Addition Of uPA Research Articles

Expression of urokinase-type plasminogen activator system in non-metastatic prostate cancer.

To investigate the prognostic role of expression of urokinase-type plasminogen activator system members, such as urokinase-type activator (uPA), uPA-receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1), in patients treated with radical prostatectomy (RP) for prostate cancer (PCa). Immunohistochemical staining for uPA system was performed on a tissue microarray of specimens from 3121 patients who underwent RP. Cox regression analyses were performed to investigate the association of overexpression of these markers alone or in combination with biochemical recurrence (BCR). Decision curve analysis was used to assess the clinical impact of these markers. uPA, uPAR, and PAI-1 were overexpressed in 1012 (32.4%), 1271 (40.7%), and 1311 (42%) patients, respectively. uPA overexpression was associated with all clinicopathologic characteristics of biologically aggressive PCa. On multivariable analysis, uPA, uPAR, and PAI-1 overexpression were all three associated with BCR (HR: 1.75, p < 0.01, HR: 1.22, p = 0.01 and HR: 1.20, p = 0.03, respectively). Moreover, the probability of BCR increased incrementally with increasing cumulative number of overexpressed markers. Decision curve analysis showed that addition of uPA, uPAR, and PAI-1 resulted in a net benefit compared to a base model comparing standard clinicopathologic features across the entire threshold probability range. In subgroup analyses, overexpression of all three markers remained associated with BCR in patients with favorable pathologic characteristics. Overexpression of uPA, uPAR, and PAI-1 in PCa tissue were each associated with worse BCR. Additionally, overexpression of all three markers is informative even in patients with favorable pathologic characteristics potentially helping clinical decision-making regarding adjuvant therapy and/or intensified follow-up.

199 EFFECT OF UROKINASE TYPE PLASMINOGEN ACTIVATOR DURING IN VITRO MATURATION OF BOVINE OOCYTES AND EARLY EMBRYO PRODUCTION

The study of molecules involved in the oocyte maturation and early embryo development is crucial to improve the conditions of in vitro embryo production. The plasminogen activation system is involved in the initial steps of reproduction and urokinase type plasminogen activator (uPA) is expressed in the granulosa cells (GC) of bovine cumulus-oocyte complexes (COC). The aim of this study was to analyse the effect of uPA in bovine oocyte in vitro maturation (IVM). We have analysed whether the addition of uPA or the inhibition of its proteolytic activity affects IVM. We have evaluated (1) nuclear and cytoplasmic maturation, (2) developmental competence, and (3) oocyte and GC gene expression. Immature COC were obtained by aspiration of ovarian follicles of slaughtered heifers. Selected COC were in vitro-matured in 4 groups: control, 10 nM uPA, dimethyl sulfoxide control, and 100 μg mL–1 amiloride, a specific inhibitor of uPA proteolytic activity (4 replicates). After 24 h of IVM, oocytes of each treatment were either fixed and stained with Hoëscht (to evaluate nuclear maturation) or LCA-FITC to analyse the cortical granules distribution as a marker of cytoplasmic maturation (n = 10/group per treatment per replicate). In addition, pools of 10 oocytes and their separated GC were snap-frozen to analyse by qRT-PCR their profile expression of genes related with apoptosis (BAX, BCL2, TP53, SHC1), cell junctions (GJA1, TJP1), cell cycle (CCNB1), oxidative stress (SOD2, GPX1), oocyte quality (BMP15, GDF9), and serpin proteases inhibitors (SRP1, SRP5), normalised respect 2 housekeeping genes (H2AFZ, ACTB). The remaining COC were fertilised (Day 0) and in vitro cultured to evaluate developmental competence in terms of cleavage rate (Day 2) and blastocyst yield (Days 7–9). All data were analysed by one-way ANOVA. In the presence of amiloride, a significant reduction in the oocyte maturation was observed at both levels; 83.33% of oocytes remained in vesicle stage, and 75.0% showed a cortical granules distribution of type I. The rest of the groups (62.67%, 62.65%, and 60.29%) reached metaphase II (MII), and 51.66%, 32.9%, and 25.44% showed granule distribution of type III. For embryo development, the amiloride group had a cleavage rate and blastocyst yield significantly lower compared with controls (23.23% v. 80.85% and 83.83%; 4.45% v. 25.21% and 25.21%, respectively), whereas uPA treatment had no effect (84.28% and 24.16%). In presence of amiloride, the transcript levels of TJP1, GJA1, and CCNB1 were up-regulated, whereas SOD2, SRP1, and SRP5 were down-regulated in GC compared with all other groups (P &lt; 0.05). No differences were observed in oocytes gene expression between treatments. In conclusion, although the addition of exogenous uPA does not alter oocyte maturation, the specific inhibition of the proteolytic activity of uPA by amiloride reduced IVM of bovine oocytes and altered the expression of genes related to cell junctions, cell cycle, oxidative stress, and serpins of GC, indicating that proteolytic activity of uPA is critical for oocyte IVM in bovines.

Novel aspects of urokinase function in the injured lung: role of α2-macroglobulin.

The level of active urokinase (uPA) is decreased in lung fluids of patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) whereas α(2)-macroglobulin (α(2)-M), a plasma proteinase inhibitor, is a major component of these fluids. Since there have been reports describing the ability of α(2)-M to form complexes with uPA in vitro, we hypothesized that α(2)-M may interact with uPA in the lung to modulate its biological activity. Pulmonary edema fluids and lung tissues from patients with ALI/ARDS were evaluated for the presence of uPA associated with α(2)-M. Complexes between α(2)-M and uPA were detected in alveolar edema fluids as well as in lungs of patients with ALI/ARDS where they were located mainly in close proximity to epithelial cells. While uPA bound to α(2)-M retains its amidolytic activity towards low-molecular-weight substrates, it is not inhibited by its main physiological inhibitor, plasminogen activator inhibitor 1. We also investigated the functional consequences of formation of complexes between uPA and α(2)-M in vitro. We found that when α(2)-M:uPA complexes were added to cultures of human bronchial epithelial cells (BEAS-2B), activation of nuclear factor-κB as well as production of interleukin-6 and -8 was substantially suppressed compared with the addition of uPA alone. Our findings indicate for the first time that the function of uPA in patients with ALI/ARDS may be modulated by α(2)-M and that the effects may include the regulation of the fibrinolytic and signaling activities of uPA.

Abstract 2456: External uPA overexpresses and interacts with Lhx-2 and induces a pancreatic cancer stem cell phenotype

Abstract Although the body of knowledge surrounding the molecular profile of pancreatic cancer has substantially increased, survival rates have not changed for over four decades. Previous studies have demonstrated that urokinase plasminogen activator (uPA) is frequently present in a higher concentration in the serum of pancreatic cancer patients. uPA is involved in extracellular matrix (ECM) degradation and has been correlated with the malignant transformation of cancer cells. Additionally, recent reports have also shown the translocation of uPA to the nucleus without binding to uPAR. However, its role in the nucleus is still unclear. Existing evidence suggest that a hypoxic environment forces cancer cells to acquire stem cell characteristics, causing recurrence after chemotherapy and radiation. In the present study, we have attempted to correlate the roles of uPA and hypoxia in cancer stem cell transformation. Initially, to determine the interaction of uPA with Lhx-2, we conducted immunoprecipitation and immunolocalization studies in MIA PaCa-2 and PANC-1 pancreatic cancer cell lines. Further, to determine the role of exogenous uPA on Lhx-2 expression in an uPA-null background, cells were downregulated for uPA and followed by supplementation with purified uPA under normoxic as well as hypoxic conditions. To further resolve whether Lhx-2 is involved in the activation of CD133 and STRO-1 expression in pancreatic cells, uPA and/or Lhx-2 were knocked down under normoxic or hypoxic conditions and the expression levels of CD133 and STRO-1 were determined. In addition, cell lysates were screened for stem cell markers using stem cell-specific antibody arrays. To determine the role of uPA in tumorigenicity after hypoxia, MIA PaCa-2 or PANC-1 hypoxic cells were subcutaneously implanted in nude mice followed by injection of siRNA for uPA or Lhx-2 and 100 nM of uPA at the tumor site, and tumor growth was determined. From the immunoprecipitation and immunolocalization studies, we observed that uPA interacts with Lhx-2 both in the cytoplasm and the nucleus of MIA PaCa-2 and PANC-1 pancreatic cancer cells. Further, we observed that the addition of external uPA caused an increase in the expression of Lhx-2. The addition of uPA under hypoxic conditions resulted in increased expression of CD133 and STRO-1 whereas suppression of Lhx-2 retarded the overexpression of CD133 and STRO-1. Antibody array analysis indicated that hypoxic cells had increased expression of stem cell markers when supplemented with uPA, which was abolished by suppression of Lhx-2. From the animal studies, we observed that hypoxic MIA PaCa-2 or PANC-1 cells supplemented with external uPA were able to form subcutaneous tumors within 30 days of implantation. Taken together, our results demonstrate that in a hypoxic environment, uPA activates the expression of stem cell markers mediated by the expression of Lhx-2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2456. doi:10.1158/1538-7445.AM2011-2456

Abstract 1534: Laminin binding integrin functions in human bone metastatic cancer

Abstract Laminin binding integrins are major determinants of cancer cell migration, invasion and metastasis. In human prostate cancer, both A6B1 and A3B1 laminin-binding integrins are expressed on tumor cell surfaces as observed in either prostatectomy or needle biopsy specimens. In human breast cancer, the A6 integrin is expressed in both primary tumors and in metastatic lesions. Integrin A6B1 is post-translationally modified on the tumor cell surface by urokinase-type plasminogen activator (uPA), producing a structural variant, A6pB1. While the A6pB1 form is devoid of the ligand binding domain, A3B1 is fully functional and not subject to cleavage. In this study, we observed the continued expression of the A6B1 integrin on tumor cell surfaces within human bone metastatic lesions as well as in human tumor xenografts growing within bone. The cleavage function of the A6 integrin was found to be constitutively expressed on nine different cancer cell lines derived from breast, prostate, lung, pancreatic tumors. Interestingly, five different normal cell types expressing the adhesion active A6B1 integrin did not have the constitutive cleavage function. However, the cleavage function in normal cells was inducible by the exogenous addition of uPA. Selection of successive bone resident cancer cells selects for constitutive cleavage of A6 integrin and increasing expression of A3B1 integrin. Taken together, these data suggest that the cleavage function is constitutively activated in cancer specimens and opens the possibility cleavage mediated release from laminin binding may be coordinated with A3 dependent adhesion. (Supported in part by CA23752 and the TACMASS core service of the Arizona Cancer Center). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1534. doi:10.1158/1538-7445.AM2011-1534

A kringle-containing protease with plasminogen-like activity in the basal chordate Branchiostoma belcheri

Plg (plasminogen), a member of the serine protease superfamily, is a key component constituting the fibrinolytic system, and its evolutionary origin remains unknown during the course of animal evolution. In the present study, we isolated a cDNA, designated BbPlgl, encoding a kringle-containing protease with plasminogen-like activity from the basal chordate Branchiostoma belcheri. The deduced protein, BbPlgl, consisted of 430 amino acids, which is structurally characterized by the presence of an N-terminal signal peptide of 16 amino acids, 2 kringle domains with a Lys-binding site structure, a serine protease domain with the putative tPA (tissue plasminogen activator)-cleavage site (between Arg297 and Val298), the catalytic triad His237-Asp288-Ser379 expected for protease function, and a potential N-linked glycosylation site, all characteristic of Plgs. Besides, the recombinant refolded BbPlgl was readily activated by human uPA (urokinase plasminogen activator), and exhibited Plg-like activity. BbPlgl was also able to auto-activate at neutral and alkaline pH at 4 degrees C without the addition of uPA, and the activation was accelerated by addition of human uPA. These results demonstrate that BbPlgl is a novel member of the Plg family, with a domain structure of K-K-SP (kringle-kringle-serine protease) lacking the PAN domain, pushing the evolutionary origin of Plg to the protochordate. In addition, BbPlgl displays a tissue-specific expression pattern in B. belcheri, with the most abundant expression in the hepatic caecum and hind-gut, agreeing with the notion that the hepatic caecum of amphioxus is the precursor of the vertebrate liver.

Plasmin-mediated Proteolysis Is Required for Hepatocyte Growth Factor Activation during Liver Repair

The physiological relevance of the activation of hepatocyte growth factor (Hgf) by the plasminogen (Plg) system of proteases and its contribution to tissue repair are largely undefined. Here, we investigated whether the defective liver repair in mice lacking Plg is due to impaired activation of Hgf. Loss of Plg in vivo suppressed Hgf activation and signaling through its Met tyrosine kinase receptor. Without Plg, hepatocytes were unresponsive to Hgf-induced proliferation and migration, with a more pronounced impairment in hepatocyte movement within the hepatic environment. Most notably, circumventing the defect in proteolytic activation of Hgf by the downstream expression of an activated Met receptor corrected the functional deficits and improved liver repair in Plg-deficient mice. These findings support a fibrinolysis-unrelated role for Plg in modulating cell proliferation and migration by activation of Hgf.

Extracellular alpha 6 integrin cleavage by urokinase-type plasminogen activator in human prostate cancer

During human prostate cancer progression, the integrin α6β1 (laminin receptor) is expressed on the cancer cell surface during invasion and in lymph node metastases. We previously identified a novel structural variant of the α6 integrin called α6p. This variant was produced on the cell surface and was missing the β-barrel extracellular domain. Using several different concentrations of amiloride, aminobenzamidine and PAI-1 and the urokinase-type plasminogen activator (uPA) function-blocking antibody (3689), we showed that uPA, acting as a protease, is responsible for production of α6p. We also showed that addition of uPA in the culture media of cells that do not produce α6p, resulted in a dose-dependent α6p production. In contrast, the addition of uPA did not result in the cleavage of other integrins. Using α2-antiplasmin and plasmin depleted media, we observed that uPA cleaves the α6 integrin directly. Further, 12- o-tetradecanoyl-phorbol-13-acetate (TPA) induced the production of α6p, and this induction was abolished by PAI-1 but not α2-antiplasmin. Finally, the α6p integrin variant was detected in invasive human prostate carcinoma tissue indicating that this is not a tissue culture phenomenon. These data, taken together, suggest that this is a novel function of uPA, that is, to remove the β-barrel ligand-binding domain of the integrin while preserving its heterodimer association.

Recycling of the urokinase receptor upon internalization of the uPA:serpin complexes.

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA) but readily internalizes and degrades uPA:serpin complexes in a process that requires the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR-LRP). This process is accompanied by the internalization of uPAR which renders it resistant to phosphatidylinositol-specific phospholipase C (PI-PLC). In this paper we show that during internalization of uPA:serpins at 37 degrees C, analysed by FACScan, immunofluorescence and immunoelectron microscopy, an initial decrease of cell surface uPAR was observed, followed by its reappearance at later times. This effect was not due to redistribution of previously intracellular receptors, nor to the surface expression of newly synthesized uPAR. Recycling was directly demonstrated in cell surface-biotinylated, uPA:PAI-1-exposed cells in which biotinylated uPAR was first internalized and subsequently recycled back to the surface upon incubation at 37 degrees C. In fact, uPAR was resistant to PI-PLC after the 4 degrees C binding of uPA:PAI-1 to biotinylated cells, but upon incubation at 37 degrees C PI-PLC-sensitive biotinylated uPAR reappeared at the cell surface. Binding of uPA:PAI-1 by uPAR, while essential to initiate the whole process, was, however, dispensable at later stages as both internalization and recycling of uPAR could be observed also after dissociation of the bound ligand from the cell surface.

Role of plasminogen activator and of 92-KDa type IV collagenase in glioblastoma invasion using an in vitro matrigel model.

The invasive nature of human gliomas represents a major factor in preventing their total resection. The exact nature of the underlying mechanisms of tumor cell invasion are still unclear. In this study, we have quantitatively assayed a glioblastoma cell line for its ability to migrate through a polycarbonate filter coated with matrigel which contains a complex of multiple basement membrane components. At 48 h the glioblastoma cell line (U251) showed a rate of invasiveness of 42% and also dependent on the concentration of matrigel. The U251 cell line produced a urokinase type plasminogen activator and a 92-KDa type IV collagenase. Both enzymes were inhibited by the addition of uPA and 92-KDa type IV collagenase antibodies. Those same antibodies reduced the invasion rate of U251 cells from 42% to 12 and 21%, respectively. Similarly, the addition of epsilon-aminocaproic acid (a plasmin inhibitor) or tissue inhibitor of metalloprotease (TIMP2, a collagenase inhibitor) reduced the invasiveness of U251 cells from 42% to 14% and 10%, respectively. Additionally, the other two glioblastoma cell lines (LG11, UWR1) and astrocytes showed a rate of invasiveness at 41%, 61% and 12%, respectively. Finally, the addition of hyaluronic acid to the matrigel, a constituent of brain extracellular matrix, enhanced the rate of invasion. These findings provide evidence for the role of serine proteases and metalloproteases in facilitating the invasion of extracellular matrix components by glioblastoma cell line and suggest a therapeutic role for protease inhibitors in attempting to minimize the invasive propensity of gliomas.