Activation of hepatocyte growth factor by urokinase-type plasminogen activator is ionic strength-dependent.
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ABSTRACT:
SUBMITTER: Mars WM
PROVIDER: S-EPMC1184584 | biostudies-other | 2005 Aug
REPOSITORIES: biostudies-other
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Project description:Plasminogen activator inhibitor-1 (PAI-1) is known to protect mice against cardiac fibrosis. It has been speculated that PAI-1 may regulate cardiac fibrosis by inactivating urokinase-type plasminogen activator (uPA) and ultimately plasmin (Pm) generation. However, the in vivo role of PAI-1 in inactivating uPA and limiting the generation of Pm during cardiac fibrosis remains to be established. The objective of this study was to determine if the cardioprotective effect of PAI-1 is mediated through its ability to directly regulate urokinase -mediated activation of plasminogen (Pg). An Angiotensin II (AngII)-aldosterone (Ald) infusion mouse model of hypertension was utilised in this study. Four weeks after AngII-Ald infusion, PAI-1-deficient (PAI-1-/-) mice developed severe cardiac fibrosis. However, a marked reduction in cardiac fibrosis was observed in PAI-1-/-/uPA-/- double knockout mice that was associated with reduced inflammation, lower expression levels of TGF-β and proteases associated with tissue remodeling, and diminished Smad2 signaling. Moreover, total ablation of cardiac fibrosis was observed in PAI-1-/- mice that express inactive plasmin (Pm) but normal levels of zymogen Pg (PAI-1-/-/PgS743A/S743A). Our findings indicate that PAI-1 protects mice from hypertension-induced cardiac fibrosis by inhibiting the generation of active Pm.
| S-EPMC5428408 | biostudies-literature
Project description:Urokinase-type plasminogen activator (uPA) participates in diverse (patho)physiological processes through intracellular signaling events that affect cell adhesion, migration, and proliferation, although the mechanisms by which these occur are only partially understood. Here we report that upon cell binding and internalization, single-chain uPA (scuPA) translocates to the nucleus within minutes. Nuclear translocation does not involve proteolytic activation or degradation of scuPA. Neither the urokinase receptor (uPAR) nor the low-density lipoprotein-related receptor (LRP) is required for nuclear targeting. Rather, translocation involves the binding of scuPA to the nucleocytoplasmic shuttle protein nucleolin through a region containing the kringle domain. RNA interference and mutational analysis demonstrate that nucleolin is required for the nuclear transport of scuPA. Furthermore, nucleolin is required for the induction smooth muscle alpha-actin (alpha-SMA) by scuPA. These data reveal a novel pathway by which uPA is rapidly translocated to the nucleus where it might participate in regulating gene expression.
| S-EPMC2435680 | biostudies-literature
Project description:uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human ?1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of ?1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from ?1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.
| S-EPMC4591041 | biostudies-literature
Project description:RationalePhagocytosis of apoptotic cells, also called efferocytosis, plays an essential role in the resolution of inflammation. Urokinase-type plasminogen activator (uPA) is a multifunctional protein that has been implicated in inflammatory conditions, including pneumonia and severe infection, which are often accompanied by the development of acute lung injury. However, the role of uPA in modulating efferocytosis of apoptotic neutrophils has not been defined.ObjectivesTo characterize the role of uPA in regulation of efferocytosis and to delineate the underlying mechanisms involved in this process.MethodsIn vitro and in vivo phagocytosis, immunoprecipitation, and Western blotting assays.Measurements and main resultsThe phagocytosis of apoptotic neutrophils by macrophages was significantly inhibited by uPA. Mutant uPA lacking the growth factor domain and catalytically inactive uPA had similar inhibitory effects on efferocytosis, as did wild-type uPA. In contrast, absence of the kringle domain abrogated the ability of uPA to diminish efferocytosis. Both the α(V)β₃ integrin and vitronectin seemed to be involved in the inhibition of efferocytosis by uPA. Incubation of macrophages with uPA also diminished activation of the small GTPase Rac-1, which normally occurs during ingestion of apoptotic neutrophils. Under in vivo conditions in the lungs, uPA decreased the uptake of apoptotic neutrophils by alveolar macrophages.ConclusionsOur data demonstrate a novel role for uPA in which it is able to diminish the uptake of apoptotic neutrophils by macrophages under both in vitro and in vivo conditions.
| S-EPMC3029937 | biostudies-literature
Project description:Plasminogen activator inhibitor-1 (PAI-1), together with its physiological target urokinase-type plasminogen activator (uPA), plays a pivotal role in fibrinolysis, cell migration, and tissue remodeling and is currently recognized as being among the most extensively validated biological prognostic factors in several cancer types. PAI-1 specifically and rapidly inhibits uPA and tissue-type PA (tPA). Despite extensive structural/functional studies on these two reactions, the underlying structural mechanism has remained unknown due to the technical difficulties of obtaining the relevant structures. Here, we report a strategy to generate a PAI-1·uPA(S195A) Michaelis complex and present its crystal structure at 2.3-? resolution. In this structure, the PAI-1 reactive center loop serves as a bait to attract uPA onto the top of the PAI-1 molecule. The P4-P3' residues of the reactive center loop interact extensively with the uPA catalytic site, accounting for about two-thirds of the total contact area. Besides the active site, almost all uPA exosite loops, including the 37-, 60-, 97-, 147-, and 217-loops, are involved in the interaction with PAI-1. The uPA 37-loop makes an extensive interaction with PAI-1 ?-sheet B, and the 147-loop directly contacts PAI-1 ?-sheet C. Both loops are important for initial Michaelis complex formation. This study lays down a foundation for understanding the specificity of PAI-1 for uPA and tPA and provides a structural basis for further functional studies.
| S-EPMC3044959 | biostudies-literature
Project description:BackgroundUrokinase-type plasminogen activator (uPA) plays a major role in extracellular proteolytic events associated with tumor cell growth, migration and angiogenesis. Consequently, uPA is an attractive target for the development of small molecule active site inhibitors. Most of the recent drug development programs aimed at nonpeptidic inhibitors targeted at uPA have focused on arginino mimetics containing amidine or guanidine functional groups attached to aromatic or heterocyclic scaffolds. There is a general problem of limited bioavailability of these charged inhibitors. In the present study, uPA inhibitors were designed on an isocoumarin scaffold containing uncharged substituents.Results4-Chloro-3-alkoxyisocoumarins were synthesized in which the 3-alkoxy group contained a terminal bromine; these were compared with similar inhibitors that contained a charged terminal functional group. Additional variations included functional groups attached to the seven position of the isocoumarin scaffold. N- [3-(3-Bromopropoxy)-4-chloro-1-oxo-1H-isochromen-7-yl]benzamide was identified as an uncharged lead inhibitor of uPA, Ki = 0.034 microM. Molecular modeling of human uPA with these uncharged inhibitors suggests that the bromine occupies the same position as positively charged arginino mimetic groups.ConclusionThis study demonstrates that potent uncharged inhibitors of uPA can be developed based upon the isocoumarin scaffold. A tethered bromine in the three position and an aromatic group in the seven position are important contributors to binding. Although the aim was to develop compounds that act as mechanism-based inactivators, these inhibitors are competitive reversible inhibitors.
| S-EPMC1479381 | biostudies-literature
Project description:Data from clinical studies, cell culture, and animal models implicate the urokinase (uPA)/Plasminogen (Plg) system in the development of atherosclerosis and aneurysms. However, the mechanisms through which uPA/Plg stimulate these diseases are not yet defined. We used genetically modified, atherosclerosis-prone mice, including mice with macrophage-specific uPA overexpression to clarify mechanisms of uPA/Plg-accelerated atherosclerosis and aneurysm formation. Microarray studies were performed to identify potential mediators of uPA-accelerated atherosclerosis. These studies identified S100A8 and S100A9 mRNA as the most highly upregulated transcripts in uPA-overexpressing macrophages; upregulation of S100A9 protein in uPA-overexpressing macrophages was confirmed by Western blotting. S100A8/A9, which are atherogenic in mice and are expressed in human atherosclerotic plaques, are also upregulated in aortae of mice with uPA-overexpressing macrophages, and macrophage S100A9 mRNA is upregulated by exposure of wild-type macrophages to medium from uPA-overexpressing macrophages. Bioinformatics analysis of the microarray data suggest significant effects of uPA overexpression on cell migration and cell-matrix interactions. Our results confirm—in a second animal model—that macrophage-expressed uPA stimulates atherosclerosis and aortic dilation. They also implicate specific pathways in uPA/Plg-accelerated atherosclerosis and aneurysmal disease.
2011-05-03 | GSE29028 | GEO
Project description:The soluble urokinase-type plasminogen activator receptor (suPAR) and the urokinase-type plasminogen activator receptor (uPAR) have been proposed as useful biomarkers of tumor progression. Recently, suPAR was associated with chemoresistance in lung cancer. However, its clinical significance in leukemia has not previously been investigated. The present study examined the plasma levels of suPAR and the expression of the uPAR on bone marrow (BM) cells in 86 patients with leukemia at diagnosis prior to chemotherapy and 26 normal subjects (control group). The plasma suPAR levels were measured using ELISA, whilst uPAR expression was assayed by flow cytometry analysis. In addition, cell surface uPAR expression on K562 and multidrug-resistant K562/ADM cell lines was studied by western blotting. On admission and follow-up, the levels of suPAR in patients with leukemia were significantly increased compared with controls. Systemic levels of suPAR were strongly associated with the numbers of white blood cells. A case was defined as uPAR-positive (uPAR+) if >20% of the gated cells expressed uPAR. In comparison with 26 healthy BM samples that were negative for uPAR expression, 48 (55.8%) of the 86 leukemia patients were uPAR+. uPAR expression on the cell surface of multidrug-resistant K562/ADM cells was increased compared with that on K562 cells. In conclusion, plasma suPAR expression may be a useful marker for subtype classification of patients with leukemia and cell surface uPAR may be associated with resistance to chemotherapy or disease progression.
| S-EPMC5494859 | biostudies-other
Project description:Small-cell lung carcinoma releases progalanin. The released progalanin is activated via a nonclassical processing pathway, being processed into an active form of galanin (1-20) by plasmin in extracellular components. Plasmin is produced from plasminogen activators. To clarify the regulation of progalanin via plasminogen activation by urokinase and tissue-plasminogen activator (t-PA), we investigated the regulation mechanism for urokinase and t-PA expression and their effect on galanin activation. Additionally, we studied the effect of activated galanin on angiogenesis. To determine the effect of cell density, we measured the expression levels of urokinase and t-PA using real-time PCR and plasminogen/gelatin zymography in a cell culture. The urokinase expression increased under both high cell density and presence of cell membrane fractions. However, urokinase increments induced by conditioned medium were low. These results indicate that expression of plasminogen activators is regulated by cell membrane factors. We used tumor-bearing mice to clarify the expression of plasminogen activators and galanin activation. Real-time PCR showed that urokinase was substantially higher in the central parts of tumors compared to the periphery, and this was confirmed by plasminogen/gelatin zymography. To evaluate the biological effect of plasminogen activators on tumor growth, we used tranexamic acid as a plasminogen inhibitor. Tranexamic acid decreased galanin (1-20) and the hemoglobin content of tumors and suppressed tumor growth. Additionally, galanin had no effect on the hemoglobin content of tumors derived from cells lacking GALR2. These results demonstrate the regulation of urokinase expression in tumors through progalanin activation in extracellular compartments, and confirm that galanin plays a role in angiogenesis.
| S-EPMC5666387 | biostudies-literature
Project description:Eosinophils migrate from the vascular circulation to the inflamed airways during asthma exacerbations. While the mechanism(s) of this process is not known, the expression of urokinase-type plasminogen activator receptor (uPAR) has been found to modulate neutrophil adhesion and migration to inflammatory sites. We hypothesized that increased expression of uPAR and its ligand, uPA, enhance eosinophil adhesion in patients with asthma. Patients with allergic asthma underwent segmental bronchoprovocation with allergen; 48 h later, peripheral blood and airway (from bronchoalveolar lavage fluid) eosinophils were isolated. uPA and uPAR protein expression were measured by flow cytometry and Western blot; mRNA was quantified by real-time PCR. Eosinophil adhesion to intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 was assessed by eosinophil peroxidase activity. Airway eosinophils expressed significantly more uPA and uPAR protein and uPAR mRNA than peripheral blood eosinophils. Removal of cell-bound uPA and/or addition of exogenous uPA had no effect on blood eosinophil adhesion to ICAM-1 or VCAM-1. In contrast, exogenous uPA stimulated ICAM and VCAM adhesion of airway eosinophils. N-formyl-methionyl-leucyl-phenylalanine-activated airway eosinophil adherence to VCAM-1 and ICAM-1 (VCAM-1, 52.8 +/- 4.7%; ICAM-1, 49.2 +/- 5.3%) was increased over blood eosinophil adhesion (VCAM-1, 38.4 +/- 3.6%; ICAM-1, 27.7 +/- 4.9%; P < 0.05). Removal of cell-bound uPA from airway eosinophils decreased adhesion to blood cell levels; reintroduction of exogenous uPA completely restored adhesion levels. These data suggest that constitutive uPA primes, and exogenous uPA can activate, airway eosinophil adhesion following segmental allergen challenge and that increased uPA expression may be a mechanism of increased eosinophil infiltration and function in asthma.
| S-EPMC2643268 | biostudies-literature