raft association of HSPG (heparan sulphate proteoglycans) modulates FGF-2 (fibroblast growth factor-2/basic fibroblast growth factor) interactions with vascular smooth-muscle cells. When lipid rafts were disrupted with sterol-binding agents, methyl-beta-cyclodextrin and filipin, FGF-2 binding to HSPG was reduced 2-5-fold, yet the amount and turnover of cell-surface HSPG were unaffected [corrected]. Approx. 50-65% of bound FGF-2 was in lipid raft-associated fractions based on insolubility in cold Triton X-100 and flotation in OptiPrep density gradients, and this level was increased with higher FGF-2 concentrations [corrected]. Less FGF-2 (50-90%) was associated in raft fractions when cholesterol was depleted or HSPG were degraded with heparinase III. To investigate how lipid raft-HSPG interactions altered binding, we compared the rates of FGF-2 dissociation with native, MbetaCD (methyl-beta-cyclodextrin)- and filipin-treated cells. We found that FGF-2 dissociation rates were increased when lipid rafts were disrupted. These results suggest that localization of HSPG within lipid rafts creates high local concentrations of binding sites such that dissociation of FGF-2 is hindered. The localization of FGF-2 and HSPG to lipid rafts also correlated with the activation of protein kinase Calpha. Thus raft association of HSPG might create growth factor traps resulting in increased binding and signal transduction to enhance cell sensitivity.
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PMID:Heparan sulphate proteoglycans modulate fibroblast growth factor-2 binding through a lipid raft-mediated mechanism. 1471 58

Smooth muscle cells (SMC) of the rat carotid arterial media proliferate and migrate in response to injury during the formation of a neointima. The interaction of fibroblast growth factor (FGF-2), which is released at the site of injury, with heparan sulfate proteoglycans (HSPGs) is necessary to induce signaling, which elicits an FGF-dependent mitogenic response by arterial smooth muscle cells, and also serves as a mechanism for storage of the growth factor within the extracellular matrix. However, whether these interactions are critical during neointimal formation has not been directly tested. In this study, a model of FGF-2-dependent medial SMC mitogenic response in balloon-injured rat carotid artery was used to test the effect of degradation of vessel wall heparan sulfate on subsequent SMC proliferation. Treatment of balloon-catheterized rat carotid arteries with chondroitin ABC lyase and/or heparin lyases eliminated heparan sulfates in the vessel wall, as determined by immunoperoxidase staining. In contrast, the distribution in the carotid vessel wall of the large core protein of perlecan, a major vessel wall HSPG that binds FGF-2, is not decreased. The effect of glycosaminoglycan digestion in situ on medial SMC proliferation in response to a bolus injection of FGF-2 after injury was determined by measuring the percentage of SMC nuclei that incorporated 5-bromo-2'-deoxyuridine (BrdU) 48 h after injury. Enzymatic removal of heparan sulfate reduced BrdU incorporation into medial SMC by 60-70% (P < 0.001) at 48 h after injury. Moreover, pre-incubation of FGF-2 with heparin prior to injection restored SMC replication to the levels present in injured vessels treated with buffer alone (P < 0.01). These experiments indicate that endogenous HSPGs are essential to promote FGF-2-driven medial SMC proliferation following injury, and that heparinase treatment can abrogate FGF-2-dependent responses in vivo.
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PMID:Removal of heparan sulfate by heparinase treatment inhibits FGF-2-dependent smooth muscle cell proliferation in injured rat carotid arteries. 1518 46

The anticoagulant serpin antithrombin acquires a potent antiangiogenic activity upon undergoing conformational alterations to cleaved or latent forms. Here we show that antithrombin antiangiogenic activity is mediated at least in part through the ability of the conformationally altered serpin to block the proangiogenic growth factors fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) from forming signaling competent ternary complexes with their protein receptors and heparan sulfate co-receptors on endothelial cells. Cleaved and latent but not native forms of antithrombin blocked the formation of FGF-2-FGF receptor-1 ectodomain-heparin ternary complexes, and the dimerization of these complexes in solution and similarly inhibited the formation of FGF-2-heparin binary complexes and their dimerization. Only antiangiogenic forms of antithrombin likewise inhibited (125)I-FGF-2 binding to its low affinity heparan sulfate co-receptor and blocked FGF receptor-1 autophosphorylation and p42/44 MAP kinase phosphorylation in cultured human umbilical vein endothelial cells (HUVECs). Moreover, treatment of HUVECs with heparinase III to specifically eliminate the FGF-2 heparan sulfate co-receptor suppressed the ability of antiangiogenic antithrombin to inhibit growth factor-stimulated proliferation. Antiangiogenic antithrombin inhibited full-length VEGF(165) stimulation of HUVEC proliferation but did not affect the stimulation of cells by the heparin-binding domain-deleted VEGF(121). Taken together, these results demonstrate that antiangiogenic forms of antithrombin block the proangiogenic effects of FGF-2 and VEGF on endothelial cells by competing with the growth factors for binding the heparan sulfate co-receptor, which mediates growth factor-receptor interactions. Moreover, the inability of native antithrombin to bind this co-receptor implies that native and conformationally altered forms of antithrombin differentially bind proangiogenic heparan sulfate domains.
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PMID:Antiangiogenic antithrombin blocks the heparan sulfate-dependent binding of proangiogenic growth factors to their endothelial cell receptors: evidence for differential binding of antiangiogenic and anticoagulant forms of antithrombin to proangiogenic heparan sulfate domains. 1704 Sep 7


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