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 effects of rabbit T lymphocytes on rabbit aortic smooth muscle cell (SMC) phenotype and proliferation were investigated in vitro. SMCs seeded at confluent density in primary culture had a volume fraction of myofilaments (Vvmyo) of 49.8 +/- 2.6% after 3 days of culture, not significantly different from that of freshly dispersed cells (Vvmyo, 54.1 +/- 2.1%). Sister cultures of SMCs to which Concanavalin A-activated T lymphocytes or T lymphocyte-conditioned medium was added had significantly lower Vvmyo (35.5 +/- 2.2% and 31.6 +/- 2.3%, respectively) at the same time point. We have previously shown that a decrease in Vvmyo could be induced by the heparan sulfate-degrading activity of living macrophages and by commercial preparations of heparinase. While activated T lymphocytes also completely degraded heparan sulfate-rich 35S-labeled extracellular matrix (an effect inhibited by the addition of 10 micrograms/mL heparin), no heparanase-like activity was detected in T lymphocyte-conditioned medium, indicating that for this cell type SMC phenotypic change is induced by a different mechanism. Incubation of the T lymphocyte-derived cytokine interferon gamma (IFN-gamma) with freshly isolated rat SMCs caused a significant reduction in Vvmyo at day 2 in primary culture from 54.3 +/- 2.1% (control) to 35.4 +/- 3.0%. Furthermore, a neutralizing antibody specific for IFN-gamma removed the effect of T lymphocytes and medium conditioned by them, thus positively identifying IFN-gamma as the T lymphocyte factor responsible for this activity. T lymphocyte-conditioned medium was mitogenic for passaged (low Vvmyo) SMCs.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:T lymphocytes affect smooth muscle cell phenotype and proliferation. 762 15

Interferon (IFN)-gamma, in common with a number of cytokines or growth factors, strongly interacts with heparan sulphate (HS). It has been shown previously that one of the C-terminal basic clusters of amino acids (a regulatory element of IFN-gamma activity) is involved in this interaction. The structural organization of the HS domain that binds to human IFN-gamma has been investigated here. IFN-gamma-affinity chromatography of HS oligosaccharides released by either enzymic or chemical cleavage showed that the binding site is not found in a domain that is resistant to either heparinase or heparitinase or exclusively N-sulphated or N-acetylated. This led us to take a 'footprinting' approach in which HS was depolymerized in the presence of IFN-gamma and the cytokine-protected sequences were separated from the digested fragments. Using this strategy we consistently isolated an IFN-gamma-protected domain (IPD; approx. 10 kDa) which displayed the same affinity as full-length HS for the cytokine. Treatment of IPD with either heparinase or heparitinase strongly reduced its affinity, confirming that the high-affinity binding site encompassed a mixture of HS structural domains. Patterns of depolymerization with either enzymic or chemical agents were consistent with IPD being composed of an extended internal domain (approx. 7 kDa) which is predominantly N-acetylated and GlcA-rich, flanked by small N-sulphated oligosaccharides (mainly hexa- to octasaccharides). This is the first description of an HS protein-binding sequence with this type of molecular organization. Furthermore, using a cross-linking strategy, we demonstrated that one HS molecule bound to an IFN-gamma dimer. Together these results lead us to propose a novel model for the interaction of HS with a protein, in which two sulphated terminal sequences of the binding domain interact directly with the two IFN-gamma C-termini and bridge the two cytokine monomers through an internal N-acetyl-rich sequence.
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PMID:Molecular organization of the interferon gamma-binding domain in heparan sulphate. 765 88

Basic fibroblast growth factor (bFGF) has been identified as an important cytokine for blood cells. To determine whether hematopoietic cells have receptors that recognize bFGF, the ability of human leukemia cell lines to bind 125I-bFGF was investigated. Specific bFGF-binding sites were identified on K562 and HL60 cells, but not on U937 cells. DAMI cells bound low amounts of 125I-bFGF specifically. Binding of 125I-bFGF to K562 cell surfaces was reduced in a dose-dependent manner by unlabeled bFGF or by heparin. Scatchard analysis of binding to K562 cells revealed two classes of binding sites: 1,650 high affinity binding sites per cell with a dissociation constant (kd) of 192 pmol/L, and 36,600 low affinity sites per cell with a kd of 9.3 nmol/L. Chemical crosslinking experiments with K562, HL60, and DAMI cells revealed receptor-growth factor complexes with molecular masses of 140 to 160 kD, similar in size to complexes formed by known receptor species. Binding of 125I-bFGF to K562 cells was sensitive to heparinase treatment but not to chondroitinase treatment, suggesting that heparan sulfate proteoglycans (HSPGs) may be responsible for the low affinity binding sites. To further investigate whether K562 cells make HSPG, the incorporation of 35SO4 into proteoglycans was assessed. Metabolically labeled cell-surface proteoglycans with molecular masses of 180 to 300 kD were identified in K562 cells. These proteoglycans were sensitive to heparinase, demonstrating that K562 cells synthesize bFGF-binding HSPG. Treatment of K562 cells with phorbol-12-myristate-13-acetate (PMA) caused a loss of bFGF-binding capacity. This decreased binding capacity reflected a rapid loss of high affinity receptors. The ability to form bFGF-receptor complexes decreased by 65% to 70% within 1 hour and declined continuously thereafter. The decrease in binding of bFGF was not due to an autocrine downregulation of bFGF receptors, because there was no increase in bFGF after PMA treatment as detected by Western blotting, and suramin, which blocks bFGF binding to receptors, did not prevent the loss of receptors after exposure to PMA. In addition, inhibitors of either protein synthesis or protease activity did not prevent the loss of bFGF receptors in PMA-treated cells. In summary, this work demonstrates that leukemia cell lines have receptors that specifically bind bFGF and supports the hypothesis that bFGF acts directly on certain blood cells to stimulate their proliferation.
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PMID:Human leukemia cell lines bind basic fibroblast growth factor (FGF) on FGF receptors and heparan sulfates: downmodulation of FGF receptors by phorbol ester. 854 48

This study examines the role of L-selectin in monocyte adhesion to arterial endothelium, a key pathogenic event of atherosclerosis. Using a nonstatic (rotation) adhesion assay, we observed that monocyte binding to bovine aortic endothelium at 4 degrees C increased four to nine times upon endothelium activation with tumor necrosis factor (TNF)-alpha. mAb-blocking experiments demonstrated that L-selectin mediates a major part (64 +/- 18%) of monocyte attachment. Videomicroscopy experiments performed under flow indicated that monocytes abruptly halted on 8-h TNF-alpha-activated aortic endothelium, approximately 80% of monocyte attachment being mediated by L-selectin. Flow cytometric studies with a L-selectin/IgM heavy chain chimeric protein showed calcium-dependent L-selectin binding to cytokine-activated and, unexpectedly, unactivated aortic cells. Soluble L-selectin binding was completely inhibited by anti-L-selectin mAb or by aortic cell exposure to trypsin. Experiments with cycloheximide, chlorate, or neuraminidase showed that protein synthesis and sulfate groups, but not sialic acid residues, were essential for L-selectin counterreceptor function. Moreover, heparin lyases partially inhibited soluble L-selectin binding to cytokine-activated aortic cells, whereas a stronger inhibition was seen with unstimulated endothelial cells, suggesting that cytokine activation could induce the expression of additional ligand(s) for L-selectin, distinct from heparan sulfate proteoglycans. Under flow, endothelial cell treatment with heparinase inhibited by approximately 80% monocyte attachment to TNF-alpha-activated aortic endothelium, indicating a major role for heparan sulfate proteoglycans in monocyte-endothelial interactions. Thus, L-selectin mediates monocyte attachment to activated aortic endothelium, and heparan sulfate proteoglycans serve as arterial ligands for monocyte L-selectin.
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PMID:Monocyte adhesion to activated aortic endothelium: role of L-selectin and heparan sulfate proteoglycans. 904 58

Inflammation is the clinical expression of chemical mediators such as the pro-inflammatory cytokine tumor necrosis factor (TNF-)-alpha produced by macrophages and other cells activated in the immune response. Hence, agents that can inhibit TNF-alpha may be useful in treating arthritis and other diseases resulting from uncontrolled inflammation. We now report that the cleavage of heparin by the enzyme heparinase I generates sulfated disaccharide (DS) molecules that can inhibit the production of TNF-alpha. Administration of nanogram amounts of the sulfated DS molecules to experimental animals inhibited delayed-type hypersensitivity to a skin sensitizer and arrested the joint swelling of immunologically induced adjuvant arthritis. Notably, the sulfated DS molecules showed a bell-shaped dose-response curve in vitro and in vivo: decreased effects were seen using amounts of the DS molecules higher than optimal. Thus, molecular regulators of inflammation can be released from the natural molecule heparin by the action of an enzyme.
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PMID:Heparin disaccharides inhibit tumor necrosis factor-alpha production by macrophages and arrest immune inflammation in rodents. 935 56

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

An emerging body of evidence suggests that type IIA secretory phospholipase A(2) (sPLA(2)-IIA) participates in the amplification of the stimulus-induced cyclooxygenase (COX)-2-dependent delayed prostaglandin (PG)-biosynthetic response in several cell types. However, the biological importance of the ability of sPLA(2)-IIA to bind to heparan sulfate proteoglycan (HSPG) on cell surfaces has remained controversial. Here we show that glypican, a glycosylphosphatidylinositol (GPI)-anchored HSPG, acts as a physical and functional adaptor for sPLA(2)-IIA. sPLA(2)-IIA-dependent PGE(2) generation by interleukin-1-stimulated cells was markedly attenuated by treatment of the cells with heparin, heparinase or GPI-specific phospholipase C, which solubilized the cell surface-associated sPLA(2)-IIA. Overexpression of glypican-1 increased the association of sPLA(2)-IIA with the cell membrane, and glypican-1 was coimmunoprecipitated by the antibody against sPLA(2)-IIA. Glypican-1 overexpression led to marked augmentation of sPLA(2)-IIA-mediated arachidonic acid release, PGE(2) generation, and COX-2 induction in interleukin-1-stimulated cells, particularly when the sPLA(2)-IIA expression level was suboptimal. Immunofluorescent microscopic analyses of cytokine-stimulated cells revealed that sPLA(2)-IIA was present in the caveolae, a microdomain in which GPI-anchored proteins reside, and also appeared in the perinuclear area in proximity to COX-2. We therefore propose that a GPI-anchored HSPG glypican facilitates the trafficking of sPLA(2)-IIA into particular subcellular compartments, and arachidonic acid thus released from the compartments may link efficiently to the downstream COX-2-mediated PG biosynthesis.
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PMID:Functional association of type IIA secretory phospholipase A(2) with the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan in the cyclooxygenase-2-mediated delayed prostanoid-biosynthetic pathway. 1051 75

Lipoprotein lipase (LPL) is a key enzyme in the hydrolysis of triglyceride-rich lipoproteins. In vascular diseases, such as atherosclerosis, inflammation plays an important role in the pathogenesis of the disease. We examined the role of LPL in modulating tumor necrosis factor-alpha (TNF-alpha)- and interferon-gamma (IFN-gamma)-mediated inflammatory cytokine signal transduction pathways in human aortic endothelial cells (HAECs). LPL significantly suppressed TNF-alpha-induced gene expression, and this suppression was reversed by tetrahydrolipstatin and heparinase. In contrast, LPL synergistically enhanced IFN-gamma-induced gene expression in HAECs. To elucidate the molecular mechanisms of LPL action, we investigated the role of transcription factors nuclear factor kappa B (NF-kappaB) and signal transducer and activator of transcription factor 1 (Stat1). The anti-inflammatory response of LPL in suppressing TNF-alpha-induced gene expression was a result of its inhibition of NF-kappaB activity by the abrogation of IkappaB-alpha degradation and phosphorylation of the p65 subunit. Although LPL alone had no effect on Stat1 activation, LPL enhanced IFN-gamma-induced phosphorylation of Stat1 on tyrosine 701 and serine 727, as well as Stat1-mediated transactivation. The synergistic effect of LPL on IFN-gamma-induced Stat1 activation was mediated by enhanced activation of the tyrosine kinase JAK2 and was abrogated by LY294002, a specific inhibitor of the phosphatidylinositol 3'-kinase pathway. Our studies indicate that LPL has differential effects on several inflammatory pathways known to be important in atherosclerosis.
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PMID:Differential effects of lipoprotein lipase on tumor necrosis factor-alpha and interferon-gamma-mediated gene expression in human endothelial cells. 1599 21

Many growth factors and cytokines are immobilized on the extracellular matrix (ECM) by binding to glycosaminoglycans and are stored in an inactive form in the cellular microenvironment. However, the mechanisms of ECM-bound growth factor or cytokine activation have not been well documented. We showed that the insulin-like growth factor type-1 receptor (IGF-1R) was rapidly phosphorylated after the addition of matrix metalloproteinase (MMP)-7 to a serum-starved human colon cancer cell line (HT29) and that phosphorylation was completely inhibited by an IGF-II neutralizing antibody. In the ECM of this cell line, IGF-II and IGF binding protein (BP)-2 coexisted, but IGFBP-2 disappeared from the ECM fraction after treatment with MMP-7 or heparinase III. On the other hand, in a cell line in which IGF-1R was overexpressed, IGF-1R was phosphorylated by supernatant from the MMP-7-treated ECM fraction of HT29 but not by that from a heparinase-III-treated ECM fraction. We also demonstrated that MMP-7 degrades IGFBP-2 in vitro at three cleavage sites (peptide bonds E(151)-L(152), G(175)-L(176) and K(181)-L(182)), which have not been documented previously. Taken together, these results demonstrate that MMP-7 generates bioactive IGF-II by degrading the IGF-II/IGFBP-2 complex binding to heparan sulfate proteoglycan in the ECM, resulting in IGF-II-induced signal transduction. This evidence indicates that some ECM-associated growth factors enhance their ability to bind to their receptors by some proteases in the tumor microenvironment. This mechanism of action ('protease-triggered matricrine') represents an attractive model for understanding ECM-tumor interactions.
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PMID:Matrix metalloproteinase-7 triggers the matricrine action of insulin-like growth factor-II via proteinase activity on insulin-like growth factor binding protein 2 in the extracellular matrix. 1735 88

Expression of angiogenic cytokines like vascular endothelial growth factor is enhanced by hypoxia. We tested the hypothesis that decreased oxygen levels up-regulate the angiogenic factor secretoneurin. In vivo, muscle cells of mouse ischemic hind limbs showed increased secretoneurin expression, and inhibition of secretoneurin by a neutralizing antibody impaired the angiogenic response in this ischemia model. In a mouse soft tissue model of hypoxia, secretoneurin was increased in subcutaneous muscle fibers. In vitro, secretoneurin mRNA and protein were up-regulated in L6 myoblast cells after exposure to low oxygen levels. The hypoxia-dependent regulation of secretoneurin was tissue specific and was not observed in endothelial cells, vascular smooth muscle cells, or AtT20 pituitary tumor cells. The hypoxia-dependent induction of secretoneurin in L6 myoblasts is regulated by hypoxia-inducible factor-1alpha, since inhibition of this factor using si-RNA inhibited up-regulation of secretoneurin. Induction of secretoneurin by hypoxia was dependent on basic fibroblast growth factor in vivo and in vitro, and inhibition of this regulation by heparinase suggests an involvement of low-affinity basic fibroblast growth factor binding sites. In summary, our data show that the angiogenic cytokine secretoneurin is up-regulated by hypoxia in muscle cells by hypoxia-inducible factor-1alpha- and basic fibroblast growth factor-dependent mechanisms.
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PMID:Hypoxia up-regulates the angiogenic cytokine secretoneurin via an HIF-1alpha- and basic FGF-dependent pathway in muscle cells. 1750 77


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