Showing papers by "Dominique Belin published in 1984"

Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages.

Abstract: The plasminogen activator (PA) produced by freshly purified human monocytes-macrophages and histiocytic, lymphoma-derived U 937 cells was analyzed by zymography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and found to migrate with an apparent Mr of 55,000, identical to that of urokinase (Uk). By immunoprecipitation with antibodies specific for the two different types of PA, the enzyme was shown to be immunologically related to urokinase, and not to tissue PA. Urokinase was secreted in the form of the inactive Mr 55,000 zymogen prourokinase , and could be converted to the active Mr 55,000 enzyme by limited proteolysis with plasmin. Conditioned media from cultures of U 937 cells and monocytes-macrophages inhibited the fibrinolytic activity of exogenously added urokinase. Using [125I]-labeled urokinase we observed the formation of an enzyme-ligand complex, which was not dissociated by boiling in SDS and migrated with an apparent Mr 40,000 daltons higher than the free enzyme; since complexed urokinase was functionally inactivated as a PA, the ligand is an inhibitor of urokinase. This inhibitor is different from fibroblast-produced protease- nexin , in that it did not interact with thrombin. These results suggest that plasminogen activation by mononuclear phagocytes can be modulated through the secretion of both (pro)enzyme and a specific inhibitor.

Tumor promoter PMA stimulates the synthesis and secretion of mouse pro-urokinase in MSV-transformed 3T3 cells: this is mediated by an increase in urokinase mRNA content.

Abstract: In mouse MSV-3T3 cells the synthesis of the urokinase form of plasminogen activator was increased 10-fold after addition of the tumor promoter phorbol-12-myristate-13-acetate (PMA). PMA also stimulated the secretion of the protein into the culture medium, mostly in the form of enzymatically inactive pro-urokinase. When assayed by injecting RNA into Xenopus laevis oocytes, the concentration of functional urokinase mRNA was found to be 6- to 10-fold higher in the PMA-treated cells; a similar increase in urokinase mRNA content was measured by hybridisation with a mouse urokinase cDNA probe. Thus, the induction of plasminogen activator by PMA in MSV-3T3 cells is accounted for by an increased content of urokinase mRNA.

Concomitant s ecretion o f p rourokinase a nd o f a plasminogen a ctivator-specif ic i nhibitor by cultured h uman m onocytes-macrop hages

Abstract: CONCOMITANT SECRETION OF PROUROKINASE AND OF A PLASMINOGEN ACTIVATOR-SPECIFIC INHIBITOR BY CULTURED HUMAN MONOCYTES-MACROPHAGES By JEAN-DOMINIQUE VASSALLI, JEAN-MICHEL DAYER,* ANNELISE WOHLWEND, AND DOMINIQUE BELIN ~ From the Institute of Histology and Embryology, *the Division of Immunology and Allergy, Department of Medicine, Sand the Department of Pathology, University of Geneva Medical School, 1211 Geneva 4, Switzerland Piasminogen activators (PA) l are thought to play a role in the proteolytic events associated with cell migration and tissue destruction and remodeling (1), in particular in the context of inflammation (2). Indeed, monocytes and macro- phages from both human and murine origin produce PA (3, 4), and the secretion of these enzymes has been linked with the participation of mononu¢lear phago- cytes in inflammatory reactions: peritoneal macrophages collected from inflam- matory exudates synthesize and secrete such enzymes (reference 3, D. Belin et al., manuscript in preparation); furthermore, PA production is enhanced by inflammatory agents (5, 6) and suppressed by antiinflammatory glucocorticoids (2). However, little is known at the molecular level about the mechanisms by which PA activity is regulated. Recently progress has been made in the characterization of PA. Two different enzymes, urokinase (Uk) and tissue activator (TA), have been identified. Their primary structures have been elucidated at the protein or mRNA level (7-9), and it is now clear that they are the products of different genes. Aspects of their biosynthesis have been investigated. Uk has been shown to be secreted by certain human and murine cells as a one-chain inactive zymogen, with a Mr of 55,000 for the human protein (10-12). Active Mr 55,000 human Uk is generated through a single intramolecular proteolytic cleavage, and consists of two disulfide- linked polypeptide chains. Mr 33,000 human Uk is a proteolytic product of the Mr 55,000 enzyme with similar specific catalytic activity. The cellular production of PA inhibitors has also been described: in particular the secretion of protease- nexin (13) by human fibroblasts has been suggested to play a role in the regulation of extracellular PA activity (14); mouse peritoneal macrophages have been reported to be a source of such inhibitors (15, 16). In view of these developments, it was of interest to determine the type of PA produced in cultures of human monocytes-macrophages, as well as to investigate This work was supported by grants 3.159-81 (to J.-D. V. and D. B.) and 3.449.0-83 (to J.-M. D.) from the Swiss National Science Foundation. Abbreviations used in this paper: BSA, bovine serum albumin; DFP, diisopropyl-fluorophosphate; FBS, fetal bovine serum; HBSS, Hank's balanced salt solution; PA, plasminogen activator; PAGE, polyacrylamide gel electrophoresis; PMA, phorbol myristate acetate; SDS, sodium dodecyl sulfate; TA, tissue activator; Uk, urokinase. J. ExP. MED. © The Rockefeller University Press • 0022-1007/84/06/1653/16 $I.00 1653 Volume 159 June 1984 1653-1668 on June 9, 2013jem.rupress.orgDownloaded from