scholarly journals alpha-2 Macroglobulin receptor/Ldl receptor-related protein(Lrp)-dependent internalization of the urokinase receptor.

1995 ◽  
Vol 131 (6) ◽  
pp. 1609-1622 ◽  
Author(s):  
M Conese ◽  
A Nykjaer ◽  
C M Petersen ◽  
O Cremona ◽  
R Pardi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Confocal Microscopy ◽  
Cell Surface ◽  
Cell Line ◽  
Plasminogen Activator ◽  
Protein Synthesis Inhibitor ◽  
Related Protein ◽  
Urokinase Plasminogen Activator Receptor ◽  
Immuno Electron Microscopy ◽  
Pai 1

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA). On the contrary, uPAR-bound complexes of uPA with its serpin inhibitors PAI-1 (plasminogen activator inhibitor type-1) or PN-1 (protease nexin-1) are readily internalized in several cell types. Here we address the question whether uPAR is internalized as well upon binding of uPA-serpin complexes. Both LB6 clone 19 cells, a mouse cell line transfected with the human uPAR cDNA, and the human U937 monocytic cell line, express in addition to uPAR also the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP/alpha 2-MR) which is required to internalize uPAR-bound uPA-PAI-1 and uPA-PN-1 complexes. Downregulation of cell surface uPAR molecules in U937 cells was detected by cytofluorimetric analysis after uPA-PAI-1 and uPA-PN-1 incubation for 30 min at 37 degrees C; this effect was blocked by preincubation with the ligand of LRP/alpha 2-MR, RAP (LRP/alpha 2-MR-associated protein), known to block the binding of the uPA complexes to LRP/alpha 2-. MR. Downregulation correlated in time with the intracellular appearance of uPAR as assessed by confocal microscopy and immuno-electron microscopy. After 30 min incubation with uPA-PAI-1 or uPA-PN-1 (but not with free uPA), confocal microscopy showed that uPAR staining in permeabilized LB6 clone 19 cells moved from a mostly surface associated to a largely perinuclear position. This effect was inhibited by the LRP/alpha 2-MR RAP. Perinuclear uPAR did not represent newly synthesized nor a preexisting intracellular pool of uPAR, since this fluorescence pattern was not modified by treatment with the protein synthesis inhibitor cycloheximide, and since in LB6 clone 19 cells all of uPAR was expressed on the cell surface. Immuno-electron microscopy confirmed the plasma membrane to intracellular translocation of uPAR, and its dependence on LRP/alpha 2-MR in LB6 clone 19 cells only after binding to the uPA-PAI-1 complex. After 30 min incubation at 37 degrees C with uPA-PAI-1, 93% of the specific immunogold particles were present in cytoplasmic vacuoles vs 17.6% in the case of DFP-uPA. We conclude therefore that in the process of uPA-serpin internalization, uPAR itself is internalized, and that internalization requires the LRP/alpha 2-MR.

scholarly journals Caveolin-1 is incorporated into mature respiratory syncytial virus particles during virus assembly on the surface of virus-infected cells

2002 ◽  
Vol 83 (3) ◽  
pp. 611-621 ◽  
Author(s):  
Gaie Brown ◽  
James Aitken ◽  
Helen W. McL. Rixon ◽  
Richard J. Sugrue
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Respiratory Syncytial Virus ◽  
Confocal Microscopy ◽  
Cell Surface ◽  
Filamentous Form ◽  
Caveolin 1 ◽  
Immuno Electron Microscopy ◽  
Infected Cells ◽  
Syncytial Virus

We have employed immunofluorescence microscopy and transmission electron microscopy to examine the assembly and maturation of respiratory syncytial virus (RSV) in the Vero cell line C1008. RSV matures at the apical cell surface in a filamentous form that extends from the plasma membrane. We observed that inclusion bodies containing viral ribonucleoprotein (RNP) cores predominantly appeared immediately below the plasma membrane, from where RSV filaments form during maturation at the cell surface. A comparison of mock-infected and RSV-infected cells by confocal microscopy revealed a significant change in the pattern of caveolin-1 (cav-1) fluorescence staining. Analysis by immuno-electron microscopy showed that RSV filaments formed in close proximity to cav-1 clusters at the cell surface membrane. In addition, immuno-electron microscopy showed that cav-1 was closely associated with early budding RSV. Further analysis by confocal microscopy showed that cav-1 was subsequently incorporated into the envelope of RSV filaments maturing on the host cell membrane, but was not associated with other virus structures such as the viral RNPs. Although cav-1 was incorporated into the mature virus, it was localized in clusters rather than being uniformly distributed along the length of the viral filaments. Furthermore, when RSV particles in the tissue culture medium from infected cells were examined by immuno-negative staining, the presence of cav-1 on the viral envelope was clearly demonstrated. Collectively, these findings show that cav-1 is incorporated into the envelope of mature RSV particles during egress.

SuPAR, an emerging biomarker in kidney and inflammatory diseases

2018 ◽  
Vol 94 (1115) ◽  
pp. 517-524 ◽  
Author(s):  
Lamiaa Hamie ◽  
Georges Daoud ◽  
Georges Nemer ◽  
Tarek Nammour ◽  
Alissar El Chediak ◽  
...  
Keyword(s):  
Cell Surface ◽  
Plasminogen Activator ◽  
Large Scale ◽  
Inflammatory Diseases ◽  
Cell Surface Receptor ◽  
Urokinase Plasminogen Activator Receptor ◽  
Surface Receptor ◽  
Treatment Plans ◽  
Plasminogen Activator Receptor ◽  
Clinical Conditions

Soluble urokinase plasminogen activator receptor (suPAR) is a circulating form of a physiological and pathophysiological important cell surface receptor, implicated in inflammation. Recent studies showed that suPAR is a promising biomarker, useful for diagnosis, assessment and prognosis of several diseases. This review summarises the majority of preliminary studies and analyses the significance and the clinical application of suPAR in various clinical conditions. SuPAR seems to have a significant value in the diagnosis as well as prognosis of many diseases; nonetheless, it merits large-scale studies to set cut-off values that help physicians in following up their patients and accordingly tailor their treatment plans.

scholarly journals Dysfunction of annexin A2 contributes to hyperglycaemia-induced loss of human endothelial cell surface fibrinolytic activity

2013 ◽  
Vol 109 (06) ◽  
pp. 1070-1078 ◽  
Author(s):  
Zhanyang Yu ◽  
Xiang Fan ◽  
Ning Liu ◽  
Min Yan ◽  
Zhong Chen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Protein Expression ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Annexin A2 ◽  
Tissue Type ◽  
Endothelial Cell Surface ◽  
Pai 1

SummaryHyperglycaemia impairs fibrinolytic activity on the surface of endothelial cells, but the underlying mechanisms are not fully understood. In this study, we tested the hypothesis that hyperglycaemia causes dysfunction of the endothelial membrane protein annexin A2, thereby leading to an overall reduction of fibrinolytic activity. Hyperglycaemia for 7 days significantly reduced cell surface fibrinolytic activity in human brain microvascular endothelial cells (HBMEC). Hyperglycaemia also decreased tissue type plasminogen activator (t-PA), plasminogen, and annexin A2 mRNA and protein expression, while increasing plasminogen activator inhibitor-1 (PAI-1). No changes in p11 mRNA or protein expression were detected. Hyperglycaemia significantly increased AGE-modified forms of total cellular and membrane annexin A2. The hyperglycemia-associated reduction in fibrinolytic activity was fully restored upon incubation with recombinant annexin A2 (rA2), but not AGE-modified annexin A2 or exogenous t-PA. Hyperglycaemia decreased t-PA, upregulated PAI-1 and induced AGE-related disruption of annexin A2 function, all of which contributed to the overall reduction in endothelial cell surface fibrinolytic activity. Further investigations to elucidate the underlying molecular mechanisms and pathophysiological implications of A2 derivatisation might ultimately lead to a better understanding of mechanisms of impaired vascular fibrinolysis, and to development of new interventional strategies for the thrombotic vascular complications in diabetes.

scholarly journals Plasmodium Niemann-Pick type C1-related protein is a druggable target required for parasite membrane homeostasis

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Eva S Istvan ◽  
Sudipta Das ◽  
Suyash Bhatnagar ◽  
Josh R Beck ◽  
Edward Owen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasmodium Falciparum ◽  
Plasma Membrane ◽  
Drug Target ◽  
Editorial Note ◽  
Editorial Process ◽  
Related Protein ◽  
Immuno Electron Microscopy ◽  
Increased Sensitivity ◽  
Niemann Pick

Plasmodium parasites possess a protein with homology to Niemann-Pick Type C1 proteins (Niemann-Pick Type C1-Related protein, NCR1). We isolated parasites with resistance-conferring mutations in Plasmodium falciparum NCR1 (PfNCR1) during selections with three diverse small-molecule antimalarial compounds and show that the mutations are causative for compound resistance. PfNCR1 protein knockdown results in severely attenuated growth and confers hypersensitivity to the compounds. Compound treatment or protein knockdown leads to increased sensitivity of the parasite plasma membrane (PPM) to the amphipathic glycoside saponin and engenders digestive vacuoles (DVs) that are small and malformed. Immuno-electron microscopy and split-GFP experiments localize PfNCR1 to the PPM. Our experiments show that PfNCR1 activity is critically important for the composition of the PPM and is required for DV biogenesis, suggesting PfNCR1 as a novel antimalarial drug target.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

Detection of Plasminogen Activator Inhibitor-1 (PAI-1) mRNA in Human Megakaryocytes by In Situ Hybridization

1994 ◽  
Vol 72 (06) ◽  
pp. 931-936 ◽  
Author(s):  
M C Alessi ◽  
N Chomiki ◽  
R Berthier ◽  
A Schweitzer ◽  
C Fossat ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Situ Hybridization ◽  
Cell Line ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Leukemia Cell Line ◽  
Allogeneic Bone ◽  
Activator Inhibitor ◽  
Pai 1

SummaryPlatelets have been described to contain a large proportion of the circulating plasminogen activator inhibitor type 1 (PAI-1) which is released on platelet activation. This protein could be taken up by platelets from the plasma or synthesized by megakaryocytes (MKs). Recently, PAI-1 mRNA has been detected in a human megakaryoblastic leukemia cell line (MEG-01) by the polymerase chain reaction (PCR). However, a direct demonstration of its presence in normal human MKs is lacking.In order to prove directly the megakaryocytic origin of platelet PAI-1, the MEG-01 cell line, human bone marrow enriched in MKs, and bone marrow smears from allogeneic bone marrow transplantation donors were investigated for the presence of PAI-1 mRNA using in situ hybridization (ISH). Specimens of bone marrow were first stained with May-Grunwald Giemsa (MGG) for cell identification according to their morphology. Subsequently, the same slides were used for ISH. PAI-1 mRNA was clearly demonstrated in the MEG-01 cell line and in MKs, and its presence correlated with the detection of PAI-1 antigen by immunocytochemistry. PAI-1 mRNA was also detected in morphologically characterized mature granulocytes of marrow samples.

Insulin Stimulates the Synthesis of Plasminogen Activator Inhibitor 1 by the Human Hepatocellular Cell Line Hep G2

1988 ◽  
Vol 60 (03) ◽  
pp. 491-494 ◽  
Author(s):  
M C Alessi ◽  
I Juhan-Vague ◽  
T Kooistra ◽  
P J Declerck ◽  
D Collen
Keyword(s):  
Endothelial Cells ◽  
Cell Line ◽  
Plasminogen Activator ◽  
Plasma Insulin ◽  
Plasminogen Activator Inhibitor ◽  
Fold Increase ◽  
Hep G2 ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

SummarySecretion of plasminogen activator inhibitor 1 (PAI-1) by cultures of human umbilical vein endothelial cells and human hepatocellular cell line Hep G2 was evaluated after insulin stimulation. The secretion of PAI-1 antigen and activity was measured in the conditioned medium and the cellular extracts after incubation of confluent cultures with 1% serum medium for 24 hours.Insulin induced a dose dependent increase of the PAI-1 secretion by Hep G2 cell line. At 10-8 M a two fold increase of PAI-1 antigen and activity were observed whereas a2 antiplasmin and fibrinogen were not significantly modified. No effect of insulin was observed on PAI-1 antigen and PAI activity production by human endothelial cells whereas endotoxin resulted in a two fold increase in PAI-1 secretion. In recent clinical studies we have demonstrated that the level of plasma insulin correlated with that of PAI-1. Thus we hypothesize that hepatocytes represent a physiological source of plasma PAI-1 which is modulated by plasma insulin level.

scholarly journals P03.17 uPA-PAI-1 heteromers promote advanced stages of breast cancer by attracting pro-tumorigenic neutrophils

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A29.2-A29
Author(s):  
B Uhl ◽  
L Mittmann ◽  
J Dominik ◽  
J Schaubächer ◽  
C Braun ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Flow Cytometry ◽  
Cell Line ◽  
Plasminogen Activator ◽  
Channel Flow ◽  
Plasminogen Activator Inhibitor 1 ◽  
Primary Mouse ◽  
Advanced Stages ◽  
Pai 1

BackgroundHigh tumor levels of urokinase-type plasminogen activator (uPA)-plasminogen activator inhibitor-1 (PAI-1) heteromers independently predict poor survival in early breast cancer. The pathogenetic role of this protein complex, however, remains largely obscure.Materials and MethodsNeutrophil trafficking was analyzed in orthotopic (multi-channel flow cytometry) and heterotopic (ear; multi-channel in vivo microscopy) mouse models of 4T1 breast cancer, in a mouse peritonitis assay (multi-channel flow cytometry), as well as in the mouse cremaster muscle (multi-channel in vivo microscopy). Cytokine expression in tumors was determined by multiplex ELISA. Phenotypic and functional properties of primary mouse neutrophils, microvascular endothelial cells (cell line bEnd.3), macrophages (cell line RAW 264.7), and breast cancer cells (cell line 4T1) were characterized in different in vitro assays. uPA/PAI-1 expression and neutrophil infiltration in human breast cancer samples were assessed by RNA sequencing, immunhistochemistry, and ELISA.ResultsHere, we demonstrate that uPA-PAI-1 heteromerization multiplies the potential of the single proteins to attract pro-tumorigenic neutrophils. To this end, tumor-released uPA-PAI-1 utilizes very low density lipoprotein receptor and ERK mitogen-activated protein kinases to initiate a pro-inflammatory program in peritumoral macrophages. This promotes neutrophil trafficking to cancerous lesions and primes these immune cells towards a pro-tumorigenic phenotype, thus supporting tumor growth and metastasis. Blockade of uPA-PAI-1 heteromerization by a novel inhibitor effectively interfered with these events and prevented tumor progression.ConclusionsOur findings identify an already therapeutically targetable interplay between hemostasis and innate immunity that drives advanced stages of breast cancer. As a personalized immunotherapeutic strategy, blockade of uPA-PAI-1 heteromerization might be particularly beneficial for patients with highly aggressive uPA-PAI-1hightumors.This study was supported by Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich (SFB) 914.Disclosure InformationB. Uhl: None. L. Mittmann: None. J. Dominik: None. J. Schaubächer: None. C. Braun: None. R. Pick: None. M. Canis: None. S. Kanse: None. W. Weichert: None. M. Sperandio: None. K. Lauber: None. F. Krombach: None. C.A. Reichel: None.

scholarly journals Regulation of urokinase plasminogen activator gene transcription in the RAW264 murine macrophage cell line by macrophage colony-stimulating factor (CSF-1) is dependent upon the level of cell-surface receptor

2000 ◽  
Vol 347 (1) ◽  
pp. 313-320 ◽  
Author(s):  
Lindsay F. FOWLES ◽  
Katryn J. STACEY ◽  
Denese MARKS ◽  
John A. HAMILTON ◽  
David A. HUME
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Plasminogen Activator ◽  
Murine Macrophage ◽  
Colony Stimulating Factor ◽  
Macrophage Cell Line ◽  
Macrophage Cell ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Macrophage colony-stimulating factor (CSF-1) binds to a receptor (CSF-1R) encoded by the c-fms proto-oncogene and activates transcription of the urokinase plasminogen activator (uPA) gene in murine bone-marrow-derived macrophages. This article demonstrates that the murine macrophage cell line RAW264 responds to CSF-1 with inducible phosphorylation of cytoplasmic proteins on tyrosine residues but fails to induce transcription of uPA. The defect was correlated with a selective failure to maintain CSF-1Rs on the cell surface, whereas all RAW264 cells contained abundant CSF-1Rs within the presumptive Golgi/endoplasmic reticulum compartment. Transfection with a CSF-1R expression plasmid permitted CSF-1-dependent activation of the signalling pathway targeting an Ets/AP1 (activator protein 1) element in the uPA promoter that has been shown previously to be a target of oncogenic ras and protein kinase C pathways. Mutation of the expressed CSF-1R at either Y807 or Y559, sites of receptor tyrosine phosphorylation implicated in signal transduction, reduced but did not abolish uPA promoter activation by CSF-1. Activation by mutant CSF-1R plasmids was additive; there was no evidence of mutual complementation. The results indicate that maintenance of elevated uPA transcription by CSF-1 requires new receptors emerging continuously on the cell surface. Parallel, partly redundant, signalling pathways arising from phosphorylated tyrosines on the CSF-1R activate multiple cis-acting elements on the complex uPA promoter.

Sign in / Sign up

Export Citation Format

Share Document