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:3.4.21.7 (plasmin)
9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transforming growth factor-beta-1 (TGF-beta1) is secreted by cells in a latent form (L-TGF-beta1) noncovalently bound to a latency-associated peptide. Activated alveolar macrophages obtained from rat lungs after bleomycin-induced pulmonary injury released increased amounts of active TGF-beta1 as well as plasmin, a protease, and thrombospondin-1 (TSP-1), a trimeric glycoprotein. Previously we had demonstrated that plasmin was critical to the activation of L-TGF- beta1. In the present study we demonstrated that TSP-1 is also important for the activation of L-TGF- beta1 because the activation can be inhibited by anti-TSP-1 monoclonal antibody. Proteins obtained from alveolar macrophage cell lysates immunoprecipitated with antibodies specific for TSP-1 were identified on immunoblots as LAP and TGF-beta1, indicating that TSP-1/L-TGF-beta1 complexes are present on alveolar macrophages. However, in the presence of plasmin both latency-associated peptide and TGF-beta1 were decreased in the same cell lysates, indicating that L-TGF-beta1 associated with TSP-1 is released by plasmin. Using immunofluorescence and antibodies to TGF-beta1 and CD36, a receptor for TSP-1, there was colocalization of TGF-beta1 with CD36. Because TSP-1 but not TGF-beta1 is a natural ligand for CD36, these findings suggest that the L-TGF-beta1 in a complex with TSP-1 localizes to the macrophage cell surface when TSP-1 interacts with its receptor, CD36. Furthermore, the association of TSP-1/L-TGF-beta1 complex with CD36 is necessary to the activation of L-TGF-beta1 because antibodies to CD36 prevent the colocalization of TGF-beta1 with CD36 as observed by immunofluorescence and inhibit activation of the L-TGF-beta1 by explanted alveolar macrophages. These findings suggest that activation of L-TGF-beta1 by plasmin occurs at the cell surface of activated alveolar macrophages and requires a TSP-1/CD36 interaction.
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PMID:Activation of rat alveolar macrophage-derived latent transforming growth factor beta-1 by plasmin requires interaction with thrombospondin-1 and its cell surface receptor, CD36. 1048 79

TGF-beta has been implicated in scarring and tissue fibrosis. Most cells secrete TGF-beta as a high molecular weight, latent complex that must be processed to a lower molecular weight, biologically active form. A number of molecules are involved in this activation step including the mannose 6-phosphate/insulin-like growth factor-II receptor, tissue transglutaminase, thrombospondin, plasmin, and others. Here we describe a rapid macrophage-based system for TGF-beta1 activation, which could be used for screening potential anti-fibrotic agents. The system employs transformed mouse peritoneal macrophages treated with lipopolysaccharide as a cell line capable of activating latent TGF-beta. The activation mechanism in our system involves mannose 6-phosphate/insulin-like growth factor-II receptor and transglutaminase. The activation of latent TGF-beta in this system can be prevented by the addition of mannose-6-phosphate but not mannose-1-phosphate. In addition, transglutaminase inhibitors, antibodies to thrombospondin, insulin-like growth factor-II in the presence of its binding protein IGFBP-2, but not IGFBP-1, suppressed the activation of TGF-beta. Anti-inflammatory molecules, such as hydrocortisone, when added to LPS-treated macrophages, inhibited TGF-beta activation by a mechanism, that may involve downregulation of transglutaminase expression. In summary, this new, rapid and reproducible system allows testing molecules for their ability to inhibit TGF-beta activation, thus providing a screening method for potential anti-scarring molecules.
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PMID:Characterization of a macrophage-based system for studying the activation of latent TGF-beta. 1073 58

The degradation of extracellular matrix (ECM) adhesive glycoproteins, fibronectin (FN), thrombospondin (TSP) and von Willebrand factor (vWF), by human leukocyte cathepsin G and elastase, and by plasmin or thrombin, was analysed by immunoblotting after incubation of physiologic doses of the proteases with confluent human umbilical vein endothelial cells. Elastase induced an almost complete disappearance of intact FN, TSP, and vWF from the ECM at 0.02 units/ml within 5 minutes of incubation at 37 degrees C. Plasmin (0.2 units/ml) was also active on all three substrates, whereas cathepsin G (0.2 units/ml) had a preferential effect on TSP. Most remarkably, these degradations occurred with no apparent change in endothelial cell morphology, as shown by phase-contrast microscopy. In contrast, thrombin (0.2 units/ml) had no apparent proteolytic action on ECM glycoproteins, where it induced cell retraction and rounding. The release of adhesive glycoproteins from the ECM was accompanied by the detection of proteolytic fragments in the conditioned medium. Kinetic studies indicated that proteolysis started within minutes and proceeded for at least 1 hour. TSP was extremely sensitive to degradation by all enzymes except thrombin, whereas vWF released from the ECM was more resistant to proteolysis than constitutively secreted vWF, and FN was poorly degraded by plasmin. Our results indicate that serine proteinases, locally produced during inflammation and/or thrombolysis, can release extracellular matrix components and generate proteolytic fragments with potential biological activities.
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PMID:Proteolysis of subendothelial adhesive glycoproteins (fibronectin, thrombospondin, and von Willebrand factor) by plasmin, leukocyte cathepsin G, and elastase. 1082 79

We have previously shown that platelet-produced thrombospondin-1 up-regulates the urokinase plasminogen activator and its receptor and promotes tumour cell invasion. Although tumour cells produce thrombospondin-1 in vivo, they produce only minimal amounts of thrombospondin-1 in vitro. To determine the effect of tumour cell-produced thrombospondin-1 in the regulation of the plasminogen/plasmin system and tumour cell invasion, we studied THBS-1-transfected MDA-MB-435 breast cancer cells that overexpress thrombospondin-1. The role of urokinase plasminogen receptor in thrombospondin-1-mediated adhesion and invasion was studied by antisense inhibition, enzymatic cleavage and antibody neutralization. Tumour cell adhesion to collagen and laminin was evaluated. Tumour cell invasion was studied in a modified Boyden chamber collagen invasion assay. Tumour cell thrombospondin-1 induced a 2-7 fold increase in urokinase plasminogen activator receptor and cell-associated urokinase plasminogen activator expression and a 50-65% increase in cell-associated urokinase plasminogen activator and plasmin activities. Furthermore, tumour cell thrombospondin-1 promoted tumour cell invasion and decreased tumour cell adhesion through up-regulation of urokinase plasminogen activator receptor-controlled urokinase plasminogen activator and plasmin activities. We conclude that tumour cell-produced thrombospondin-1 may play a critical role in the regulation of tumour cell adhesion and tumour cell invasion.
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PMID:Tumour cell thrombospondin-1 regulates tumour cell adhesion and invasion through the urokinase plasminogen activator receptor. 1091 42

Transforming growth factor (TGF)-beta1 is an important regulator of inflammation and fibrosis. TGF-beta1 is usually secreted as a biologically latent protein called latent TGF-beta1 (L-TGF-beta1). L-TGF-beta1 has no biologic effect unless L-TGF-beta1 is converted to its active form. Using a well-recognized model of lung injury induced by the antineoplastic antibiotic bleomycin (Blm), we demonstrated that 7 d after intratracheal Blm administration, total lung TGF-beta was maximally increased. This induction was due to TGF-beta1 production by alveolar macrophages that, when explanted, generated increased quantities of L-TGF-beta1 complexed with the glycoprotein thrombospondin (TSP)-1. The TSP-1/L-TGF-beta1 complex was associated with CD36, a receptor for TSP-1. The association of TSP-1/L-TGF-beta1 to CD36 was critical for plasmin-mediated release of mature TGF-beta1. In this paper we show that, compared with administration of Blm by itself, when a synthetic peptide of CD36 between amino acids 93 and 110 is given concomitantly with Blm to rats, alveolar macrophages generate markedly less active TGF-beta1, the rats gain weight more rapidly, and there is less inflammation, collagen I and III, and fibronectin synthesis. These findings demonstrate a novel in vivo mechanism of activation of L-TGF-beta1 in lung injury and the importance of alveolar macrophage- derived active TGF-beta1 in the pathogenesis of pulmonary inflammation and fibrosis.
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PMID:A CD36 synthetic peptide inhibits bleomycin-induced pulmonary inflammation and connective tissue synthesis in the rat. 1091 87

Glucose is a key factor in the development of diabetic complications, including diabetic nephropathy. The development of diabetic glomerulosclerosis is dependent on the fibrogenic growth factor, transforming growth factor-beta (TGF-beta). Previously we showed that thrombospondin-1 (TSP-1) activates latent TGF-beta both in vitro and in vivo. Activation occurs as the result of specific interactions of latent TGF-beta with TSP-1, which potentially alter the conformation of latent TGF-beta. As glucose also up-regulates TSP-1 expression, we hypothesized that the increased TGF-beta bioactivity observed in rat and human mesangial cells cultured with high glucose concentrations is the result of latent TGF-beta activation by autocrine TSP-1. Glucose-induced bioactivity of TGF-beta in mesangial cell cultures was reduced to basal levels by peptides from two different sequences that antagonize activation of latent TGF-beta by TSP, but not by the plasmin inhibitor, aprotinin. Furthermore, glucose-dependent stimulation of matrix protein synthesis was inhibited by these antagonist peptides. These studies demonstrate that glucose stimulation of TGF-beta activity and the resultant matrix protein synthesis are dependent on the action of autocrine TSP-1 to convert latent TGF-beta to its biologically active form. These data suggest that antagonists of TSP-dependent TGF-beta activation may be the basis of novel therapeutic approaches for ameliorating diabetic renal fibrosis.
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PMID:Glucose stimulation of transforming growth factor-beta bioactivity in mesangial cells is mediated by thrombospondin-1. 1102 38

Fibrinogen (FBG), together with its polymerized form fibrin, modulates cellular responses during wound repair and tissue remodeling. Thus, we sought to determine whether A549 lung epithelial type II-like cells would endocytose insoluble, surface-bound FBG as a potential mechanism of alveolar matrix remodeling. Surface-bound FBG was endocytosed into either lysosomes or late endosomes by A549 cells through arg-gly-asp-dependent binding to alphavbeta3 but not alpha5beta1 integrin receptors. Soluble FBG added to confluent monolayers of A549 cells was not endocytosed. Unlike the uptake of the extracellular matrix glycoproteins vitronectin and thrombospondin by other cell types, endocytosis of FBG by A549 cells was neither inhibited by heparin nor dependent on binding to cell-surface heparan sulfate proteoglycans. FBG did not colocalize with endocytosed transferrin, whereas dextran showed partial colocalization with FBG in endocytic vesicles, suggesting nonclathrin-mediated endocytosis. Inhibition of actin filament polymerization blocked endocytosis of both dextran and FBG but not transferrin, providing further support that FBG is endocytosed via a nonclathrin pathway. Disruption of actin polymerization inhibited integrin-mediated cell spreading, which contributed to an overall reduction in FBG clearance that was most likely due to reduced cell migration and associated pericellular proteolysis. Trasylol inhibition of extracellular plasmin activity did not inhibit endocytosis of FBG. The endocytosed FBG was degraded to trichloroacetic acid-soluble fragments that showed an electrophoretic pattern distinctly different from plasmin-degraded FBG. Together, these results suggest that endocytosis of matrix-associated FBG by alveolar epithelial cells may be involved in the processes of alveolar tissue repair and matrix remodeling.
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PMID:Integrin alphavbeta3-mediated endocytosis of immobilized fibrinogen by A549 lung alveolar epithelial cells. 1115 45

Serine proteases are implicated in a variety of processes during neurogenesis, including cell migration, axon outgrowth, and synapse elimination. Tissue-type plasminogen activator and urokinase-type activator are expressed in the floor plate during embryonic development. F-spondin, a gene also expressed in the floor plate, encodes a secreted, extracellular matrix-attached protein that promotes outgrowth of commissural axons and inhibits outgrowth of motor axons. F-spondin is processed in vivo to yield an amino half protein that contains regions of homology to reelin and mindin, and a carboxyl half protein that contains either six or four thrombospondin type I repeats (TSRs). We have tested F-spondin to see whether it is subjected to processing by plasmin and to determine whether the processing modulates its biological activity. Plasmin cleaves F-spondin at its carboxyl terminus. By using nested deletion proteins and mutating potential plasmin cleavage sites, we have identified two cleavage sites, the first between the fifth and sixth TSRs, and the second at the fifth TSR. Analysis of the extracellular matrix (ECM) attachment properties of the TSRs revealed that the fifth and sixth TSRs bind to the ECM, but repeats 1-4 do not. Structural functional experiments revealed that two basic motives are required to elicit binding of TSR module to the ECM. We demonstrate further that plasmin releases the ECM-bound F-spondin protein.
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PMID:Plasmin-mediated release of the guidance molecule F-spondin from the extracellular matrix. 1135 77

Angiogenesis, the formation of new capillary blood vessels, occurs almost exclusively in the microcirculation. This process is controlled by the interaction between factors with positive and negative regulatory activity. In this study, we have compared the effect of two well described positive regulators, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2) on bovine adrenal cortex-derived microvascular endothelial (BME) and bovine aortic endothelial (BAE) cells. The parameters we assessed included (a) cellular reorganization and lumen formation following exposure of the apical cell surface to a three-dimensional collagen gel; (b) organization of the actin cytoskeleton; (c) expression of thrombospondin-1 (TSP-1), an endogenous negative regulator of angiogenesis; and (d) extracellular proteolytic activity mediated by the plasminogen activator (PA)/plasmin system. We found that (a) collagen gel overlay induces rapid reorganization and lumen formation in BME but not BAE cells; (b) FGF-2 but not VEGF induced dramatic reorganization of actin microfilaments in BME cells, with neither cytokine affecting BAE cells; (c) FGF-2 decreased TSP-1 protein and mRNA expression in BME cells, an effect which was specific for FGF-2 and BME cells, since TSP-1 protein levels were unaffected by VEGF in BME cells, or by FGF-2 or VEGF in BAE cells; (d) FGF-2 induced urokinase-type PA (uPA) in BME and BAE cells, while VEGF induced uPA and tissue-type PA in BME cells with no effect on BAE cells. Taken together, these findings reveal endothelial cell-type specific responses to FGF-2 and VEGF, and point to the greater specificity of these cytokines for endothelial cells of the microvasculature than for large vessel (aortic) endothelial cells. Furthermore, when viewed in the context of our previous observation on the synergistic interaction between VEGF and FGF-2, our present findings provide evidence for complementary mechanisms which, when acting in concert, might account for the synergistic effect.
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PMID:Response of bovine endothelial cells to FGF-2 and VEGF is dependent on their site of origin: Relevance to the regulation of angiogenesis. 1150 Sep 40

Porcine thyrocytes in vitro in the presence of TSH adopt follicular-like morphology. Epidermal growth factor, phorbol esters or transforming growth factor beta-1 (TGFbeta-1) induce a rapid spreading of the cells and dedifferentiation. In addition to thyroglobulin, dedifferentiated thyrocytes secreted into the culture medium three proteins in abundant quantities. Two of them have been previously identified as thrombospondin-1 and clusterin, respectively. Using the microsequencing method we identified the third one, a M(r) 45,000 glycosylated protein, as plasminogen activator inhibitor-1 (PAI-1). EGF, phorbol esters or TGF-beta1 predominantly increased PAI-1 protein expression in TSH-treated cells. The maximal increase of PAI-1 mRNA steady-state level was observed 6 h after EGF treatment and sustained up to 48 h. Recombinant PAI-1 inhibited cell-associated plasmin activity and delayed cell spreading. Enhanced synthesis and secretion of PAI-1 upon treatment with different growth factors during dedifferentiation process and spreading may be considered a feed-back defence mechanism of the cells to harmful extracellular stimuli.
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PMID:Enhanced expression of plasminogen activator inhibitor-1 by dedifferentiated thyrocytes. 1209 1


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