Role Of uPAR Research Articles

Pro-urokinase up-regulates the expression of urokinase-type plasminogen activator (u-PA) in human pulmonary arterial endothelial cells

The fibrinolytic function of endothelial cells plays an important role in the pathophysiology of pulmonary vascular diseases. In this study, the effects of pro-urokinase, a new thrombolytic drug that is currently being tested for the treatment of pulmonary embolism, on the expression of urokinase-type plasminogen activator (u-PA) and u-PA receptor (u-PAR) were assessed. The role of u-PAR was also investigated. Immunocytofluorescence and RT-PCR techniques were employed. In normal human pulmonary arterial endothelial cells (HPAECs), the expression levels of u-PA and u-PAR were very low. Incubation with pro-urokinase up-regulated u-PA expression at both the mRNA level and the protein level; however, the expression of u-PAR was not affected. The effect of pro-urokinase induction was totally inhibited by the release of u-PAR from the HPAECs' surface with PLC. This result suggests that the combination of u-PA with u-PAR may be a critical pathway for the induction of u-PA expression.

Down-regulation of uPAR and uPA activates caspase-mediated apoptosis and inhibits the PI3K/AKT pathway

Urokinase plasminogen activator (uPA) and its receptor (uPAR) play a major role in invasion and proliferation. A growing body of evidence has suggested that the uPA system promotes tumor metastasis by several different mechanisms, and not just solely by breaking down the ECM. In this study we have used RNAi-mediated simultaneous down-regulation of uPAR and uPA to determine the signaling pathway molecules and caspase-mediated apoptosis. From our in vitro experiments, we have observed that plasmid-based RNAi-mediated down-regulation of uPAR and uPA in SNB19 human glioma cells caused a decrease in the levels of uPAR protein and uPA enzyme activities. In addition, we observed a decrease in the phosphorylation of the Ras-activated pathway molecules such as FAK, p38MAPK, JNK and ERK1/2, as well as the MEK-activated phosphatidylinositol 3-kinase (PI3k) pathway, and also retarded the dephosphorylation of p-AKTser473 and p-mTORser2448, indicative of a feedback signaling mechanism of the uPAR-uPA system. Activation of caspase 8 accompanied by the release of cytochrome c and cleavage of PARP was also observed and indicative of Fas-mediated apoptosis. The use of FMK-VAD-FAK peptides coupled with FITC indicated activation of polycaspases, which was accompanied by the presence of fragmented nuclei. Our studies provide evidence for the presence of a feedback response of the uPAR-uPA system indicative of the multifaceted role of uPAR, and also the therapeutic potential of simultaneously targeting uPAR and uPA in cancer patients.

The uPA receptor and the somatomedin B region of vitronectin direct the localization of uPA to focal adhesions in microvessel endothelial cells

Vitronectin is a plasma protein which can deposit into the extracellular matrix where it supports integrin and uPA dependent cell migration. In earlier studies, we have shown that the plasma protein, vitronectin, stimulates focal adhesion remodeling by recruiting urokinase-type plasminogen activator (uPA) to focal adhesion sites [Wilcox-Adelman, S. A., Wilkins-Port, C. E., McKeown-Longo, P. J., 2000. Localization of urokinase-type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors. Cell. Adhes. Commun.7, 477–490]. In the present study, we used a variety of vitronectin constructs to demonstrate that the localization of uPA to adhesion sites requires the binding of both vitronectin integrin receptors and the uPA receptor (uPAR) to vitronectin. A recombinant fragment of vitronectin containing the connecting sequence (VN CS) was able to support integrin-dependent adhesion, spreading and focal adhesion assembly by human microvessel endothelial cells. Cells adherent to this fragment were not able to localize uPA to focal adhesions. A second recombinant fragment containing both the amino-terminal SMB domain and the CS domain was able to restore the localization of uPA to adhesion sites. This fragment, which contains a uPAR binding site, also resulted in the localization of uPAR to adhesion sites. uPAR blocking antibodies as well as phospholipase C treatment of cells inhibited uPA localization to adhesion sites confirming a role for uPAR in this process. The SMB domain alone was unable to direct either uPAR or uPA to adhesion sites in the absence of the CS domain. Our results indicate that vitronectin-dependent localization of uPA to adhesion sites requires the sequential binding of vitronectin integrins and uPAR to vitronectin.

Rosuvastatin regulates vascular smooth muscle cell phenotypic modulation in vascular remodeling: Role for the urokinase receptor

The urokinase (uPA)/urokinase receptor (uPAR) multifunctional system is an important mediator of migration and proliferation of vascular smooth muscle cells (VSMC). However, whether uPA/uPAR-directed mechanisms are involved in the beneficial effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors on vascular remodeling remains unexplored. In this study, we have investigated the effect of the hydrophilic statin rosuvastatin on neointimal remodeling, and the role of uPAR. Using an ex vivo organ and in vitro cell culture models we demonstrate that rosuvastatin decreases injury-induced neointima formation and proliferation of medial VSMC in porcine coronary arteries, as well as migration and proliferation of human coronary VSMC. Studies on the underlying mechanisms show that rosuvastatin impairs VSMC transition from their physiological contractile to the pathophysiologal synthetic phenotype. These effects are mediated, at least in part, via uPAR, as confirmed by means of rosuvastatin-directed uPAR expression and uPAR silencing in both models. Our findings provide evidence that rosuvastatin modulates VSMC phenotypic changes and subsequently their proliferation and migration, and indicate the important role for uPAR in these processes. This mechanism contributes to the beneficial non-lipid lowering effect of rosuvastatin on negative vascular remodeling

The urokinase plasminogen activator receptor is crucially involved in host defense during acute pyelonephritis

The urokinase plasminogen activator receptor (uPAR) is expressed at the cell surface of inflammatory cells and plays an important role in neutrophil migration. To investigate the in vivo role of uPAR during urinary tract infection, acute pyelonephritis was induced in uPAR-/- and wild-type (WT) mice by intravesical inoculation with 1 x 10(9) colony-forming units (CFU) of uropathogenic Escherichia coli. Mice were killed after 24 and 48 h, after which bacterial outgrowth and cytokine levels in kidney homogenates were determined. Influx of neutrophils was quantified by myeloperoxidase-enzyme-linked immunosorbent assay. uPAR-/- kidneys had significantly higher numbers of E. coli CFU, accompanied by higher levels of interleukin-1beta (IL-1beta), IL-6, keratinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2), and tumor necrosis factor-alpha (TNF-alpha). However, the number of infiltrating neutrophils was similar in uPAR-/- and WT mice at both time points, suggesting that uPAR-/- neutrophils have a lower ability to eliminate E. coli. To further investigate this, neutrophil oxidative burst and phagocytosis was measured. The generation of reactive oxygen species upon stimulation with E. coli was not diminished in uPAR-/- neutrophils compared with WT. Interestingly, uPAR-/- neutrophils displayed significantly impaired phagocytosis of E. coli organisms compared with WT neutrophils. We conclude that uPAR is crucially involved in host defense through phagocytosis during E. coli induced acute pyelonephritis.

Inhibition of urokinase-type plasminogen activator receptor induces apoptosis in melanoma cells by activation of p53

The urokinase-type plasminogen activator receptor (uPAR) is involved in several biological processes, including proteolysis, adhesion, migration and inflammation. Increased expression of uPAR is associated with metastasis in several tumor types. We studied the biological role of uPAR in melanoma and found that inhibition of uPAR via RNA interference induced massive death in three different metastatic cell lines. Annexin-V staining and caspase activation analysis revealed induction of the mitochondrial apoptotic pathway. The expression of members of the Bcl-2 family (Bax, Bcl-2, Bak and Bcl-x(L)) was changed in a pro-apoptotic manner. uPAR inhibition induced the expression of the tumor suppressor p53 and of its downstream target gene p21. Inhibition of p53 rescued cells from apoptosis indicating that p53 was critical for apoptosis induction. Apoptosis was observed in melanoma cells carrying activating BRAF mutations and occurred in the presence of extracellular signal-regulated kinase (ERK) phosphorylation. uPAR can activate focal adhesion kinase (FAK), which is implicated in adhesion-dependent tumor cell survival. However, inhibition of FAK did not induce apoptosis. Our data suggest a new function of uPAR acting as a survival factor for melanoma by downregulating p53. Inhibition of uPAR induces a pro-apoptotic signalling pathway via p53 that is independent of ERK or FAK signalling. These findings may offer new treatment strategies for metastatic melanoma.

ID: 269 The Role of uPAR and Alpha 3-Beta 1 Integrin in the Modulation of Integrin Function

ID: 269 The Role of uPAR and Alpha 3-Beta 1 Integrin in the Modulation of Integrin Function

Functional Relevance of Urinary-type Plasminogen Activator Receptor-α3β1 Integrin Association in Proteinase Regulatory Pathways

Squamous cell carcinoma of the oral cavity is characterized by persistent, disorganized expression of integrin alpha3beta1 and enhanced production of urinary-type plasminogen activator (uPA) and its receptor (uPAR) relative to normal oral mucosa. Because multivalent aggregation of alpha3beta1 integrin up-regulates uPA and induces a dramatic co-clustering of uPAR, we explored the hypothesis that lateral ligation of alpha3beta1 integrin by uPAR contributes to uPA regulation in oral mucosal cells. To investigate mechanisms by which uPAR/alpha3beta1 binding enhances uPA expression, integrin-dependent signal activation was assessed. Both Src and ERK1/2 were phosphorylated in response to integrin aggregation, and blocking Src kinase activity completely abrogated ERK1/2 activation and uPA induction, whereas inhibition of epidermal growth factor receptor tyrosine kinase activity did not alter uPA expression. Proteinase up-regulation occurred at the transcriptional level and mutation of the AP1 (-1967) site in the uPA promoter blocked the uPAR/integrin-mediated transcriptional activation. Because uPAR is redistributed to clustered alpha3beta1 integrins, the requirement for uPAR/alpha3beta1 interaction in uPA regulation was assessed. Clustering of alpha3beta1 in the presence of a peptide (alpha325) that disrupts uPAR/alpha3beta1 integrin binding prevented uPA induction. Depletion of cell surface uPAR using small interfering RNA also blocked uPA induction following integrin alpha3beta1 clustering. These results were confirmed using a genetic strategy in which alpha3 null epithelial cells reconstituted with wild type alpha3 integrin, but not a mutant alpha3 unable to bind uPAR, induced uPA expression upon integrin clustering, confirming the critical role of uPAR in integrin-regulated proteinase expression. Disruption of uPAR/alpha3beta1 binding using peptide alpha325 or small interfering RNA blocked filopodia formation and matrix invasion, indicating that this interaction stimulates invasive behavior. Together these data support a model wherein matrix-induced clustering ofalpha3beta1 integrin promotes uPAR/alpha3beta1 interaction, thereby potentiating cellular signal transduction pathways culminating in activation of uPA expression and enhanced uPA-dependent invasive behavior.

Coordinate regulation of fibronectin matrix assembly by the plasminogen activator system and vitronectin in human osteosarcoma cells

BackgroundPlasminogen activators are known to play a key role in the remodeling of bone matrix which occurs during tumor progression, bone metastasis and bone growth. Dysfunctional remodeling of bone matrix gives rise to the osteoblastic and osteolytic lesions seen in association with metastatic cancers. The molecular mechanisms responsible for the development of these lesions are not well understood. Studies were undertaken to address the role of the plasminogen activator system in the regulation of fibronectin matrix assembly in the osteoblast-like cell line, MG-63.ResultsTreatment of MG-63 cells with P25, a peptide ligand for uPAR, resulted in an increase in assembly of fibronectin matrix which was associated with an increase in the number of activated β1 integrins on the cell surface. Overexpression of uPAR in MG-63 cells increased the effect of P25 on fibronectin matrix assembly and β1 integrin activation. P25 had no effect on uPAR null fibroblasts, confirming a role for uPAR in this process. The addition of plasminogen activator inhibitor Type I (PAI-1) to cells increased the P25-induced fibronectin polymerization, as well as the number of activated integrins. This positive regulation of PAI-1 on fibronectin assembly was independent of PAI-1's anti-proteinase activity, but acted through PAI-1 binding to the somatomedin B domain of vitronectin.ConclusionThese results indicate that vitronectin modulates fibronectin matrix assembly in osteosarcoma cells through a novel mechanism involving cross-talk through the plasminogen activator system.

Direct interaction of the kringle domain of urokinase-type plasminogen activator (uPA) and integrin αvβ3 induces signal transduction and enhances plasminogen activation

It has been questioned whether there are receptors for urokinase-type plasminogen activator (uPA) that facilitate plasminogen activation other than the high affinity uPA receptor (uPAR/CD87) since studies of uPAR knockout mice did not support a major role of uPAR in plasminogen activation. uPA also promotes cell adhesion, chemotaxis, and proliferation besides plasminogen activation. These uPA-induced signaling events are not mediated by uPAR, but mediated by unidentified, lower-affinity receptors for the uPA kringle. We found that uPA binds specifically to integrin alpha v beta 3 on CHO cells depleted of uPAR. The binding of uPA to alpha v beta 3 required the uPA kringle domain. The isolated uPA kringle domain binds specifically to purified, recombinant soluble, and cell surface alpha v beta 3, and other integrins (alpha 4 beta 1 and alpha 9 beta 1), and induced migration of CHO cells in an alpha v beta 3-dependent manner. The binding of the uPA kringle to alpha v beta 3 and uPA kringle-induced alpha v beta 3-dependent cell migration were blocked by homologous plasminogen kringles 1-3 or 1-4 (angiostatin), a known integrin antagonist. We studied whether the binding of uPA to integrin alpha v beta 3 through the kringle domain plays a role in plasminogen activation. On CHO cell depleted of uPAR, uPA enhanced plasminogen activation in a kringle and alpha v beta 3-dependent manner. Endothelial cells bound to and migrated on uPA and uPA kringle in an alpha v beta 3-dependent manner. These results suggest that uPA binding to integrins through the kringle domain plays an important role in both plasminogen activation and uPA-induced intracellular signaling. The uPA kringle-integrin interaction may represent a novel therapeutic target for cancer, inflammation, and vascular remodeling.

Role of Plasmin and uPAR in Bone Marrow HSC/HPC Retention and Mobilization.

We previously identified the plasmin protease family as a critical determinant of the mobilization of hematopoietic stem and progenitor cells (HSC/HPC), but the role of the urokinase receptor uPAR remained unclear. uPAR is a membrane-anchored glycoprotein, which not only localizes its ligand urokinase (uPA) to the cell surface via its GPI-anchor but also regulates β1-integrin dependent cell adhesion and migration.Following 5-FU myeloablation or G-CSF treatment, mice lacking uPAR (uPAR−/−) had impaired hematopoietic recovery and HSC/HPC mobilization as compared to wild type (WT) mice. However, this phenotype was not mimicked in mice lacking uPA, suggesting a role of uPAR in mobilization independent of uPA-mediated proteolysis.The impaired mobilization in uPAR−/− mice was reversed upon pre-transplantation with WT BM cells (BMC), suggesting functional expression of uPAR on transplantable BMCs. Conversely, loss or inhibition of uPAR on transplanted BMCs impaired homing to the BM but not to the spleen, and compromised survival of myeloablated WT recipients. In vitro experiments revealed that loss or inhibition of uPAR impaired BMC adhesion to stromal cells and fibronectin. Anti-α4-β1 antibodies blocked adhesion of WT but not uPAR−/− BMCs. Thus, uPAR appears to regulate BM homing and α4-β1 dependent retention of transplantable BMCs, possibly HSC/HPCs.If uPAR mediates retention of HSC/HPCs, then this signal should be inactivated upon mobilization. Indeed, in 5-FU or G-CSF-treated WT mice, we found increased uPAR cleavage, and elevated levels of soluble uPAR (suPAR) in BM plasma. These processes failed to occur in mice lacking plasminogen, suggesting that plasmin cleaves uPAR during mobilization. Cleavage of uPAR appeared critical as the inactivation of the retention signals membrane-bound Kit ligand and SDF-1α was normal in uPAR−/− mice. Moreover, the generated suPAR may also affect the BM, as administration of recombinant suPAR in WT mice enhanced hematopoietic recovery and HSC/HPC mobilization after 5-FU or G-CSF. In vitro and transplantation experiments revealed that suPAR blocked α4-β1 dependent adhesion.Thus, in steady state, membrane-anchored uPAR appears to function as a BM retention signal for transplantable BMCs, possibly HSC/HPCs. In conditions of mobilization, the uPAR retention signal is cleaved, which weakens α4-β1 dependent adhesion and allows mobilization out of the BM. Soluble uPAR may then additionally amplify mobilization, in part by further attenuating α4-β1 dependent adhesion to the BM. Currently, we are investigating the role of uPAR on subsets of HSC/HPCs, and in the different BM niches. We are also performing long-term competitive repopulation experiments to further delineate the therapeutic potential of uPAR.

Outside-In Signaling upon Assembly on Endothelial Cells of the Proteins of the Plasma Kallikrein/Kinin System.

Investigations from our laboratory have shown that there is a multiprotein receptor system on endothelial cells (HUVEC) consisting of gC1qR, uPAR, and cytokeratin 1 for high molecular weight kininogen (HK) and factor XII (XII) (Blood 97:2342; 99:3585) that serves as a presentation receptor for prekallikrein (PK) activation. When PK binds to HK on HUVEC, it is activated to plasma kallikrein by the serine protease prolylcarboxypeptidase (JBC 277:17962; Blood 103:4554). HK is also known to have anti-proliferative and anti-angiogenic activity. We asked if there is outside-in signaling through this HUVEC receptor complex. Initial investigations determined if single chain urokinase (ScuPA) or XII stimulates Erk 1 and 2 (MAPK42 and 44) (MAPK) in HUVEC. Independently ScuPA (5–200 nM) or XII (15–200 nM) in the presence of 0.05 mM zinc ion stimulates HUVEC MAPK expression and it is blocked by 0.1 mM PD98059, 30 nM wortmann, or 0.05 mM LY294002. Two chain uPA or APMSF-treated ScuPA upregulates MAPK to the same extent as ScuPA. Similarly, XIIa or APMSF-treated XII upregulates MAPK like XII. Since HK, ScuPA, XII and vitronectin (VN) all bind to the same region on uPAR (JBC 279:16621), studies focused on the role of uPAR in these activities. Mab3B10 to the HK, ScuPA, XII, and VN binding site on uPAR's Domain 2 (D2) blocks ScuPA or XII upregulation of MAPK. Peptides LRG20, YLP20, PGS20, or FHN20 from uPAR's D2 (L166-N200) block ScuPA or XII upregulation of MAPK. Similarly, HKa (1 micromolar) and peptides from the HK Domain 5 (G469-H498) HUVEC binding region (GGH18, HKH20) (JBC 270:19256) block ScuPA- or XII-induced MAPK expression. Treatment of HUVEC with 5-20 mM methyl-beta-cyclodextran (MBCD) or fillipin (1 microgram/ml), agents known to disrupt lipid rafts, do not interfere with ScuPA- or XII-induced MAPK upregulation. These combined data indicate that zymogen ScuPA or XII, two proteins with growth factor regions, directly signal through uPAR to upregulate MAPK to possibly induce cellular proliferative activities. HK or its activated fragments bound to uPAR inhibits these potential growth promoting activities. These studies imply that HKa's anti-proliferative and anti-angiogenic activities are mediated through its interaction with uPAR. These investigations also indicate that, in addition to a presentation complex for PK activation, the HUVEC multiprotein receptor complex for HK, XII, and ScuPA has an auto-regulating outside-in signaling system in endothelials cells.

Urokinase plasminogen activator receptor promotes macrophage infiltration into the vascular wall of ApoE deficient mice

The urokinase plasminogen activator receptor (uPAR) regulates macrophage adhesion and migration by binding directly to matrix proteins and signaling through integrin complexes. In this study, we examined the role of uPAR on macrophage infiltration into the vascular wall. Stable murine macrophage (Raw264.7) cell lines expressing high levels of human uPAR, human urokinase plasminogen activator (uPA), or both were established using expression vectors driven by the human CD68 promoter. Stimulation with human uPA specifically induced phosphorylation of early response regulated kinase (ERK) in cells expressing human uPAR but not in sham transfected cells. The human uPAR expressing Raw264.7 cells showed increased adhesion to both human uPA and vitronectin (Vn). Raw264.7 cells expressing human uPAR or both human uPAR and uPA, but not uPA alone, were detected in the aortic wall of ApoE(-/-) mice, and no cells were detected in that of age-matched C57BL/6J mice after intravenous infusion of the cells. Blocking of Mac-1/ICAM-1 interaction by anti-alphaM antibody (M1/70) significantly reduced the infiltration of huPAR-expressing Raw264.1 cells into aorta of ApoE(-/-) mice. Treatment of C57BL/6J mice with angiotensin II resulted in infiltration of Raw264.7 cells expressing human uPAR. These data demonstrate that uPAR plays a key role in promoting macrophage infiltration into the arterial wall of ApoE(-/-) mice.

Vascular Endothelial Growth Factor (VEGF)-Induced Endothelial Cell Migration Requires Urokinase Receptor (uPAR) for Integrin Redistribution.

VEGF activates endothelial cells to migrate and invade surrounding tissues, an initial event in the angiogenic process. For invasion, the coordinated localized formation of a proteolytic repertoir is necessary. Focusing the urokinase receptor towards the leading edge of migrating cells provides such armor and inhibition of uPA binding to its receptor inhibits invasion of endothelial cells. In addition integrins continuously have to form focal contacts at the leading edge. Thus the spatial proximity between the localized proteases and the matrix seems to be essential for matrix degradation. In order to allow cell locomotion integrins have to release their ligands when they reach the trailing end and are subsequently endocytosed and redistributed to newly formed focal adhesions in a repetitive process.We here describe a new role of uPAR in regulating integrin redistribution. We have previously reported that stimulation of human endothelial cells by VEGF (50ng/ml) via its receptor flk-1 induces pro-uPA activation, when bound to uPAR. Subsequently a uPA/PAI-1/uPAR-complex is formed, which thereafter is endocytosed via a LDL-R family member. We now show that by this process beta-1 integrins are co-internalized in clathrin coated vesicles via a uPAR dependent mechanism. Subsequently, endocytosed uPAR recycles to focal adhesions where it co-localizes with integrin alpha-v/beta-3. Disrupting this chain of events, either by (1) RAP - a specific inhibitor of the LDL-R family - or by (2) uPAR depletion (using uPAR−/− cells or cleaving the GPI-anchor of uPAR by PI-PLC), beta-1 integrins are no longer internalized after VEGF stimulation. Under the same circumstances the migratory response of endothelial cells toward VEGF is impaired in vitro as shown by video-based migration assays and in vivo as demonstrated by matrigel angiogenesis assays. Next, we generated synthetic peptides interfering with uPAR/integrin interaction, which inhibit not only VEGF-induced integrin redistribution, but also diminish VEGF-induced endothelial cell migration, significantly.These data suggest that in VEGF-induced cell migration uPAR plays a central role not only in focusing proteolytic activity, but also in initial integrin redistribution. Interference with this process could be a therapeutic target for diseases depending on VEGF-induced angiogenesis.

Novel Role of Plasmin in Mobilization and of uPAR in Retention of Hematopoietic Stem/Progenitor Cells in the Bone Marrow Niche: Therapeutic Implications.

Hematopoietic stem/progenitor cells (HSC/HPCs) are retained in the bone marrow (BM) niche via receptor-ligand interactions and mobilized from the BM after proteolytic degradation of these retention complexes. Yet, the proteinases and retention signals involved remain incompletely identified. Here, we studied the role of the plasminogen proteinase system with its plasminogen activators PA) tPA and uPA, and active plasmin (Pli) in chemo- and G-CSF-induced mobilization of HPCs and HSCs.Therefore, 5-fluorouracil (5-FU) or G-CSF were administered to mice lacking plasminogen (Plg−/−), tPA (tPA−/−), uPA (uPA−/−), both activators (tPA−/−uPA−/−), uPAR (uPAR−/−), PAI-1 (PAI-1−/−), or α2-antiplasmin (α2-AP−/−).5-FU treatment in WT mice elevated Pli activity in BM plasma 5-fold, killed 10% of WT mice, and increased number/proliferation of HSC/HPCs in the surviving mice, with full hematopoietic recovery after 3 weeks. In contrast, up to 75% of 5-FU-treated Plg−/−and tPA−/−uPA−/− demised in the early phase of recovery, with reduced number/proliferation of HSC/HPCs and delayed hematopoietic recovery. Following G-CSF, deficiency of Plg or inhibition of Pli by tranexamic acid reduced HPC expansion and HSC translocation in the BM, resulting in impaired HPC/HSC mobilization, by up to 55% and 75%, respectively.Further analysis using uPA−/− and tPA−/− mice revealed that uPA was critical for 5-FU-induced mobilization, whereas tPA was crucial for G-CSF-induced mobilization. In addition, analysis of mice lacking MMP-2, -3, -9, and -12 revealed that 5-FU- and G-CSF-induced mobilization required predominantly MMP-9 and -3, respectively. MMP-3 and MMP-9 activities upon mobilization were reduced in Plg−/− mice, suggesting that Pli activates these MMPs. In the absence of Plg, degradation of fibronectin in the BM and production of soluble Kit ligand (but not SDF1 α) were impaired, indicating additional downstream targets of Pli.uPAR is a membrane-anchored receptor for uPA, which is cleaved into a soluble form (suPAR) by Pli and other proteinases. Interestingly, uPAR was expressed on BM-derived HPC/HSCs, and uPAR deficiency reduced their retention within the BM niche in vitro and in vivo. Furthermore, uPAR−/− mice showed poor HSC/HPC mobilization in response to 5-FU and G-CSF, while suPAR administration in WT mice amplified G-CSF-induced mobilization. Moreover, suPAR levels in the BM were increased in WT but not in Plg−/− mice during mobilization, indicating, all together, that uPAR might be a novel retention signal for HSC/HPCs in the BM niche.Finally, increased Pli activity in PAI-1−/− and α2-AP−/− mice, and WT mice treated with tenecteplase (i.e. recombinant tPA variant used for clinical thrombolysis) enhanced G-CSF-induced mobilization. Importantly, initial results suggest that thrombolytic treatment of individuals after acute myocardial infarction also seemed to stimulate HPC mobilization, extrapolating our findings to man.In conclusion, these genetic and pharmacological data reveal, for the first time, a novel role for uPAR as a retention signal for HSC/HPCs in the BM and suggest that strategies to increase PA or Pli activity might offer novel therapeutic opportunities for HSC/HPC mobilization.

Mitogenic signaling of urokinase receptor-deficient kidney fibroblasts: actions of an alternative urokinase receptor and LDL receptor-related protein.

The urokinase receptor (uPAR) attenuates myofibroblast recruitment and fibrosis in the kidney. This study examined the role of uPAR and its co-receptor LDL receptor-related protein (LRP) in the regulation of kidney fibroblast proliferation and extracellular signal-regulated kinase (ERK) signaling. Compared with uPAR+/+ cells, uPAR-/- kidney fibroblasts were hyperproliferative. UPAR-/- fibroblast proliferation was 60% inhibited by an ERK kinase inhibitor. LRP protein was reduced and extracellular accumulation of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) proteins were greater in uPAR-/- cultures. Addition of functional uPA protein or LRP antisense RNA significantly increased ERK signaling and cell mitosis in both genotypes. Enhanced uPAR-/- fibroblast proliferation was reversed by a recombinant nonfunctional uPA peptide. The density of cell-bound fluor-uPA was similar between uPAR-/- and uPAR+/+ fibroblasts (78 +/- 6 versus 92 +/- 16 units). These data suggest that uPAR-deficient kidney fibroblasts express lower levels of its scavenger co-receptor LRP, resulting in greater extracellular accumulation of uPA and PAI-1. Enhanced proliferation of uPAR-/- fibroblasts seems to be mediated by uPA-dependent ERK signaling via an alternative urokinase receptor.

Species-specific urokinase receptor ligands reduce glioma growth and increase survival primarily by an antiangiogenesis mechanism

Species-specific urokinase receptor (uPAR) ligands with improved pharmacokinetics were generated by site-specific mutagenesis and amino-terminal pegylation. These molecules were used to probe the role of uPAR in brain tumor progression and angiogenesis. The ligands blocked endothelial cell tube formation in Matrigel in a species-specific manner and reduced both baseline and uPA amino-terminal fragment-stimulated cell migration on vitronectin gradients. Treatment of U87MG gliomas implanted orthotopically in mice with single species-specific or combination uPAR ligands resulted in significant decreases in tumor size, which translated to increases in survival time, and which were most significant when the murine-specific ligand was included. Further analysis of tumors showed that the reduced sizes were correlated with a decrease in tumor cell proliferation and mean vessel density and an increase in tumor cell apoptosis. In addition, a large increase in collagen deposition was observed in the treated groups. Statistical analysis showed that the combination therapy demonstrated a clear synergy as compared to the individual agent treatments. These results suggest that the major role of the uPAR system in brain tumor progression is in the stromal compartment and particularly in neovascularization, a hallmark of invasive brain tumors.

Thrombospondin-1 up-regulates tumor cell invasion through the urokinase plasminogen activator receptor in head and neck cancer cells

Background We have previously demonstrated that thrombospondin-1 (TSP-1) is expressed in squamous cell carcinomas of the head and neck. We have also shown that TSP-1 promotes tumor cell invasion through up-regulation of the urokinase plasminogen activator receptor (uPAR), in adenocarcinoma models. We now determined the role of TSP-1 in the regulation of uPAR expression and tumor cell invasion in squamous cell carcinoma of the head and neck cells. Materials and methods KB squamous cell carcinoma of the head and neck cells were used. The effect of TSP-1 on uPAR and its ligand, urokinase plasminogen activator (uPA), expression were determined by ELISA. The effect of TSP-1 on KB tumor cell invasion was determined in a modified Boyden chamber collagen invasion assay. To determine the role of uPAR on TSP-1-mediated KB tumor cell invasion, we used the three following different strategies: (a) blocking uPAR or its ligand, uPA, with neutralizing antibodies; (b) enzymatic cleavage of uPAR with glycosylphosphatidylinositol (GPI)-specific phospholipase C; and (c) inhibition of plasminogen binding by using ϵ-aminocaproic acid. Results TSP-I up-regulated uPAR and uPA expression 3- and 4-fold, respectively. TSP-1 up-regulated KB tumor cell invasion 5-fold. Inhibition of uPAR blocked the TSP-1-mediated up-regulation of KB tumor cell invasion. Conclusions Our data support a central role for TSP-1 in the regulation of uPAR and tumor cell invasion in squamous cell carcinomas of the head and neck cells. Furthermore, uPAR seems to play a crucial role in TSP-1-mediated squamous cell carcinoma of the head and neck tumor cell invasion.

Urokinase-type plasminogen activator receptor is involved in mediating the apoptotic effect of cleaved high molecular weight kininogen in human endothelial cells.

Cleaved high molecular weight kininogen (HKa) has been shown to inhibit in vivo neovascularization and induce apoptosis of endothelial cells. We have shown that HKa-induced apoptosis correlated with its antiadhesive effect and was regulated by extracellular matrix (ECM) proteins. In this study, we identified the urokinase-type plasminogen activator receptor (uPAR) as a target of HKa activity at the endothelial cell surface. Anti-uPAR antibodies blocked the apoptotic effect of HKa. Further studies revealed that uPAR formed a signaling complex containing integrin alpha(v)beta3 or alpha5beta1, caveolin, and Src kinase Yes in endothelial cells. HKa physically disrupted the formation of this complex in a manner that paralleled its apoptotic effect. For the first time, our results provide a mechanistic explanation for the previous observation that HKa selectively induces apoptosis of endothelial cells grown on vitronectin, but not cells grown on fibronectin. These data also resolve the controversial role of uPAR in mediating the apoptotic and antiadhesive activities of HKa.

Clinical Significance of Urokinase‐Type Plasminogen Activator Receptor (uPAR) Expression in Cancer

AbstractFor Abstract see ChemInform Abstract in Full Text.