Degradation of internalized αvβ5 integrin is controlled by uPAR bound uPA: effect on β1 integrin activity and α-SMA stress fiber assembly.
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ABSTRACT:
SUBMITTER: Wang L
PROVIDER: S-EPMC3309951 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The repair of injured tissue is a highly complex process that involves cell proliferation, differentiation, and migration. Cell migration requires the dismantling of intercellular contacts in the injured zone and their subsequent reconstitution in the wounded area. Urokinase-type plasminogen activator (uPA) is a serine proteinase found in multiple cell types including endothelial cells, smooth muscle cells, monocytes, and macrophages. A substantial body of experimental evidence with different cell types outside the central nervous system indicates that the binding of uPA to its receptor (uPAR) on the cell surface prompts cell migration by inducing plasmin-mediated degradation of the extracellular matrix. In contrast, although uPA and uPAR are abundantly found in astrocytes and uPA binding to uPAR triggers astrocytic activation, it is unknown if uPA also plays a role in astrocytic migration. Neuronal cadherin is a member of cell adhesion proteins pivotal for the formation of cell-cell contacts between astrocytes. More specifically, while the extracellular domain of neuronal cadherin interacts with the extracellular domain of neuronal cadherin in neighboring cells, its intracellular domain binds to β-catenin, which in turn links the complex to the actin cytoskeleton. Glycogen synthase kinase 3β is a serine-threonine kinase that prevents the cytoplasmic accumulation of β-catenin by inducing its phosphorylation at Ser33, Ser37, and Ser41, thus activating a sequence of events that lead to its proteasomal degradation. The data discussed in this perspective indicate that astrocytes release uPA following a mechanical injury, and that binding of this uPA to uPAR on the cell membrane induces the detachment of β-catenin from the intracellular domain of neuronal cadherin by triggering its extracellular signal-regulated kinase 1/2-mediated phosphorylation at Tyr650. Remarkably, this is followed by the cytoplasmic accumulation of β-catenin because uPA-induced extracellular signal-regulated kinase 1/2 activation also phosphorylates lipoprotein receptor-related protein 6 at Ser1490, which in turn, by recruiting glycogen synthase kinase 3β to its intracellular domain abrogates its effect on β-catenin. The cytoplasmic accumulation of β-catenin is followed by its nuclear translocation, where it induces the expression of uPAR, which is required for the migration of astrocytes from the injured edge into the wounded area.
| S-EPMC9120704 | biostudies-literature
Project description:Macrophages play a crucial role in maintaining homeostasis in the intestine, but the underlying mechanisms have not yet been elucidated fully. Here, we show for the first time that mature intestinal macrophages in mouse intestine express high levels of αvβ5 integrin, which acts as a receptor for the uptake of apoptotic cells and can activate molecules involved in several aspects of tissue homeostasis such as angiogenesis and remodeling of the ECM. αvβ5 is not expressed by other immune cells in the intestine, is already present on intestinal macrophages soon after birth, and its expression is not dependent on the microbiota. In adults, αvβ5 is induced during the differentiation of monocytes in response to the local environment and it confers intestinal macrophages with the ability to promote engulfment of apoptotic cells via engagement of the bridging molecule milk fat globule EGF-like molecule 8. In the absence of αvβ5, there are fewer monocytes in the mucosa and mature intestinal macrophages have decreased expression of metalloproteases and IL 10. Mice lacking αvβ5 on haematopoietic cells show increased susceptibility to chemical colitis and we conclude that αvβ5 contributes to the tissue repair by regulating the homeostatic properties of intestinal macrophages.
| S-EPMC6207937 | biostudies-literature
Project description:Mounting evidence suggests that the urokinase plasminogen activator (uPA) and its receptor (uPAR) play a central role in tumor progression. The goal of this study was to develop an 89Zr-labeled, antibody-based positron emission tomography (PET) tracer for quantitative imaging of the uPA/uPAR system. An anti-uPA monoclonal antibody (ATN-291) was conjugated with a deferoxamine (Df) derivative and subsequently labeled with 89Zr. Flow cytometry, microscopy studies, and competitive binding assays were conducted to validate the binding specificity of Df-ATN-291 against uPA. PET imaging with 89Zr-Df-ATN-291 was carried out in different tumors with distinct expression levels of uPA. Biodistribution, histology examination, and Western blotting were performed to correlate tumor uptake with uPA or uPAR expression. ATN-291 retained uPA binding affinity and specificity after Df conjugation. 89Zr-labeling of ATN-291 was achieved in good radiochemical yield and high specific activity. Serial PET imaging demonstrated that, in most tumors studied (except uPA- LNCaP), the uptake of 89Zr-Df-ATN-291 was higher compared to major organs at 120 h post-injection, providing excellent tumor contrast. The tumor-to-muscle ratio of 89Zr-Df-ATN-291 in U87MG was as high as 45.2 ± 9.0 at 120 h p.i. In vivo uPA specificity of 89Zr-Df-ATN-291 was confirmed by successful pharmacological blocking of tumor uptake with ATN-291 in U87MG tumors. Although the detailed mechanisms behind in vivo 89Zr-Df-ATN-291 tumor uptake remained to be further elucidated, quantitative PET imaging with 89Zr-Df-ATN-291 in tumors can facilitate oncologists to adopt more relevant cancer treatment planning.
| S-EPMC5342023 | biostudies-literature
Project description:Pleural fibrosis (PF) is a chronic and progressive lung disease which affects approximately 30,000 people per year in the United States. Injury and sustained inflammation of the pleural space can result in PF, restricting lung expansion and impairing oxygen exchange. During the progression of pleural injury, normal pleural mesothelial cells (PMCs) undergo a transition, termed mesothelial mesenchymal transition (MesoMT). While multiple components of the fibrinolytic pathway have been investigated in pleural remodeling and PF, the role of the urokinase type plasminogen activator receptor (uPAR) is unknown. We found that uPAR is robustly expressed by pleural mesothelial cells in PF. Downregulation of uPAR by siRNA blocked TGF-β mediated MesoMT. TGF-β was also found to significantly induce uPA expression in PMCs undergoing MesoMT. Like uPAR, uPA downregulation blocked TGF-β mediated MesoMT. Further, uPAR is critical for uPA mediated MesoMT. LRP1 downregulation likewise blunted TGF-β mediated MesoMT. These findings are consistent with in vivo analyses, which showed that uPAR knockout mice were protected from S. pneumoniae-mediated decrements in lung function and restriction. Histological assessments of pleural fibrosis including pleural thickening and α-SMA expression were likewise reduced in uPAR knockout mice compared to WT mice. These studies strongly support the concept that uPAR targeting strategies could be beneficial for the treatment of PF.
| S-EPMC8551303 | biostudies-literature
Project description:Cervical cancer remains a major health threat. Urokinase serves as a marker of metastatic tumors. The present study aimed to determine whether the expression levels of urokinase plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR), before and during the course of radiotherapy, serve as prognostic markers for patients with cervical cancer. Cervical tumor tissue biopsies were collected from 72 patients before radiotherapy and after the completion of external beam radiotherapy (EBRT) before intracavitary brachytherapy. The levels of uPA and uPAR were determined using ELISA assays. The significance of the associations between the protein expression levels and the clinical outcomes of patients was determined. Although irradiation enhanced uPA and uPAR expression in cervical cancer cell lines, average uPA levels significantly decreased in tumors, and uPAR levels significantly increased after EBRT. The levels of uPA increased in 12 patients and decreased in 26 patients; and those of uPAR increased in 13 patients and decreased in two patients. Cox regression analysis revealed that increased expression of uPAR was significantly associated with 5-year overall survival rate [hazard ratio (HR), 3.65; 95% confidence interval (CI), 1.18-11.30]. However, the levels of both proteins before radiotherapy failed to predict clinical outcomes. Other significant predictive factors were partial response (HR 7.22; 95% CI 1.17-44.73) and disease progression (HR, 13.41; 95% CI, 1.17-153.07). These findings indicated that increased expression of uPAR in cervical tumor tissue during radiotherapy may serve as a prognostic marker for patients with cervical cancer.
| S-EPMC8025075 | biostudies-literature
Project description:BackgroundLoss of intestinal epithelial barrier integrity is a critical component of Inflammatory Bowel Disease (IBD) pathogenesis. Co-expression regulation of ligand-receptor pairs in IBD mucosa has not been systematically studied. Targeting ligand-receptor pairs which are induced in IBD mucosa and function in intestinal epithelial barrier integrity may provide novel therapeutics for IBD.MethodsWe performed transcriptomic meta-analysis on public IBD datasets combined with cell surface protein-protein-interaction (PPI) databases. We explored primary human/mouse intestinal organoids and Caco-2 cells for expression and function studies of uPA-uPAR (prime hits from the meta-analysis). Epithelial barrier integrity was measured by Trans-Epithelial Electrical Resistance (TEER), FITC-Dextran permeability and tight junction assessment. Genetic (CRISPR, siRNA and KO mice) and pharmacological (small molecules, neutralizing antibody and peptide inhibitors) approaches were applied. Mice deficient of uPAR were studied using the Dextran Sulfate Sodium (DSS)-induced colitis model.FindingsThe IBD ligand-receptor meta-analysis led to the discovery of a coordinated upregulation of uPA and uPAR in IBD mucosa. Both genes were significantly upregulated during epithelial barrier breakdown in primary intestinal organoids and decreased during barrier formation. Genetic inhibition of uPAR or uPA, or pharmacologically blocking uPA-uPAR interaction protects against cytokine-induced barrier breakdown. Deficiency of uPAR in epithelial cells leads to enhanced EGF/EGFR signalling, a known regulator of epithelial homeostasis and repair. Mice deficient of uPAR display improved intestinal barrier function in vitro and during DSS-induced colitis in vivo.InterpretationOur findings suggest that blocking uPA-uPAR interaction via pharmacological agents protects the epithelial barrier from inflammation-induced damage, indicating a potential therapeutic target for IBD.FundingThe study was funded by Boehringer Ingelheim.
| S-EPMC8688562 | biostudies-literature
Project description:The role of integrins, in particular αv integrins, in regulating insulin resistance is incompletely understood. We have previously shown that the αvβ5 integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) regulates cellular uptake of fatty acids. In this work, we evaluated the impact of MFGE8 on glucose homeostasis. We show that acute blockade of the MFGE8/β5 pathway enhances while acute augmentation dampens insulin-stimulated glucose uptake. Moreover, we find that insulin itself induces cell-surface enrichment of MFGE8 in skeletal muscle, which then promotes interaction between the αvβ5 integrin and the insulin receptor leading to dampening of skeletal-muscle insulin receptor signaling. Blockade of the MFGE8/β5 pathway also enhances hepatic insulin sensitivity. Our work identifies an autoregulatory mechanism by which insulin-stimulated signaling through its cognate receptor is terminated through up-regulation of MFGE8 and its consequent interaction with the αvβ5 integrin, thereby establishing a pathway that can potentially be targeted to improve insulin sensitivity.
| S-EPMC8106306 | biostudies-literature
Project description:BackgroundIn our earlier reports, we showed that downregulation of uPA and uPAR inhibited glioma tumor angiogenesis in SNB19 cells, and intraperitoneal injection of a hairpin shRNA expressing plasmid targeting uPA and uPAR inhibited angiogenesis in nude mice. The exact mechanism by which inhibition of angiogenesis takes place is not clearly understood.Methodology/principal findingsIn the present study, we have attempted to investigate the mechanism by which uPA/uPAR downregulation by shRNA inhibits angiogenesis in endothelial and glioblastoma cell lines. uPA/uPAR downregulation by shRNA in U87 MG and U87 SPARC co-cultures with endothelial cells inhibited angiogenesis as assessed by in vitro angiogenesis assay and in vivo dorsal skin-fold chamber model in nude mice. Protein antibody array analysis of co-cultures of U87 and U87 SPARC cells with endothelial cells treated with pU2 (shRNA against uPA and uPAR) showed decreased angiogenin secretion and angiopoietin-1 as well as several other pro-angiogenic molecules. Therefore, we investigated the role of angiogenin and found that nuclear translocation, ribonucleolytic and 45S rRNA synthesis, which are all critical for angiogenic function of angiogenin, were significantly inhibited in endothelial cells transfected with uPA, uPAR and uPA/uPAR when compared with controls. Moreover, uPA and uPAR downregulation significantly inhibited the phosphorylation of Tie-2 receptor and also down regulated FKHR activation in the nucleus of endothelial cells via the GRB2/AKT/BAD pathway. Treatment of endothelial cells with ruPA increased angiogenin secretion and angiogenin expression as determined by ELISA and western blotting in a dose-dependent manner. The amino terminal fragment of uPA down regulated ruPA-induced angiogenin in endothelial cells, thereby suggesting that uPA plays a critical role in positively regulating angiogenin in glioblastoma cells.Conclusions/significanceTaken together, our results suggest that uPA/uPAR downregulation suppresses angiogenesis in endothelial cells induced by glioblastoma cell lines partially by downregulation of angiogenin and by inhibition of the angiopoietin-1/AKT/FKHR pathway.
| S-EPMC2929192 | biostudies-literature
Project description:The identification of cell surface markers specific to pancreatic beta cells is important for both the study of islet biology and for investigating the pathophysiology of diseases in which this cell type is lost or damaged. Following analysis of publicly available RNAseq data, we identified specific integrin subunits, integrin αv and integrin β5, that were expressed in beta cells. This finding was further elaborated using immunofluorescence analysis of histological sections derived from donor human pancreas. Despite the broad expression of specific integrin subunits, we found that expression of integrin αvβ5 heterodimers was restricted to beta cells and that this complex persisted in islet remnants of some type 1 diabetic individuals from which insulin expression had been lost. This study identifies αvβ5 heterodimers as a novel cell surface marker of human pancreatic beta cells, a finding that will aid in the identification and characterisation of this important cell type.
| S-EPMC8050092 | biostudies-literature
Project description:The vascular leakage in diabetic retinopathy leads to macular edema and vision loss. Although astrocyte play an important role in regulating blood-brain barrier integrity in the brain, the precise role of astrocyte in blood-retinal barrier was yet to be elucidated. This study aimed to investigate the role of angiopoietin 2 (Ang2) in astrocyte loss and vascular leakage in the early streptozotocin-induced diabetic retinopathy. We demonstrated that vascular leakage occurred with astrocyte loss in early diabetic mice retina as Ang2 increased. The astrocyte loss and vascular leakage were inhibited by intravitreal injection of Ang2-neutralizing antibody. In vitro, Ang2 aggravated high glucose-induced astrocyte apoptosis via GSK-3β activation. Ang2 directly bound to αvβ5 integrin, which was abundant in astrocyte, and the blockade of αvβ5 integrin, in vitro, effectively attenuated Ang2-induced astrocyte apoptosis. In vivo, intravitreal injection of anti-αvβ5-integrin antibody inhibited astrocyte loss in early diabetic retinopathy. Taken together, Ang2 induced astrocyte apoptosis under high glucose via αvβ5-integrin/GSK-3β/β-catenin pathway. Therefore, we suggest that Ang2/integrin signaling could be a potential therapeutic target to prevent the vascular leakage by astrocyte loss in early diabetic retinopathy.
| S-EPMC5399183 | biostudies-literature