scholarly journals Urokinase-type plasminogen activator-mediated crosstalk between N-cadherin and β-Catenin promotes wound healing

2021 ◽  
Author(s):  
Ariel Diaz ◽  
Cynthia Martin-Jimenez ◽  
Yang Xu ◽  
Paola Merino ◽  
Yena Woo ◽  
...  
Keyword(s):  
Wound Healing ◽  
Plasminogen Activator ◽  
Nuclear Translocation ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Serine Proteinase ◽  
Cytoplasmic Tail ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Density Lipoprotein Receptor

Urokinase-type plasminogen activator (uPA) is a serine proteinase that in the central nervous system induces astrocytic activation. β-Catenin is a protein that links the cytoplasmic tail of cadherins to the actin cytoskeleton, thus securing the formation of cadherin-mediated cell adhesion complexes. Disruption of cell-cell contacts leads to the detachment of β-Catenin from cadherins, which is then degraded by the proteasome following its phosphorylation by GSK3β. Here we show that astrocytes release uPA following a scratch injury, and that this uPA promotes wound healing via a plasminogen-independent mechanism. We found that uPA induces the detachment of β-Catenin from the cytoplasmic tail of N-Cadherin (NCAD) by triggering its phosphorylation at Tyr654. Surprisingly, this is not followed by degradation of β-Catenin because uPA also induces the phosphorylation of the low density lipoprotein receptor-related protein 6 (LRP6) at Ser1490, which then blocks the kinase activity of GSK3β. Our work indicates that the ensuing cytoplasmic accumulation of β-Catenin is followed by its nuclear translocation and β-Catenin-triggered transcription of uPA's receptor (Plaur), which in turn is required for uPA to induce astrocytic wound healing.

The Role of the Low-Density Lipoprotein Receptor-Related Protein (LRP) in the Plasma Clearance and Liver Uptake of Recombinant Single-chain Urokinase-type Plasminogen Activator in Rats

1997 ◽  
Vol 77 (04) ◽  
pp. 710-717 ◽  
Author(s):  
Marieke E van der Kaaden ◽  
Dingeman C Rijken ◽  
J Kar Kruijt ◽  
Theo J C van Berkel ◽  
Johan Kuiper
Keyword(s):  
Plasminogen Activator ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Single Chain ◽  
Liver Uptake ◽  
Type Plasminogen Activator ◽  
Parenchymal Liver ◽  
Urokinase Type Plasminogen Activator ◽  
Density Lipoprotein Receptor

SummaryUrokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, recombinant single-chain u-PA (rscu-PA) expressed in E.coli is recognized by the Low-Density Lipoprotein Receptor-related Protein (LRP) on rat parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA in rats. A preinjection of the LRP inhibitor GST-RAP reduced the maximal liver uptake of 125I-rscu-PA at 5 min after injection from 50 to 30% of the injected dose and decreased the clearance of rscu-PA from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10% of the liver uptake, respectively. The reduction in liver uptake of rscu-PA by the preinjection of GST-RAP was caused by a 91 % and 62% reduction in the uptake by parenchymal and Kupffer cells, respectively. In order to investigate the part of rscu-PA that accounted for the interaction with LRP, experiments were performed with a mutant of rscu-PA lacking residues 11-135 (= deltal25- rscu-PA). Deletion of residues 11-135 resulted in a 80% reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for respectively 60, 33 and 7% of the liver uptake of 125I-deltal25-rscu-PA. Preinjection of GST-RAP completely reduced the liver uptake of delta 125-rscu-PA and reduced its clearance to 0.79 ml/min. Treatment of isolated Kupffer cells with PI-PLC reduced the binding of rscu-PA by 40%, suggesting the involvement of the urokinase-type Plasminogen Activator Receptor (u-PAR) in the recognition of rscu-PA. Our results demonstrate that in vivo LRP is responsible for more than 90% of the parenchymal liver cell mediated uptake of rscu-PA and for 60% of the Kupffer cell interaction. It is also suggested that u-PAR is involved in the Kupffer cell recognition of rscu-PA.

scholarly journals Fibulin-5 binds urokinase-type plasminogen activator and mediates urokinase-stimulated β1-integrin-dependent cell migration

2012 ◽  
Vol 443 (2) ◽  
pp. 491-503 ◽  
Author(s):  
Alexander Kapustin ◽  
Victoria Stepanova ◽  
Natalia Aniol ◽  
Douglas B. Cines ◽  
Alexei Poliakov ◽  
...  
Keyword(s):  
Cell Migration ◽  
Plasminogen Activator ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Β1 Integrin ◽  
Upa Receptor ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Density Lipoprotein Receptor ◽  
Wild Type Mefs

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 (FblnRGE/RGE MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human β1-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 β1-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 β1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.

Analysis of a two-domain binding site for the urokinase-type plasminogen activator–plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein

2001 ◽  
Vol 357 (1) ◽  
pp. 289-296 ◽  
Author(s):  
Olav M. ANDERSEN ◽  
Helle H. PETERSEN ◽  
Christian JACOBSEN ◽  
S⊘ren K. MOESTRUP ◽  
Michael ETZERODT ◽  
...  
Keyword(s):  
Binding Site ◽  
Plasminogen Activator ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Plasminogen Activator Inhibitor ◽  
Plasminogen Activator Inhibitor 1 ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Density Lipoprotein Receptor ◽  
Pai 1

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each ≈ 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca2+ cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. α2-macroglobulin (α2M)–proteinase complexes, lipoprotein-containing particles and serine proteinase–inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA–PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3–CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA–PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp958,CR5, Asp999,CR6, Trp953,CR5 and Trp994,CR6), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP)–the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA–PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA–PAI-1 as well as for the binding of RAP.

Cellular Degradation of Free and Inhibitor-bound Tissue-type Plasminogen Activator

2000 ◽  
Vol 83 (02) ◽  
pp. 290-296 ◽  
Author(s):  
Chantal Camani ◽  
Olivier Gavin ◽  
Egbert Kruithof
Keyword(s):  
Plasminogen Activator ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Density Lipoprotein Receptor ◽  
Activator Inhibitor Type ◽  
Pai 1

SummaryThe low density lipoprotein receptor-related protein (LRP) is a multiligand clearance receptor that removes free tissue-type plasminogen activator (t-PA) or complexes of t-PA with plasminogen activator inhibitor type 1 (PAI-1) from the blood circulation or the pericellular space. Co-receptors are essential for LRP-mediated clearance of several ligands (e.g. glycosaminoglycans for thrombin/protease nexin and lipoprotein lipase, and the urokinase receptor for urokinase/PAI-1 complexes). The present study was undertaken to investigate whether LRP-mediated t-PA clearance requires a co-receptor as well.In five cell lines from different organs and species degradation of t-PA and t-PA/PAI-1 was mediated by LRP (or LRP-like receptors). No degradation of t-PA and t-PA/PAI-1 occurred in THP-1 or U-937 human monocyte-like cells, despite the presence of functional LRP. As glycosaminoglycans can bind t-PA and PAI-1 we investigated whether they are involved in t-PA/PAI-1 degradation. Pre-treatment of COS cells or HT1080 cells with chlorate, an inhibitor of glycosaminoglycan sulfation, did not decrease t-PA/PAI-1 degradation. Furthermore, CHO cells genetically deficient in glycosaminoglycans efficiently degraded t-PA/PAI-1. Thus it is unlikely that glycosaminoglycans are co-receptors for degradation of t-PA or t-PA/PAI-1.This study indicates that THP-1 and U-937 cells lack a critical component (co-receptor?) for the LRP-mediated degradation of t-PA. Abbreviations: LRP, low density lipoprotein receptor-related protein; PAI-1, plasminogen activator inhibitor type 1; RAP, receptor-associated protein; t-PA, tissue-type plasminogen activator; u-PA, urokinase; u-PAR, urokinase receptor.

scholarly journals Nuclear translocation of urokinase-type plasminogen activator

Blood ◽  
2008 ◽  
Vol 112 (1) ◽  
pp. 100-110 ◽  
Author(s):  
Victoria Stepanova ◽  
Tatiana Lebedeva ◽  
Alice Kuo ◽  
Serge Yarovoi ◽  
Sergei Tkachuk ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Intracellular Signaling ◽  
Nuclear Translocation ◽  
Mutational Analysis ◽  
Density Lipoprotein ◽  
Single Chain ◽  
Nuclear Targeting ◽  
Proteolytic Activation ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Abstract 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 α-actin (α-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.

Native and Non-glycosylated Recombinant Single-chain Urokinase-type Plasminogen Activator Are Recognized by Different Receptor Systems on Rat Parenchymal Liver Cells

1995 ◽  
Vol 74 (02) ◽  
pp. 722-729 ◽  
Author(s):  
Marieke E van der Kaaden ◽  
Dingeman C Rijken ◽  
Eleonore Groeneveld ◽  
Theo J C van Berkel ◽  
Johan Kuiper
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Liver Cells ◽  
Single Chain ◽  
Type Plasminogen Activator ◽  
Parenchymal Liver ◽  
Urokinase Type Plasminogen Activator ◽  
Density Lipoprotein Receptor ◽  
Parenchymal Cells ◽  
Lipoprotein Receptor Related Protein

SummaryThe recognition systems mediating the clearance of glycosylated high molecular weight single-chain urokinase-type plasminogen activator (HMW-scu-PA, produced in human embryonic kidney cells) and recombinant non-glycosylated scu-PA (rscu-PA, produced in E. coli) were analyzed by studying their binding charactaristics to freshly isolated rat parenchymal liver cells.The binding of 125I-HMW-scu-PA at 4° C was calcium-dependent and of high affinity (Kd = 37.6 nM) and could be inhibited by low molecular weight two-chain u-PA (LMW-tcu-PA) and lactose, but not by the low density lipoprotein receptor-related protein (LRP)-associated 39-kDa protein (RAP), rscu-PA or a mutant form lacking amino acids 11-135 (Delta 125-rscu-PA). Removal of the carbohydrate side chain of HMW-scu-PA by treatment with N-glycosidase F, completely reduced the specific binding to the parenchymal cells and strongly reduced its competition with 125I-HMW-scu-PA in cell binding.Recombinant scu-PA also bound with high affinity (Kd= 38.7 nM) to the parenchymal liver cells. The binding of 125I-rscu-PA could be competed for by unlabeled rscu-PA while Delta 125-rscu-PA, LMW-tcu-PA or lactose were ineffective. In contrast to HMW-scu-PA, binding of 125I-rscu-PA could be effectively inhibited by RAP (Ki = 1.1 nM), while also its association and degradation, as determined at 37° C, were inhibited by RAP. Pretreatment of the parenchymal cells with proteinase K supplied further evidence for the involvement of two different receptor systems. The binding of rscu-PA was decreased for 91%, while that of HMW-scu-PA showed a decrease of 51%.It is suggested that native HMW-scu-PA is bound and degraded by the rat parenchymal liver cells via a lectin-like recognition site, while non-glycosylated recombinant scu-PA is bound and degraded by rat parenchymal liver cells via the low density lipoprotein receptor-related protein (LRP). The differences in recognition system for native and recombinant proteins by liver cells suggest that the glycosylation of recombinant proteins, as obtained in mammalian expression systems, can be important for their physiological fate and their pharmacological application.

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