Plasminogen activator inhibitor from human fibrosarcoma cells binds urokinase-type plasminogen activator, but not its proenzyme.
15 Jun 1986-Journal of Biological Chemistry (American Society for Biochemistry and Molecular Biology)-Vol. 261, Iss: 17, pp 7644-7651
TL;DR: An approximately 75% pure form of a human Mr approximately 54,000 plasminogen activator inhibitor from conditioned culture fluid of the fibrosarcoma cell line HT-1080 was obtained by a single step of chromatography on concanavalin A-Sepharose.
About: This article is published in Journal of Biological Chemistry.The article was published on 1986-06-15 and is currently open access. It has received 235 citations till now. The article focuses on the topics: Plasminogen activator & Urokinase.
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TL;DR: Findings show that u-PA is synthesized and released from mammalian cells as a proenzyme with little or no activity (pro-u-PA), that cells possess a specific binding site for u- PA and pro-u -PA, and that they can also synthesize different rapidly acting, high affinity inhibitors of PAs.
Abstract: N 'Ew information has recently become available that we believe may be of value for a more precise focusing of research on the role of plasminogen activators (PAs) in cellular physiology and pathology. PAs convert the abundant extracellular zymogen plasminogen into plasmin, an active protease that, directly or indirectly, can promote degradation of all components of the extracellular matrix. The urokinase-type PA (u-PA) is one of two activators with similar catalytic specificities that have been described in each mammalian species so far studied. Many cell types synthesize and secrete u-PA at different stages in their life cycle; it has been a consistent finding that the expression of u-PAcatalyzed proteolysis is under the control of hormonal-type mediators, the nature of which depends on the cell population considered, u-PA is thought to play a central role in regulating extracellular proteolysis in a variety of normal and pathological processes involving tissue destruction and cell migration, e.g., organ involution, inflammatory reactions, and invasive growth of trophoblasts and cancer cells (for reviews, see 8, 18, 44, 47, 49, 50). Recent findings show that u-PA is synthesized and released from mammalian cells as a proenzyme with little or no activity (pro-u-PA), that cells possess a specific binding site for u-PA and pro-u-PA, and that they can also synthesize different rapidly acting, high affinity inhibitors of PAs. Thus, activation of pro-u-PA, u-PA receptor occupancy, and PA-inhibitors may finely regulate the location and extent of plasminogen activation.
622 citations
Cites background from "Plasminogen activator inhibitor fro..."
...These three PA inhibitors react with u-PA, but not with pro-u-PA (2, 20, 64)....
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...One of these, PAI type-l, is released by endothelial cells and some neoplastic cell lines (2, 21, 23, 32, 34, 62, 65); it is also present in thrombocytes and blood plasma (22, 68)....
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...plasminogen, does not react with macromolecular antiproteases, lacks amidolytic activity against synthetic plasminogen activator substrates, and is unable to react with the active site titrant diisopropylfluorophosphate (2, 18, 26, 29, 39, 55, 64, 70)....
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TL;DR: A dynamic regulatory role for PAI-1 and uPA in uPAR-mediated cell adhesion and release is suggested, and may help to explain why high PAi-1 levels indicate a poor prognosis for many cancers.
Abstract: Induction of the urokinase type plasminogen activator receptor (uPAR) promotes cell adhesion through its interaction with vitronectin (VN) in the extracellular matrix, and facilitates cell migration and invasion by localizing uPA to the cell surface. We provide evidence that this balance between cell adhesion and cell detachment is governed by PA inhibitor-1 (PAI-1). First, we demonstrate that uPAR and PAI-1 bind to the same site in VN (i.e., the amino-terminal somatomedin B domain; SMB), and that PAI-1 competes with uPAR for binding to SMB. Domain swapping and mutagenesis studies indicate that the uPAR-binding sequence is located within the central region of the SMB domain, a region previously shown to contain the PAI-1-binding motif. Second, we show that PAI-1 dissociates bound VN from uPAR and detaches U937 cells from their VN substratum. This PAI-1 mediated release of cells from VN appears to occur independently of its ability to function as a protease inhibitor, and may help to explain why high PAI-1 levels indicate a poor prognosis for many cancers. Finally, we show that uPA can rapidly reverse this effect of PAI-1. Taken together, these results suggest a dynamic regulatory role for PAI-1 and uPA in uPAR-mediated cell adhesion and release.
463 citations
Cites background from "Plasminogen activator inhibitor fro..."
...In this regard, it should be noted that if the bound uPA is in its proenzyme form, PAI-1 will not be able to bind to and inactivate it (Andreasen et al., 1986)....
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TL;DR: The results indicate that growth factors modulate the proteolytic balance of cultured cells by altering the amounts of PAs and their inhibitors.
Abstract: Cultured human embryonic lung fibroblasts were used as a model to study the effects of transforming growth factor-beta (TGF beta) on the plasminogen activator (PA) activity released by nontumorigenic cells into the culture medium. The cells were exposed to TGF beta under serum-free conditions, and the changes in PA activity and protein metabolism were analyzed by caseinolysis-in-agar assays, zymography, and polypeptide analysis. Treatment of the cells with TGF beta caused a significant decrease in the PA activity of the culture medium as analyzed by the caseinolysis-in-agar assays. The quantitatively most prominent effect of TGF beta on confluent cultures of cells was the induction of an Mr 47,000 protein, as detected by metabolic labeling. The Mr 47,000 protein was a PA inhibitor as judged by reverse zymography. It was antigenically related to a PA inhibitor secreted by HT-1080 tumor cells as demonstrated with monoclonal antibodies. The induced Mr 47,000 inhibitor was deposited into the growth substratum of the cells, as detected by metabolic labeling, immunoblotting analysis, and reverse zymography assays of extracellular matrix preparations. TGF beta also decreased the amounts of urokinase-type and tissue-type PAs accumulated in the conditioned medium, as detected by zymography. Epidermal growth factor antagonized the inhibitory effects of TGF beta by enhancing the amounts of the PAs. These results indicate that growth factors modulate the proteolytic balance of cultured cells by altering the amounts of PAs and their inhibitors.
434 citations
Journal Article•
TL;DR: It is suggested that PAI-1 plays a role in protecting the tumor against degrading itself and may be a biochemical marker of tumor angiogenesis, in good agreement with the supposed function of uPA in cancer invasion.
Abstract: The urokinase pathway of plasminogen activation is supposed to be involved in proteolytic degradation of the extracellular matrix during cancer invasion. The prognostic value of urokinase-type plasminogen activator (uPA) and type 1 plasminogen activator inhibitor (PAI-1) levels in cytosolic extracts of ductal breast carcinomas was studied, retrospectively, in 118 premenopausal and 72 postmenopausal high-risk patients entered into the protocol of Danish Breast Cancer Cooperative Group trials for adjuvant treatment of breast cancer. The median observation time was 8.5 years. uPA and PAI-1 levels were determined by sandwich enzyme-linked immunosorbent assays. There is a strong correlation between these levels (P
421 citations
TL;DR: Etude de la regulation hormonale des enzymes appartenant a la famille de serine protease inhibiteur (plasminogen activator et urokinase)
404 citations
References
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TL;DR: This chapter describes two types of plasminogen activators—namely, the urokinase-type plasMinogen activator (u-PA) and the tissue- type plasmineg activator(t-PA), which are essentially different gene products.
Abstract: Publisher Summary This chapter discusses the role of plasminogen activators in various biological processes. In specific, it describes two types of plasminogen activators—namely, the urokinase-type plasminogen activator (u-PA) and the tissue-type plasminogen activator (t-PA), which are essentially different gene products. The amino acid sequences of these activators and nucleotide sequences of the corresponding cDNAs have largely been determined, and the cDNAs have been cloned using recombinant techniques. A variety of enzymatic as well as immunological assay and detection methods have also been developed that allows a precise quantification of the activators, a distinction between u-PA and t-PA, determination of whether an activator is present in its active or zymogen form, analysis of the kinetics of different steps of the cascade reaction, and immunocytochemical identification of u-PA and t-PA in tissue sections. Much of the studies on plasminogen activators and cancer has been guided by the hypothesis that proteolysis of the components of extracellular matrix, initiated by the release of plasminogen activator from the cancer cells, plays a decisive role for the degradation of normal tissue, and thereby for invasive growth and metastases.
2,545 citations
TL;DR: This paper presents a meta-analyses of the proton-probes of ATP and its role in the building blocks of proteinase Inhibitor and shows how the role of ATP in the design of proteinases changes with age and disease progression.
Abstract: PERSPECTIVES AND SUMMARy 655 INTRODUCTION 657 MECHANISM OF ACTION OF PROTEINASE INHIBITORS 659 The Standard Mechanism 660 Deviations Jrom the Standard M echanism 661 ALPHA I-PROTEINASE INHIBITOR 663 Polymorphysm oj (x, Proteinase Inhibitor 665 Mechanism Studies 667 Physiological Role of (x, Proteinase Inhibitor 672 ANTITHROMBIN III ...... 674 Effect oj Heparin 674 Mechanism Studies 675 Physiological Role of Antithrombin III 676 ALPHAz-ANTIPLASMIN 676 ALPHA,-ANTICHYMOTRYPSIN 678 CI-INHIBITOR 681 ALPHA2-MACROGLOBULIN 684 The Trapping Reaction ..... 685 The Covalent-Linking Reaction 690 The Adherence Reaction 693 Physiological Role oJ(Xz-Macroglobulin 693 INTER-ALPHA-TRYPSIN INHIBITOR 695 BETA,-ANTICOLLAGENASE 696 ALPHA-CYSTEINE PROTEINASE INHIBITOR 698 OTHER INHIBITORS 699 CONCLUDING REMARKS 700
2,066 citations
TL;DR: Plasminogen was prepared from human plasma by affinity chromatography on L-lysine-substituted Sepharose with a specific activity of 100 caseinolytic units per milligram of nitrogen.
Abstract: Plasminogen was prepared from human plasma by affinity chromatography on L-lysine-substituted Sepharose. Thirty milligrams of plasminogen, with a specific activity of 100 caseinolytic units (Committee on Thrombolytic Agents) per milligram of nitrogen, were obtained from 340 milliliters of plasma. This corresponds to over 200-fold purification from plasma. Disc-gel electrophoresis at pH 8.3 indicated seven distinct bands, all of which contained activity.
2,051 citations
TL;DR: The identification of such a cell line, Sp2/0-Ag14, is reported here the identification of a tumour cell fusion partner that makes no Ig but which can nevertheless be fused with spleen cells to obtain hybrids secreting only the specific antibody.
Abstract: FUSION of myeloma cells which grow in tissue culture with spleen cells from an immunised mouse provides a general method for obtaining cell lines (hybridomas) which make antibody of the desired specificity1–3. Hybrids derived from these myelomas make the immunoglobulin (Ig) heavy and light chains of the myeloma parent as well as the antigen-specific heavy and light chains of the spleen cell parent. In conditions in which the two heavy and two light chains associate randomly, a hybridoma would make 10 distinct Ig molecules, and the specific antibody would comprise only 1/16 of the total Ig4,5. To obtain hybridomas making only the specific antibodies requires a tumour cell fusion partner that itself makes no Ig but which can nevertheless be fused with spleen cells to obtain hybrids secreting only the specific antibody. We report here the identification of such a cell line, Sp2/0-Ag14.
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