uPAR Activity Research Articles

Urokinase-Type Plasminogen Activator Receptor (uPAR) Cooperates with Mutated KRAS in Regulating Cellular Plasticity and Gemcitabine Response in Pancreatic Adenocarcinomas.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers. Given the currently limited therapeutic options, the definition of molecular subgroups with the development of tailored therapies remains the most promising strategy. Patients with high-level gene amplification of urokinase plasminogen activator receptor (uPAR/PLAUR) have an inferior prognosis. We analyzed the uPAR function in PDAC to understand this understudied PDAC subgroup's biology better. A total of 67 PDAC samples with clinical follow-up and TCGA gene expression data from 316 patients were used for prognostic correlations. Gene silencing by CRISPR/Cas9, as well as transfection of uPAR and mutated KRAS, were used in PDAC cell lines (AsPC-1, PANC-1, BxPC3) treated with gemcitabine to study the impact of these two molecules on cellular function and chemoresponse. HNF1A and KRT81 were surrogate markers for the exocrine-like and quasi-mesenchymal subgroup of PDAC, respectively. High levels of uPAR were correlated with significantly shorter survival in PDAC, especially in the subgroup of HNF1A-positive exocrine-like tumors. uPAR knockout by CRISPR/Cas9 resulted in activation of FAK, CDC42, and p38, upregulation of epithelial makers, decreased cell growth and motility, and resistance against gemcitabine that could be reversed by re-expression of uPAR. Silencing of KRAS in AsPC1 using siRNAs reduced uPAR levels significantly, and transfection of mutated KRAS in BxPC-3 cells rendered the cell more mesenchymal and increased sensitivity towards gemcitabine. Activation of uPAR is a potent negative prognostic factor in PDAC. uPAR and KRAS cooperate in switching the tumor from a dormant epithelial to an active mesenchymal state, which likely explains the poor prognosis of PDAC with high uPAR. At the same time, the active mesenchymal state is more vulnerable to gemcitabine. Strategies targeting either KRAS or uPAR should consider this potential tumor-escape mechanism.

(-)-Epigallocatechin-3-Gallate Prevents IL-1β-Induced uPAR Expression and Invasiveness via the Suppression of NF-κB and AP-1 in Human Bladder Cancer Cells.

(-)-Epigallocatechin-3-O-gallate (EGCG), a primary green tea polyphenol, has powerful iron scavengers, belongs to the family of flavonoids with antioxidant properties, and can be used to prevent cancer. Urokinase-type plasminogen activator receptors (uPARs) are glycosylphosphatidylinositol (GPI)-anchored cell membrane receptors that have crucial roles in cell invasion and metastasis of several cancers including bladder cancer. The mechanism of action of EGCG on uPAR expression has not been reported clearly yet. In this study, we investigated the effect of EGCG on interleukin (IL)-1β-induced cell invasion and uPAR activity in T24 human bladder cancer cells. Interestingly, nuclear factor (NF)-κB and activator protein (AP)-1 transcription factors were critically required for IL-1β-induced high uPAR expression, and EGCG suppressed the transcriptional activity of both the ERK1/2 and JNK signaling pathways with the AP-1 subunit c-Jun. EGCG blocked the IL-1β-stimulated reactive oxygen species (ROS) production, in turn suppressing NF-κB signaling and anti-invasion effects by inhibiting uPAR expression. These results suggest that EGCG may exert at least part of its anticancer effect by controlling uPAR expression through the suppression of ERK1/2, JNK, AP-1, and NF-κB.

ALG3 Promotes Peritoneal Metastasis of Ovarian Cancer through Increasing Interaction of α1,3-mannosylated uPAR and ADAM8.

Peritoneal metastasis is the main cause of poor prognoses and high mortality in ovarian cancer patients. Abnormal protein glycosylation modification is associated with cancer malignancy. Elevated α1,3-mannosyltransferase 3 (ALG3), which catalyzes the α1,3-mannosylation of glycoproteins, has been found in some malignant tumors. However, the pathological significance of ALG3 and its regulatory mechanism in ovarian cancer metastasis is unclear. The results showed that the level of ALG3/α1,3-mannosylation was higher in human ovarian cancer tissues compared with normal ovarian tissues, as measured by Lectin chip, Western blot and Lectin blot analyses, as well as ovarian tissue microarray analysis. ALG3 was also correlated with the poor prognosis of ovarian cancer patients, according to survival analysis. The downregulation of ALG3 decreased the proliferation, stemness and peritoneal metastasis of ovarian cancer cells. The increase in urokinase plasminogen activator receptor (uPAR) α1,3-mannosylation catalyzed by ALG3 enhanced urokinase plasminogen activator (uPA)/uPAR activation and the interaction of uPAR with a disintegrin and metalloproteinase 8 (ADAM8), which promoted ovarian cancer peritoneal metastasis via the ADAM8/Ras/ERK pathway. Furthermore, decreased ALG3 suppressed ascites formation and the peritoneal metastasis of ovarian cancer cells in mice. This study highlights ALG3 as a potential diagnostic biomarker and prospective therapeutic target for ovarian cancer.

Role of Urokinase-Type Plasminogen Activator Receptor in the Regulation of Angiogenic Properties of Sca1+ Vasculogenic Progenitor Cells

Neoangiogenesis is the key process determining myocardial regeneration after infarction. The urokinase-type plasminogen activator receptor (uPAR) is known to play an important role in the regulation of endothelial cell function and postnatal angiogenesis. However, uPAR its involvement in the regulation of the properties of vascular progenitor cells remains poorly studied. Aim: to evaluate uPAR expression on the surface of resident cardiac vascular progenitor cells (rcVPCs) and its impact on angiogenic cell properties in vitro as well as postinfarction cardiac vascularization. Materials and Methods. We used immunofluorescent analysis of cryosections of a murine myocardial infarction model to characterize vessels and rcVPCs, and evaluatedв the angiogenic properties potential of vasculogenic progenitor cells using the «tube assay» and induction ofinducing differentiation in a specialized medium. Results. We have found that the majority of Sca-1+ rcVPCs express the urokinase receptor and endothelial cell markers on their surface and are capable of proliferation and integration into the newly formed vessels in the injured area, indicating their possible involvement in thecontribution to vascularization process after infarction. After acute ischemic injury, the accumulation of vasculogenic progenitor cells (8+2 and 27+7 cells per visual field, respectively; P = 0.032) and vascularization processes (85+11 and 166+25 capillaries per visual field, respectively; P = 0.033) were observed in myocardium of uPAR-/- animals, compared with wild-type animals. Our studies demonstrated that Sca-1+ rcVCPs derived from uPAR-/- murine hearts demonstrated a reduced ability to form capillary-like structures and endothelial differentiation compared with Sca-1+ rcVCPs from hearts of wild-type mice. Conclusion. Thus, uPAR deficiency may lead to impaired vasculogenic properties of Sca-1+ rcVCPs, which is likely due to the loss of regulatory influence of specific ligands and the ability to interact with signaling mediators such as integrins. From the viewpoint of regenerative medicine, the modulation of uPAR activity can be considered as a potential targetpromising approach for targeted regulation of vasculogenic progenitor cells properties and postnatal angiogenesis.Highlight The urokinase-type plasminogen activator receptor is involved in the regulation of the angiogenic properties of Sca1+ vasculogenic progenitor cells.

Modulation of Cellular Function by the Urokinase Receptor Signalling: A Mechanistic View.

Urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycosyl-phosphatidyl-inositol anchored (GPI) membrane protein. The uPAR primary ligand is the serine protease urokinase (uPA), converting plasminogen into plasmin, a broad spectrum protease, active on most extracellular matrix components. Besides uPA, the uPAR binds specifically also to the matrix protein vitronectin and, therefore, is regarded also as an adhesion receptor. Complex formation of the uPAR with diverse transmembrane proteins, including integrins, formyl peptide receptors, G protein-coupled receptors and epidermal growth factor receptor results in intracellular signalling. Thus, the uPAR is a multifunctional receptor coordinating surface-associated pericellular proteolysis and signal transduction, thereby affecting physiological and pathological mechanisms. The uPAR-initiated signalling leads to remarkable cellular effects, that include increased cell migration, adhesion, survival, proliferation and invasion. Although this is beyond the scope of this review, the uPA/uPAR system is of great interest to cancer research, as it is associated to aggressive cancers and poor patient survival. Increasing evidence links the uPA/uPAR axis to epithelial to mesenchymal transition, a highly dynamic process, by which epithelial cells can convert into a mesenchymal phenotype. Furthermore, many reports indicate that the uPAR is involved in the maintenance of the stem-like phenotype and in the differentiation process of different cell types. Moreover, the levels of anchor-less, soluble form of uPAR, respond to a variety of inflammatory stimuli, including tumorigenesis and viral infections. Finally, the role of uPAR in virus infection has received increasing attention, in view of the Covid-19 pandemics and new information is becoming available. In this review, we provide a mechanistic perspective, via the detailed examination of consolidated and recent studies on the cellular responses to the multiple uPAR activities.

Abstract 2847: Human CYP1B1 induces cancer cell metastasis through Sp1-mediated activation of uPA-uPAR signaling pathway

Abstract Human CYP1B1 is known as a major metabolizer for estrogen and shows tumor-specific hyper-expression. To explore the role of CYP1B1 on metastasis of human cancer cells, we studied the effects of CYP1B1 inhibition and activation in MCF-10A, MCF-7, MDA-MB-231, and HeLa cells. CYP1B1 significantly induced expression of urokinase-type plasminogen activator receptor (uPAR) as well as uPA. Treatment with DMBA, a well-known CYP1B1 inducer, enhanced uPAR expression while CYP1B1 siRNA and TMS (tetramethoxystilbene), a specific CYP1B1 inhibitor, suppressed uPAR. We also found that CYP1B1 activated uPAR signaling through regulation of related factors including integrin β1 and α5. Interestingly, uPAR overexpression only caused a significant increase of integrin β1 and α5 in protein levels, indicating protein degradation may play an important role in regulating integrin protein level. Surprisingly, CYP1B1 down-regulated p53 expression through MDM2 activation and nutlin-3a, an inhibitor for MDM2, blocked the promoting effects of DMBA on uPAR, which explains that CYP1B1 induced uPAR expression through regulation of MDM2-p53 system. In addition, we revealed that CYP1B1 regulates uPAR through activation of Sp1 transcription factor. CYP1B1 and its enzymatic product, 4-OHE2, promote Sp1 expression levels in both mRNA and protein levels and subsequently Sp1 elevates the transcriptional activity of uPAR through binding to promoter region of uPAR. Taken together, our data suggest that CYP1B1 promotes cancer cell metastasis via activating uPAR pathway which is one of the targets of p53 and Sp1 signaling. Citation Format: Yeo-Jung Kwon, Sangyun Shin, Young-Jin Chun. Human CYP1B1 induces cancer cell metastasis through Sp1-mediated activation of uPA-uPAR signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2847.

D2A sequence of the urokinase receptor induces cell growth through αvβ3 integrin and EGFR.

The urokinase receptor (uPAR) stimulates cell proliferation by forming a macromolecular complex with αvβ3 integrin and the epidermal growth factor receptor (EGFR, ErbB1 or HER1) that we name the uPAR proliferasome. uPAR transactivates EGFR, which in turn mediates uPAR-initiated mitogenic signal to the cell. EGFR activation and EGFR-dependent cell growth are blocked in the absence of uPAR expression or when uPAR activity is inhibited by antibodies against either uPAR or EGFR. The mitogenic sequence of uPAR corresponds to the D2A motif present in domain 2. NMR analysis revealed that D2A synthetic peptide has a particular three-dimensional structure, which is atypical for short peptides. D2A peptide is as effective as EGF in promoting EGFR phosphorylation and cell proliferation that were inhibited by AG1478, a specific inhibitor of the tyrosine kinase activity of EGFR. Both D2A and EGF failed to induce proliferation of NR6-EGFR-K721A cells expressing a kinase-defective mutant of EGFR. Moreover, D2A peptide and EGF phosphorylate ERK demonstrating the involvement of the MAP kinase signalling pathway. Altogether, this study reveals the importance of sequence D2A of uPAR, and the interdependence of uPAR and EGFR.

Moesin and merlin regulate urokinase receptor-dependent endothelial cell migration, adhesion and angiogenesis

The glycosyl-phosphatidyl-inositol (GPI)-anchored urokinase receptor (uPAR) has no intracellular domain, but nevertheless initiates signalling through proximal interactions with other membrane receptors including integrins. The relationships between uPAR and ezrin/radixin/moesin (ERM) proteins, moesin and merlin have never been explored. Moesin and merlin are versatile membrane-actin links and regulators of receptors signalling, respectively. We show that uPAR controls moesin and merlin, which propagate uPAR-initiated signals and modulate integrin functions, thereby regulating uPAR activity. uPAR rapidly de-phosphorylates moesin and phosphorylates merlin inactivating both proteins, and enhancing cell migration and angiogenesis. Moesin behaves as a molecular switch turning either on or off uPAR signalling through cycles of de-activation/activation, or sustained activation, respectively. Furthermore, moesin is at the crossroads of uPAR-initiated outside-in and inside-out signalling promoting integrin-dependent cell adhesion suggesting that uPAR also activates integrins distally through moesin. Knocking down merlin expression enhanced cell migration and adhesion through different regulation of fibronectin- and vitronectin-binding integrins.

Urokinase-type plasminogen activator receptor interaction with β1 integrin is required for platelet-derived growth factor-AB-induced human mesenchymal stem/stromal cell migration.

IntroductionMesenchymal stem cells (MSC) are well described for their role in tissue regeneration following injury. Migratory properties of endogenous or administrated MSC are critical for tissue repair processes. Platelet-derived growth factor (PDGF) is a chemotactic growth factor that elicits mesenchymal cell migration. However, it is yet to be elucidated if signaling pathways other than direct activation of PDGF receptor (PDGF-R) are involved in PDGF-induced cell migration.MethodsKnocking down and co-immunoprecipitation approaches were used to evaluate urokinase-type plasminogen activator receptor (uPAR) requirement and its interactions with proteins involved in migration mechanisms, in human MSC induced to migrate under PDGF-AB effect.ResultsWe demonstrated that uPAR activation and its association with β1-integrin are required for PDGF-AB-induced migration. This phenomenon takes place in MSC derived from bone marrow and from adipose tissue.ConclusionsWe showed that PDGF-AB downstream signaling requires other effector molecules in MSC such as the uPA/uPAR system and β1 integrin signaling pathway known for their role in migration. These findings provide new insights in molecular mechanisms of PDGF-AB-induced migration of human MSC that may be relevant to control MSC function and tissue remodeling after injury.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0163-5) contains supplementary material, which is available to authorized users.

AduPARE1A and gemcitabine combined treatment trigger synergistic antitumor effects in pancreatic cancer through NF-κB mediated uPAR activation.

BackgroundCombined treatment of oncolytic adenoviruses with chemotherapeutic agents is foreseen as a therapeutic option for cancer. Here we have investigated the potential to use gemcitabine in combination with the oncolytic adenovirus AduPARE1A to treat pancreatic cancer and evaluate the underlying mechanism.MethodsWe treated pancreatic cancer cell lines BxPC-3 and PANC-1 with AduPARE1A and gemcitabine individually or in combination and analyzed cell viability, combination index, apoptosis and viral production. We also investigated the effects of the combination on tumor growth and mice survival in two xenograft models. Furthermore, we analyzed uPAR promoter activity from different uPAR-controlled adenovirus and studied NF-κB mediated effects.ResultsSynergistic cell killing from the combination AduPARE1A/Gemcitabine was observed in BxPC-3 and PANC-1 cells. Moreover, the combination treatment produced therapeutic benefits over either individual modality in two mouse models bearing orthotopic tumors, showing reduced tumor progression and significant prolonged mouse survival. Mechanistic studies showed that the synergistic cell death was not due to an increase in viral replication but occurred through an enhancement of apoptotic cell death. Gemcitabine stimulation increased the transcription of uPAR-controlled transgenes through the induction of NF-κB acting on the uPAR promoter. Interestingly, NF-κB gemcitabine-mediated induction of AduPAR adenoviruses interfered with the activation of NF-κB regulated genes, probably as a result of an intracellular competition for NF-κB DNA binding. Consequently, AduPARE1A infection sensitized cells to gemcitabine-induced apoptosis in the combined treatment.ConclusionsThese data highlights the potential of the combination as a treatment modality for pancreatic cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0413-2) contains supplementary material, which is available to authorized users.

Urokinase receptor and resistance to targeted anticancer agents.

The urokinase receptor (uPAR) is a GPI-anchored membrane protein, which regulates protease activity at the cell surface and, in collaboration with a system of co-receptors, triggers cell-signaling and regulates gene expression within the cell. In normal tissues, uPAR gene expression is limited; however, in cancer, uPAR is frequently over-expressed and the gene may be amplified. Hypoxia, which often develops in tumors, further increases uPAR expression by cancer cells. uPAR-initiated cell-signaling promotes cancer cell migration, invasion, metastasis, epithelial-mesenchymal transition, stem cell-like properties, survival, and release from states of dormancy. Newly emerging data suggest that the pro-survival cell-signaling activity of uPAR may allow cancer cells to “escape” from the cytotoxic effects of targeted anticancer drugs. Herein, we review the molecular properties of uPAR that are responsible for its activity in cancer cells and its ability to counteract the activity of anticancer drugs.

Activation of uPAR is required for cigarette smoke extract-induced epithelial-mesenchymal transition in lung epithelial cells.

Cigarette smoke is a major risk factor for lung cancer, which may contribute to lung cancer invasion and metastasis. However, the mechanism remains unclear. Epithelial-mesenchymal transition (EMT) is a critical phenotypic alteration of cells that triggers invasion and metastasis. The urokinase-type plasminogen activator receptor (uPAR) is originally thought to assist the directional invasion of migrating cells, and increasing evidences show that overexpression of uPAR in cancer cells promotes EMT. Therefore, we intend to study the role of uPAR in cigarette smoke extract (CSE)-induced EMT in lung epithelial cells. In this study, we showed that lung epithelial cells cultured after CSE treatment demonstrated changes consistent with EMT. E-cadherin was decreased, while vimentin, N-cadherin, and α-SMA expression was increased in both A549 and BEAS-2B cells. Cells acquired a mesenchymal-like morphology and increased cell motility and invasion. In addition, CSE-induced EMT was accompanied by increased expression of uPAR and activation of AKT downstream of uPAR. CSE-induced EMT and activation of AKT were blocked by uPAR gene silencing. Antagonizing PI3K also inhibits development of CSE-induced EMT. We conclude that CSE can induce EMT, and the activity of uPAR-dependent signal pathway in EMT is recapitulated in lung epithelial cells in vitro.

Clinical relevance and cellular source of elevated soluble urokinase plasminogen activator receptor (suPAR) in acute liver failure

Acute liver failure (ALF) is a life-threatening condition with a high mortality rate. The expression of urokinase plasminogen activator receptor (uPAR, CD87) and release of its shedded receptor into serum as soluble uPAR (suPAR) have been closely related to immune activation and prognosis in systemic inflammation and cirrhosis. We now aimed at investigating the clinical relevance and cellular source of uPAR and circulating suPAR in ALF. Serum suPAR concentrations were measured in 48 ALF patients and 62 healthy controls from a German liver transplantation centre. Hepatic immune cell subsets and uPAR expression were studied by FACS, qPCR and immunohistochemistry. Circulating suPAR levels were significantly increased in ALF patients, independent from the underlying aetiology, in comparison to controls. Serum suPAR concentrations were closely correlated with parameters reflecting liver cell injury, decreased liver function and the model of end-stage liver disease (MELD) score in ALF patients. By immunohistochemistry from explanted livers, ALF was associated with distinct immune cell accumulation and strong up-regulation of intrahepatic uPAR mRNA expression. CD87 (uPAR) expression was specifically detected on intrahepatic 'non-classical' monocytes (CD14(+) CD16(+) ), NKT and CD56(dim) NK cells isolated from human liver, but not on parenchymal or other non-parenchymal hepatic cell types. Membrane-bound uPAR was rapidly cleaved from monocytes upon inflammatory stimulation by lipopolysaccharide (LPS) and partially by co-cultured lymphocytes. Similar to its prognostic properties in patients with sepsis or cirrhosis, intrahepatic uPAR activation and serum suPAR concentrations might serve as an interesting biomarker in ALF.

Urokinase-type plasminogen activator receptor in IgA nephropathy

Urokinase-type plasminogen activator receptor in IgA nephropathy

Direct evidence of the importance of vitronectin and its interaction with the urokinase receptor in tumor growth

AbstractExtensive evidence implicates the urokinase plasminogen activator receptor (uPAR) in tumor growth, invasion, and metastasis. Recent studies have substantiated the importance of the interaction between uPAR and the extracellular matrix protein vitronectin (VN) for the signaling activity of the receptor in vitro, however, the possible relevance of this interaction for the activity of uPAR in tumor growth and metastasis has not been assessed. We generated a panel of HEK293 cell lines expressing mouse uPAR (muPARWT), an uPAR mutant specifically deficient in VN binding (muPARW32A), and a truncation variant (muPARΔD1) deficient in both VN and uPA binding. In vitro cells expressing muPARWT display increased cell adhesion, spreading, migration, and proliferation associated with increased p130Cas and MAPK signaling. Disruption of VN binding or ablation of both VN and uPA binding specifically abrogates these activities of uPAR. When xenografted into SCID (severe combined immunodeficiency) mice, the expression of muPARWT, but not muPARW32A or muPARΔD1, accelerates tumor development, demonstrating that VN binding is responsible for the tumor-promoting activity of uPAR in vivo. In an orthotopic xenograft model using MDA-MB-231 cells in RAG1−/−/VN−/− mice, we document that host deficiency in VN strongly impairs tumor formation. These 2 lines of in vivo experimentation independently demonstrate an important role for VN in tumor growth even if the uPAR dependence of the effect in the MDA-MB-231 model remains to be ascertained.

Single Amino Acid Substitutions in the Chemotactic Sequence of Urokinase Receptor Modulate Cell Migration and Invasion

The receptor for urokinase-type plasminogen activator (uPAR) plays an important role in controlling cell migration. uPAR binds urokinase and vitronectin extracellular ligands, and signals in complex with transmembrane receptors such as Formyl-peptide Receptors (FPR)s and integrins. Previous work from this laboratory has shown that synthetic peptides, corresponding to the uPAR88–92 chemotactic sequence, when carrying the S90P or S90E substitutions, up- or down-regulate cell migration, respectively. To gain mechanistic insights into these opposite cell responses, the functional consequences of S90P and S90E mutations in full-length uPAR were evaluated. First, (HEK)-293 embryonic kidney cells expressing uPARS90P exhibit enhanced FPR activation, increased random and directional cell migration, long-lasting Akt phosphorylation, and increased adhesion to vitronectin, as well as uPAR/vitronectin receptor association. In contrast, the S90E substitution prevents agonist-triggered FPR activation and internalization, decreases binding and adhesion to vitronectin, and inhibits uPAR/vitronectin receptor association. Also, 293/uPARS90P cells appear quite elongated and their cytoskeleton well organized, whereas 293/uPARS90E cells assume a large flattened morphology, with random orientation of actin filaments. Interestingly, when HT1080 cells co-express wild type uPAR with uPAR S90E, the latter behaves as a dominant-negative, impairing uPAR-mediated signaling and reducing cell wound repair as well as lung metastasis in nude mice. In contrast, signaling, wound repair and in vivo lung metastasis of HT1080 cells bearing wild type uPAR are enhanced when they co-express uPARS90P. In conclusion, our findings indicate that Ser90 is a critical residue for uPAR signaling and that the S90P and S90E exert opposite effects on uPAR activities. These findings may be accommodated in a molecular model, in which uPARS90E and uPARS90P are forced into inactive and active forms, respectively, suggesting important implications for the development of novel drugs targeting uPAR function.

Abstract 2407: Snail 1 regulates invasion through uPA-uPAR and MMP-9 signaling in prostate cancer cells

Abstract Epithelial-mesenchymal transition (EMT) is a process by which cancer cells acquire mesenchymal properties, such as induction of N-cadherin, while epithelial-associated genes like E-cadherin are lost. This enables the cells to be more invasive, migratory and metastatic. Factors that can induce EMT include growth factors like transforming growth factor -α (TGF-β) and epidermal growth factor (EGF), and transcription factors like Snail1. Snail1-induced EMT promotes migration and invasion and we hypothesized that this may be mediated by urokinase (uPA) and its receptor (uPAR) activities. uPA, a serine protease, may be activated by integrin engagement to bind to its receptor, uPAR, and initiate a signaling cascade that regulates a variety of biological pathways including invasion. Since Snail1 can induce matrix metalloproteinases (MMP 2 and 9) which are also involved in cell invasion, we also examined MMP activity. Androgen-dependent LNCaP, and 22Rv1 and androgen-independent ARCaP human prostate cancer (PC) cells were stably transfected with empty vector control (Neo) or constitutively active Snail1 which led to increased cell migration and invasion. The levels of Snail1, uPA, and uPAR were measured by Western Blot analysis. uPA activity in conditioned media was measured using the Elisa uPA activity assay kit. Additionally, cells were treated with MAPK inhibitor, UO126, at set time points. Gelatin zymography analysis was utilized to detect MMP-9 activity in the conditioned media. Currently, ARCaP-Snail cells are being transiently transfected with uPAR siRNA to assay for the effects of uPAR knockdown on uPA and MMP activity. The data suggests that overexpression of Snail1 increased uPA and uPAR protein levels, while uPA activity was elevated in conditioned media from LNCaP, 22Rv1 and ARCaP cells. Also, MAPK inhibited MAPK expression in the cell and decreased uPA activity. In addition gelatin zymography showed higher MMP 9 activity in ARCaP-Snail overexpressing cells that was inhibited by MAPK inhibitor. Therefore, Snail-mediated cell migration and invasion in human prostate cancer cells may occur via the regulation of uPA and uPAR activities in addition to MMPs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2407. doi:1538-7445.AM2012-2407

Urokinase Receptor Associates With Myocardin to Control Vascular Smooth Muscle Cells Phenotype in Vascular Disease

The urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR) are a potent multifunctional system involved in vascular remodeling. The goal of the study was to unravel the mechanisms of uPA/uPAR-directed vascular smooth muscle cell (VSMC) differentiation. Using cultured human primary VSMCs, we identified a new molecular mechanism controlling phenotypic modulation in vitro and in vivo. We found that the urokinase-type plasminogen activator receptor (uPAR) acts together with the transcriptional coactivator myocardin to regulate the VSMC phenotype. uPAR, a glycosylphosphatidylinositol-anchored cell-surface receptor family member, undergoes ligand-induced internalization and nuclear transport in VSMCs. Platelet-derived growth factor receptor β and SUMOylated RanGAP1 mediate this trafficking. Nuclear uPAR associates with myocardin, which is then recruited from the promoters of serum response factor target genes and undergoes proteasomal degradation. This chain of events initiates the synthetic VSMC phenotype. Using mouse carotid artery ligation model, we show that this mechanism contributes to adverse vascular remodeling after injury in vivo. We then cultured cells on a microstructured biomaterial and found that substrate topography induced uPAR-mediated VSMC differentiation. These findings reveal the transcriptional activity of uPAR, controlling the differentiation of VSMCs in a vascular disease model. They also suggest a new role for uPAR as a therapeutic target and as a marker for VSMC phenotyping on prosthetic biomaterials.

Urokinase plasminogen activator receptor activation of invasion and migration in H460 cells requires Na+/H+ transporter activity and expression

Non‐small cell lung cancer (NSCLC) is an aggressive cancer where 10% of patients survive beyond five years of initial diagnosis. One of the reasons for the poor survival rate is the robust invasion and metastatic properties of NSCLC. Urokinase plasminogen activator (uPA) and its receptor are directly associated with a poor prognosis in NSCLC. Na+/H+ transporter (NHE1) has been implicated in regulation of cell motility in a number of cancer cells. We wished to identify the role NHE1 plays in uPA activation of tumor progression in NSCLC. We used both uPA and the inactive‐receptor binding amino terminal fragment of uPA (ATF) to stimulate human lung H460 carcinoma cells. 10 nM uPA or ATF stimulated proliferation 1.4 ‐ 1.2 fold over non‐treated cells. Inhibition of NHE1 with 10 μM EIPA abrogated agonist‐stimulated proliferation. Tumor formation size and number was significantly increased by addition of either uPA or ATF, while NHE1 inhibition blocked the ability of either agonist to induce tumors in soft agar assays. Migration of H460 cells was inhibited in the presence of EIPA. Both uPA and ATF enhanced MMP9 activity but not in cells treated with EIPA. Only ATF increased MMP9 expression. We also show the effect of uPA in NHE1 and uPAR knockdown cell lines where we investigated MMP9 activity, and the location of NHE1 and uPAR in lipid rafts. Our results indicate that uPAR activation of cancer cell properties requires NHE1. Support from NIH‐1‐R15‐CA135616‐01

Participation of the urokinase receptor in neutrophil efferocytosis

AbstractThe urokinase receptor (uPAR) plays an important role in regulation of fibronolysis, cell migration, and adhesion. In this study, we examined whether uPAR plays a role in modulating efferocytosis of neutrophils. Macrophages from uPAR−/− mice demonstrated enhanced ability to engulf viable wild-type (WT) neutrophils in vitro and in vivo in the lungs. The increased phagocytic activity of uPAR−/− macrophages was abrogated by incubation with soluble uPAR (suPAR), arginine-glycine-aspartic acid (RGD)–containing peptides, or anti-integrin antibodies. There was increased uptake of viable uPAR−/− neutrophils by WT macrophages. Incubation of uPAR−/− neutrophils with suPAR or anti-integrin antibodies diminished uptake by WT macrophages to baseline. Uptake of uPAR−/− neutrophils by uPAR−/− macrophages was not enhanced. However, incubation of uPAR−/− neutrophils or uPAR−/− macrophages, but not both, with suPAR enhanced the uptake of viable uPAR−/− neutrophils by uPAR−/− macrophages. The adhesion of WT neutrophils to uPAR−/− macrophages was higher than to WT macrophages. uPAR−/− neutrophils demonstrated increased adhesion to suPAR, which was abrogated by blocking of low-density lipoprotein related protein and integrins. Expression of uPAR on the surface of apoptotic neutrophils was reduced compared with levels on viable neutrophils. These results demonstrate a novel role for uPAR in modulating recognition and clearance of neutrophils.