uPA mRNA Research Articles

Unequal tissue expression of proteins from the PA/PAI system, myoma necrosis, and uterus survival after uterine artery occlusion

Objective To further the understanding of the mechanism behind myoma necrosis and uterus survival after uterine artery occlusion. Method Myomal and myometrial gene transcription and protein expression of tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), and plasminogen activator inhibitor type 1 (PAI-1) were investigated in 30 uteri. Results Compared with myometrial expression, myomal expression was less for uPA mRNA (1.34% ± 2.18% vs 2.84% ± 2.97%; P < 0.05) and greater for PAI-1 mRNA (2.67% ± 1.82% vs 1.27% ± 1.99%; P < 0.05), but the difference in tPA mRNA expression was not significant. Accordingly, compared with myometrial expression, myomal expression was less for the uPA protein ( P < 0.05) and greater for the PAI-1 protein ( P < 0.05), but the difference in tPA protein was not significant. Conclusion The PA/PAI system was found to be expressed differently in myomas and the myometrium, which may play a role in uterus survival and myoma necrosis after uterine artery occlusion.

Urokinase Expression by Tumor Suppressor Protein p53

Lung carcinoma (H1299) cells deficient in p53 (p53(-/-)) express large amounts of urokinase-type plasminogen activator (uPA) protein and uPA mRNA, and exhibit slower degradation of uPA mRNA than that of p53-expressing nonmalignant Beas2B human airway epithelial cells. Expression of p53 protein in H1299 cells, upon transfection with p53 cDNA, suppressed basal as well as uPA-induced expression of uPA protein in both conditioned media and cell lysates, and decreased the level of steady-state uPA mRNA primarily due to increased uPA mRNA turnover. Inhibition of p53 expression by RNA silencing (SiRNA) in Beas2B cells enhanced basal and uPA-mediated uPA protein and mRNA expression with stabilization of uPA mRNA. Purified p53 binds to the uPA mRNA 3' untranslated region (UTR) in a sequence-specific manner and endogenous uPA mRNA associates with p53 protein isolated from Beas2B cytosolic extracts. p53 binds to a 35-nucleotide uPA 3'UTR sequence and insertion of this sequence into beta-globin mRNA accelerates degradation of otherwise stable beta-globin mRNA. These observations confirm a new role for p53 as a uPA mRNA binding protein that down-regulates uPA mRNA stability and decreases cellular uPA expression.

H‐Ras increases urokinase expression and cell invasion in genetically modified human astrocytes through Ras/Raf/MEK signaling pathway

Previous study reported that the activation of Ras pathway cooperated with E6/E7-mediated inactivation of p53/pRb to transform immortalized normal human astrocytes (NHA/hTERT) into intracranial tumors strongly resembling human astrocytomas. The mechanism of how H-Ras contributes to astrocytoma formation is unclear. Using genetically modified NHA cells (E6/E7/hTERT and E6/E7/hTERT/Ras cells) as models, we investigated the mechanism of Ras-induced tumorigenesis. The overexpression of constitutively active H-RasV12 in E6/E7/hTERT cells robustly increased the levels of urokinase plasminogen activator (uPA) mRNA, protein, activity and invasive capacity of the E6/E7/hTERT/Ras cells. However, the expressions of MMP-9 and MMP-2 did not significantly change in the E6/E7/hTERT and E6/E7/hTERT/Ras cells. Furthermore, E6/E7/hTERT/Ras cells also displayed higher level of uPA activity and were more invasive than E6/E7/hTERT cells in 3D culture, and formed an intracranial tumor mass in a NOD-SCID mouse model. uPA specific inhibitor (B428) and uPA neutralizing antibody decreased uPA activity and invasion in E6/E7/hTERT/Ras cells. uPA-deficient U-1242 glioblastoma cells were less invasive in vitro and exhibited reduced tumor growth and infiltration into normal brain in xenograft mouse model. Inhibitors of Ras (FTA), Raf (Bay 54-9085) and MEK (UO126), but not of phosphatidylinositol 3-kinase (PI3K) (LY294002) and of protein kinase C (BIM) pathways, inhibited uPA activity and cell invasion. Our results suggest that H-Ras increased uPA expression and activity via the Ras/Raf/MEK signaling pathway leading to enhanced cell invasion and this may contribute to increased invasive growth properties of astrocytomas.

Regulation of the Plasminogen Activator System in the MDA‐MB‐231 Breast Cancer Cell Line

The objective of this study was to determine the contribution of the urokinase‐type plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI‐1) to invasiveness of the MDA‐MB‐231 breast cancer cell line, and to study control of uPA and PAI‐1 expression. Amiloride, a small molecule inhibitor of uPA, and a uPA blocking antibody inhibited invasion of the 231 cells by 30% while a PAI‐1 blocking antibody increased invasion by 50%. Treatment with the PI3K inhibitor LY294002 increased PAI‐1 protein levels in conditioned media while differences in uPA were not detected. However, another PI3K inhibitor, wortmannin, decreased uPA in conditioned media. Semi‐quantitative RT‐PCR indicated LY294002 and wortmannin increased PAI‐1 mRNA levels with a concomitant decrease in uPA mRNA. Amiloride treatment decreased PI3K activity resulting in increased PAI‐1 in conditioned media. Treatment with the receptor associated peptide (RAP), which blocks activity of the low density like lipoprotein receptor responsible for binding and clearing uPA‐PAI‐1 complexes from the cell surface, decreased uPA mRNA and protein levels. These results confirm uPA and PAI‐1 are important contributors to MDA‐MB‐231 invasiveness, PI3K positively regulates uPA while negatively regulating PAI‐1, and the PI3K pathway which regulates uPA and PAI‐1 expression may be activated by the recognition of uPA‐PAI‐1 complexes at the cell surface.

Prognostic Significance of Growth Factors and the Urokinase-Type Plasminogen Activator System in Pancreatic Ductal Adenocarcinoma

To determine the diagnostic and prognostic significance of growth factors and the urokinase-type plasminogen activator (uPA) system in pancreatic ductal adenocarcinoma (PDAC) using a multigene assay. Messenger RNA (mRNA) expression of 15 genes from epidermal growth factor receptor, insulin-like growth factor (IGF), and uPA families were measured in 46 PDAC tissue samples using quantitative real-time reverse transcription-polymerase chain reaction. These results were compared with those of the uninvolved adjacent (AP) tissue and benign mucinous cystadenomas (BMC). The mRNA expression was evaluated using logistic regression and receiver operating characteristic area under the curve (ROC AUC) analyses. Their relationship with prognosis was tested by Cox regression multivariate analysis. All genes were overexpressed in most of the PDAC tissue. When compared with AP tissue, the median expression values for IGF-binding protein 3 (IGFBP-3) and uPA receptor (uPAR) was 9.8- and 9.6-fold, respectively. Expression levels of uPA, uPAR, IGF-I, and IGFBP-3 mRNA were significantly greater in PDAC than in BMC. The IGFBP-3 mRNA expression demonstrated greatest ROC AUC values for PDAC versus AP tissue (ROC AUC, 0.745; 95% confidence interval [CI], 0.65-0.86); whereas ROC AUC values were greatest for uPAR when PDAC was compared with BMC (ROC AUC, 0.846; 95% CI, 0.76-0.94). The combination of uPA, uPAR, and IGF-I significantly improved discriminatory power (ROC AUC, 0.965; 95% CI, 0.93-1.00). The IGFBP-3, uPA, plasminogen activator inhibitor-2, and International Union Against Cancer stage had a significant influence on survival, but the effect of IGFBP-3 was lost after multivariate stepwise analysis. These results indicate that there is an influence of IGF system in tumor progression from BMC to PDAC, whereas the uPA/uPAR system has the greater influence on survival in PDAC.

Quantitative RT-PCR assays for the determination of urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 mRNA in primary tumor tissue of breast cancer patients: Comparison to antigen quantification by ELISA

Urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor type 1 (PAI-1) play a key role in tumor-associated processes such as the degradation of extracellular matrix proteins, tissue remodeling, cell adhesion, migration, and invasion. High antigen levels of uPA and PAI-1 in tumor tissue of various solid malignant tumors, including breast cancer, are associated with poor patient prognosis. In the present study, we examined whether analysis of uPA and PAI-1 mRNA expression represents an alternative to the measurement of the respective antigen levels in breast cancer. Highly sensitive quantitative real-time PCR (QPCR) assays, based on the LightCycler technology, were established to quantify uPA and PAI-1 mRNA expression in breast cancer cell lines as well as in tumor tissue of breast cancer patients. mRNA concentrations were normalized to the expression level of the housekeeping gene h-G6PDH. The respective uPA and PAI-1 antigen concentrations were determined by established ELISA formats. QPCR mean interassay variation coefficients were 11% (uPA) and 8% (PAI-1). In breast cancer cell lines, mRNA and antigen values were highly correlated for both uPA and PAI-1 (each: rs=0.95; p<0.001). In contrast, correlations between uPA/PAI-1 mRNA and protein in the breast cancer samples were found to be distinctly weaker or not significant. Thus, quantitative determination of mRNA expression for both factors does not mirror antigen levels in breast cancer tissue, possibly due to posttranscriptional regulation. Except for nodal status being inversely correlated with uPA mRNA levels, no significant interrelations were observed between uPA or PAI-1 mRNA expression and clinicopathological parameters. On the protein level, elevated uPA and PAI-1 values were associated with a negative steroid hormone receptor status. In conclusion, the implementation of mRNA quantification of uPA and PAI-1 in breast tumors is unable to serve as a one-to-one substitution for antigen determination by ELISA.

Protein Kinase C Synergistically Stimulates Tumor Necrosis Factor-α–Induced Secretion of Urokinase-Type Plasminogen Activator in Human Dental Pulp Cells

Plasminogen activator (PA) is the enzyme converting plasminogen to its active form, plasmin, involved in various physiological and pathological phenomena. The conversion is catalyzed by two types of PA, urokinase-type PA (uPA) and tissue-type PA (tPA). When human dental pulp cells were stimulated by the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), PA activity in the conditioned medium was increased, indicating that TNF-alpha provoked PA secretion. The TNF-alpha-induced PA release was significantly enhanced in the presence of phorbol-12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator. The PKC inhibitor Ro31-8220 abolished the effect of PMA on the PA release. The activity of PA secreted from the cells stimulated by TNF-alpha and PMA was reduced by immunoprecipitation using anti-uPA antibody. PMA failed to enhance the TNF-alpha-induced expression of uPA mRNA. These results suggest that protein kinase C synergistically enhances the secretion of uPA in TNF-alpha-stimulated human dental pulp cells.

Detailed Technical Analysis of Urine RNA-Based Tumor Diagnostics Reveals ETS2/Urokinase Plasminogen Activator to Be a Novel Marker for Bladder Cancer

The noninvasive detection of RNA tumor markers in body fluids represents an attractive diagnostic option, but diagnostic performance of tissue-derived markers is often poorer when measured in body fluids rather than in tumors. We aimed to develop a procedure for measurement of tumor RNA in urine that would minimize donor-dependent influences on the results. RNA isolated from urinary cell pellet, cell-depleted fraction, and whole urine was quantified by reverse transcription quantitative-PCR. The donor-dependent influence of urine background on individual steps of the standardized procedure was analyzed using an external RNA standard. Using a test set of samples from 61 patients with bladder cancer and 37 healthy donors, we compared 4 putative RNA tumor markers identified in whole urine with 5 established, tissue-derived RNA tumor markers for the detection of bladder cancer. Of the markers analyzed by this system, the RNA ratio of v-ets erythroblastosis virus E26 oncogene homolog 2 (avian; ETS2) to urokinase plasminogen activator (uPA) enabled the most specific (100%) and sensitive (75.4%) detection of bladder cancer from whole urine, with an area under the curve of 0.929 (95% CI 0.882-0.976). The described methodology for RNA marker detection in urine appears to be clinically applicable. The ratio of ETS2 mRNA to uPA mRNA in urine is a potential marker for bladder cancer.

Abstract 2316: Myocardial PAI-1 Over-expression Mediates Graft Sclerosis in Cardiac Transplant Recipients with Idiopathic Dilated Cardiomyopathy

Idiopathic dilated cardiomyopathy (DCM) is associated with reduced myocardial vascular density and increased collagen deposition with subsequent myocardial fibrosis and heart failure. The urokinase plasminogen activator (uPA)/plasminogen activator inhibitor (PAI) system plays an important role in extracellular matrix homeostasis as well as in preserving the integrity of the vascular endothelial cell (EC) intimal layer and EC regeneration, proliferation and motility. However, the involvement of the uPA/PAI system in DCM and graft remodeling following cardiac transplantation remains unclear. Gene expression of uPA, the uPA receptor uPAR and PAI-1 was examined in left ventricular (LV) biopsies of explanted hearts in 24 ischemic cardiomyopathy (ICM) and 60 DCM patients and 12 controls by real time RT-PCR and Western blotting. Vascular endothelial growth factor (VEGF) and uPA/PAI interplay was examined in stimulated DCM and ICM myocardium in vitro. Expression of uPA and PAI-1 mRNA was evaluated in endomyocardial biopsies (EMB) at 1, 2, 3, 4, 7, 12, 24 and 52 weeks post-transplant. The mRNA levels of uPA and uPAR were unchanged in both ICM and DCM patients compared to controls. PAI-1 mRNA and protein were up-regulated in DCM compared to ICM and controls (p&lt;0.01). Active and inactive uPA protein was down-regulated in DCM and ICM compared to controls (p&lt;0.05). VEGF stimulation in vitro did not affect uPA, uPAR or PAI-1 expression. Following transplantation, PAI-1 expression returned to baseline control levels in EMB from both patient groups, although PAI-1 expression increased at 24 and 52 weeks post-transplant in DCM patients (p&lt;0.01 compared to post-transplant weeks 6 and 12). These data indicate a significant PAI-1 up-regulation associated with uPA down-regulation in DCM, independent of VEGF, which was temporarily corrected by cardiac transplantation. However, recurring up-regulation of myocardial PAI-1 levels at 6 and 12 months post-transplant suggest a persistent PAI-1-related molecular pathology in DCM. We have previously shown increased endothelin-1 expression and collagen deposition in grafts of DCM patients at 1 year post-transplant. The present data point to a previously unknown role of PAI-1 in mediating graft sclerosis.

Altered expression of urokinase-type plasminogen activator and plasminogen activator inhibitor in high-risk soft tissue sarcomas

In recent years, classification of soft-tissue sarcomas (STS) has improved with cytogenetic analyses, but their clinical behavior is still not easily predictable. The aim of this study was to detect alterations in the urokinase-type plasminogen system, involved in tumor growth and invasion, by comparing mRNA levels of its components with those of paired normal tissues, and relating them with patient clinical course. Real-time PCR was performed on human STS cell lines and tissues from highly malignant STS, including leiomyosarcomas and malignant fibrous histiocytomas, to evaluate the expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1). Immunohistochemistry of gene products was also performed. Median mRNA values of all genes studied were higher in tumors than in paired normal tissues. In agreement with data on STS cell lines, significant up-regulation for uPA and PAI-1 genes compared to reference values was seen. Moreover, different levels of expression were related to histotype and metastatic phenotype. There was accordance between uPA mRNA and protein expression, while immunodetection of PAI-1 product was weak and scattered. Clearly, the controversial role of PAI-1 protein requires further biological analyses, but evident involvement of uPA/PAI-1 gene overexpression in STS malignancy may highlight a molecular defect useful in discriminating STS high-risk patients.

Gene expression changes of urokinase plasminogen activator and urokinase receptor in rat testes at postnatal stages

To investigate the gene expression changes of urokinase plasminogen activator (uPA)/urokinase receptor (uPAR) in rat testes at postnatal stages and explore the effects of uPA/uPAR system on the rat spermatogenesis. The mRNAs of uPA and uPAR in rat testes were measured by using real-time quantitative polymerase chain reaction (PCR) at postnatal days 0, 5, 10, 15, 21, 28, 35, 42, 49 and 56, respectively. The tendencies of uPA and uPAR mRNA expression were similar at most postnatal stages except for D(0). The expression of uPAR mRNA in rats testes was relatively higher than that of uPA at postnatal D(0), and both were decreased until D(21), increased obviously at postnatal D(28), reached a peak at postnatal D(35), then declined sharply at postnatal D(42) and retained at a low level afterwards. The uPA/uPAR system may be strongly linked to spermiation and spermatogenesis via regulating germ cell migration and proliferation, as well as promoting the spermiation and detached residual bodies from the mature spermatids.

A role for the urokinase-type plasminogen activator system in amyotrophic lateral sclerosis

There is substantial evidence, implicating extracellular matrix (ECM) regulating enzymes in the pathogenesis of motor neuron degeneration in amyotrophic lateral sclerosis (ALS). The most important ECM-degrading proteases are serine proteases (plasminogen activators, PA) and matrix metalloproteinases (MMPs). Since the role of MMPs in ALS has been addressed recently, we investigated the expression of the serine protease urokinase-type plasminogen activator (uPA) and its receptor in ALS. Employing rtPCR, zymography and immunohistochemistry we analyzed the expression of uPA and its receptor uPAR in spinal cord tissue of ALS cases and in the G93A SOD1 transgenic mouse. In the ventral horn of the spinal cord of ALS cases we found increased uPAR staining of motor neurons. In G93A mice, the expression profile of uPA and uPAR mRNA was significantly increased starting at the age of 90 days as compared to non-transgenic littermates. The uPA-dependent plasminogen activation in G93A mice at endstage increased markedly compared with controls and immunostaining of the spinal cord from G93A mice revealed increased uPAR immunostaining in neurons. To determine the functional role of uPA, we investigated the effect of intraperitoneal (i.p.) administration of the uPA inhibitor WX-340 (10 mg/kg), starting at the age of 30 days ( n = 18). Treatment with WX-340 prolonged ( p < 0.05) survival of the animals (135 ± 2 vs. 126 ± 3) as well as improving rotarod performance. Our experiments demonstrate that uPA and its receptor are expressed in ALS patients and in an animal model of ALS. Early inhibition with a synthetic uPA inhibitor prolonged the life of the transgenic animals. These findings indicate that the urokinase-type plasminogen activator system may play a role in the complex pathogenesis of ALS.

EXPRESSION OF EXTRACELLULAR MATRIX DEGRADING PROTEASES AND VEGF IN FOLLICLES BEFORE AND AFTER GnRH APPLICATION AND AFTER OVULATION IN THE COW

There is evidence that extracellular matrix (ECM) proteases like matrix metalloproteases (MMP) and plasminogen activator (PA) as well as angiogenic factors VEGF are implicated in tissue remodeling during final follicle development and ovulation in the ovary. The objective of this study was to evaluate the expression pattern of MMP, PA and VEGF system members in time defined follicle classes before and after GnRH application and after ovulation in cow. We characterized in our study the mRNA patterns of the members of MMP (MMP-1, MMP-2, MMP-14, MMP-19, TIMP-1, TIMP-2), PA (tPA, uPA, uPAR, PAI-1) and VEGF family (VEGF-A Isoform 120, 164 and 188 and VEGFR-1 and VEGFR-2) after FSH follicle stimulation and GnRH induced ovulation. Ovaries containing preovulatory follicles or new corpora lutea (CL) were collected at 0, 4, 10, 20, 25 (follicles) and 60h (CL day 1–2) relative to injection of GnRH to induce an LH surge (n=5/group). The expression of MMP-1 mRNA increased rapidly (610-fold upregulation) 4h after GnRH injection (during LH surge) and remained high during the whole experimental period. The MMP-2, MMP-14 and TIMP-2 mRNA expression did not change throughout the experiment when compared with the time before GnRH application. In contrast MMP-19 mRNA expression did not change in follicle classes during periovulation but increased significantly only after ovulation (CL day 1–2). The TIMP-1 mRNA increased significantly 4h after GnRH application (during LH surge) in order to increase again after ovulation. From the PA family, tPA mRNA increased 4h after GnRH (during LH surge) and remained high during the whole experimental period. The uPA mRNA expression did not change in follicle classes during periovulation but increased significantly only after ovulation. The uPAR and PAI-1 mRNA expression increased in follicle group at 4h after GnRH application (during LH surge), in order to increase again after ovulation. The VEGF120 and VEGF164 mRNA decreased 20h after GnRH and periovulation phase. VEGF188 mRNA already decreased at 10h after GnRH followed by a further decline during periovulation. All VEGF isoform transcripts increased again after ovulation. The mRNA of both VEGF receptors decreased significantly during periovulation phase, followed by a significant increase in early CL. All VEGF isoforms are upregulated after ovulation suggesting an active participation in regulation of angiogenesis in early bovine CL. In conclusion, the temporal and spatial expression pattern of ECM proteases (MMP and PA systems) and VEGF during periovulation suggest them to be important mediators of LH dependent rupture of bovine follicle and early CL formation (angiogenesis). Supported by DFG, BE 3189/2–1. (poster)

THE PLASMINOGEN ACTIVATOR SYSTEM IS REGULATED BY PROSTAGLANDINS IN THE PRIMATE PERIOVULATORY FOLLICLE

Successful ovulation requires prostaglandin (PG) production by periovulatory granulosa cells. Administration of a PG synthesis inhibitor prevented periovulatory events such as follicle rupture, while coadministration of PG synthesis inhibitor and PGE2 restored ovulation in primates. Follicle rupture requires proteolytic degradation of the follicle wall. The plasminogen activators (PA) tPA and uPA degrade extracellular matrix and likely contribute to follicle rupture. Plasminogen activator inhibitor type 1 (PAI-1) inactivates tPA and uPA. In several species, follicular PA activity increases in response to the ovulatory gonadotropin surge, with maximal PA activity measured just before ovulation. We hypothesized that 1) the ovulatory gonadotropin surge increases PA activity in the primate periovulatory follicle and 2) PGs mediate the ability of gonadotropin to increase ovarian PA activity, leading to follicle rupture in primates. Female cynomolgus macaques received gonadotropins to stimulate the development of multiple follicles. Granulosa cells, follicular fluid, and whole ovaries (n=3-4/time point) were obtained at 0, 12, 24 and 36 hours (h) after hCG administration; ovulation occurs about 40 hours after hCG. In some animals, hCG was coadministered with the PG synthesis inhibitor celecoxib to block follicular PG production during the periovulatory interval. mRNAs for tPA, uPA and PAI-1 were quantitated by real time RT-PCR in granulosa cells obtained throughout the periovulatory period. tPA and uPA mRNA levels tended to peak 12 h after hCG administration; celecoxib administration had no effect on tPA or uPA mRNA levels. PAI-1 mRNA was low 0-24 h after hCG but increased 17-fold above 0 h levels to peak 36 h after hCG administration; celecoxib treatment decreased PAI-1 mRNA to a level intermediate between levels measured at 0 h and 36 h hCG. Using immunofluorescence, granulosa cell tPA protein was low/non-detectable in ovaries obtained 0 h after hCG. Intense staining for tPA was present in ovaries obtained 12-36 h after hCG. In contrast, tPA protein was low/nondetectable in the ovaries obtained from animals treated for 36 h with hCG + celecoxib. uPA was not detected in monkey follicles by immunofluorescence. Similar to tPA, PAI-1 protein was non-detectable before hCG administration, was present 12-36 h after hCG administration, and was non-detectable in hCG + celecoxib-treated animals. Follicular PA activity as detected by in situ zymography was low before hCG administration and readily detectable 12, 24 and 36 h after hCG administration; PA activity was considerably attenuated by preincubation with an anti-tPA antibody. Animals treated for 36 h with hCG + celecoxib had low ovarian PA activity. In conclusion, tPA and PAI-1 expression by primate periovulatory follicles increased after exposure to an ovulatory dose of gonadotropin; hCG also increased ovarian PA activity. The PG synthesis inhibitor celecoxib reduced hCG-stimulated tPA and PAI-1 expression as well as PA activity. These data support the hypothesis that PGs mediate the ability of the ovulatory gonadotropin surge to increase PA activity in the granulosa cell layer, contributing to follicle rupture at ovulation. (platform)

Cell Surface-Bound Plasminogen Regulates Hepatocyte Proliferation Through a uPA-Dependent Mechanism

Plasminogen and plasminogen activators play important roles in liver regeneration. Previously, we found that plasminogen potentiates hepatocyte proliferation in the primary culture of rat hepatocytes. Here, we examined how exogenous plasminogen affects the downstream events leading to cell proliferation. The addition of plasminogen to hepatocytes increased urokinase-type plasminogen activator (uPA) activity, but did not affect matrix metalloproteinase (MMP)-9 or MMP-2 activities. To increase uPA activity, plasminogen was required to bind the hepatocyte surface through the lysine-binding site of plasminogen molecule, but neither uPA mRNA nor uPA receptor (uPAR) mRNA was affected by the exogenous plasminogen. In addition, treatment of hepatocytes with an uPA inhibitor, p-aminobenzamidine, inhibited the plasminogen-induced and even EGF-induced hepatocyte proliferation. These results suggest that plasminogen-related control of hepatocyte proliferation is exerted topically by producing a hyperfibrinolytic state on the cellular surface involving the activation of uPA.

Iron Stimulates Urokinase Plasminogen Activator Expression and Activates NF-kappa B in Human Prostate Cancer Cells

: Urokinase-type plasminogen activator (uPA) on prostate cancer cell surfaces mediates pericellular proteolysis and destruction of extracellular matrix barriers to tumor invasion and metastasis. Increased expression of tumor-associated uPA leads to enhanced tumor dissemination and poor cancer outcomes in men with prostate cancer. Expression of uPA is regulated in part by the oxidant-sensitive transcription factor, NF-kappa B (NF-κB), which is activated by intracellular reactive oxygen intermediates (ROI). This study examined the effect of iron on the production of ROI, activation of NF-κB and expression of uPA in the human prostate cancer cell line, PC-3. Treatment of PC-3 cells with iron in the form of ferric nitrilotriacetate (FeNTA) in the absence of added transferrin resulted in a dose-dependent increase in cellular ferritin content in both the presence and absence of neutralizing antibody to the transferrin receptor. Cellular uptake of iron resulted in stimulation of intracellular ROI production, and increases in uPA mRNA, antigen, and activity. Concurrent treatment with the iron chelator, desferrioxamine (DFO) abrogated these effects, and treatment with DFO alone inhibited constitutive uPA production. Finally, we observed nuclear translocation, and therefore activation of NF-κB in response to iron exposure. We conclude that iron enters PC-3 cells via a non-transferrin dependent pathway and increases uPA expression. Our data indicate that one mechanism by which iron may stimulate uPA production is through the generation of intracellular ROI and activation of NF-κB-mediated signaling pathways.

Inhibition of the Urokinase-Type Plasminogen Activator by Triplex-Forming Oligonucleotides in Rat Sertoli Cells: A New Contraceptive Alternative?

Urokinase-type plasminogen activator (uPA), expressed in Sertoli cells in the testis, is closely related with tight junctions of blood-testis barrier (BTB), and it has been considered as a potential contraceptive target. In the present study, the antigene effects of triplex-forming oligodeoxynucleotides (TFO) targeting uPA in rat Sertoli cells were investigated in vitro. The stable triplexes, formed by uPA specific TFOs under physiological conditions, were tested by means of electrophoretic mobility shift assays (EMSA). Although tPA, another form of plasminogen activators (PAs), partially compensated the lose of PAs activities, uPA mRNA and protein were significantly reduced as demonstrated by real-time reverse transcription PCR and a chromogenic assay, after the treatment of Sertoli cells with uPA specific TFOs at a concentration of 330 nM. The capacity of TFOs resistance to nuclease degradation was enhanced by the phosphorothioated on the backbone of the oligonucleotides. Our results indicated that the TFOs can downregulate uPA expression and uPA might be an alternative contraceptive target.

Effects of hypoxia and reoxygenation on the expression levels of the urokinase-type plasminogen activator, its inhibitor plasminogen activator inhibitor type-1 and the urokinase-type plasminogen activator receptor in human head and neck tumour cells

One aim during oncological radiation therapy is to induce reoxygenation in hypoxic tumours in order to enhance radiosensitivity and ultimately increase cell death. In squamous cell carcinomas of the head and neck (SCCHN), hypoxia is considered a pivotal physiological modulator for malignant progression, whereby the plasminogen activation system is involved in overlapping functions such as the shaping of the extracellular matrix, cell proliferation and signal transduction. Since little is known about reoxygenation and the plasminogen activation system in SCCHN, three human SCCHN cell lines (BHY, FaDu, and CAL27) and a non-transformed control cell line (VH7) were exposed to hypoxic (<0.5% O2) conditions for up to 72 h and subsequently reoxygenated for 24 h at normoxic conditions. The mRNA expression of the urokinase-type plasminogen activator (uPA), the plasminogen activator inhibitor type-1 (PAI-1) and the urokinase-type plasminogen activator receptor (uPAR) was assessed by means of real-time semi-quantitative RT-PCR, and the protein expression was determined by immunoenzymometric quantification (ELISA). Both hypoxia and reoxygenation induced statistically significant changes in uPA, PAI-1 and uPAR mRNA and protein levels in the various cell lines investigated, showing that oxygen tension is a strong modulator of the plasminogen activation system in vitro. However, no uniform correlation pattern was found between the mRNA and protein levels analysed over all three time-points (24, 48, and 72 h) and oxygen treatment variants (N, H, R) nor according to oxygen treatment conditions over all three time-points. Changes in oxygen tension could therefore be modulating the fragile balance between the various components of the plasminogen activation system in SSCHN ultimately leading to an increased tumour matrix disruption, alterations in cell invasiveness, and the dissemination of tumour cells to distant organs.

Progestin inhibits and thrombin stimulates the plasminogen activator/inhibitor system in term decidual stromal cells: implications for parturition

Labor is associated with 'decidual activation' with increased proteolysis and extracellular matrix degradation. The balance between plasminogen activator inhibitor-1 (PAI-1) and urokinase (uPA) and tissue-type plasminogen activator (tPA) is an important determinant of proteolytic activity at the maternal-fetal interface. Thrombin released at the time of placental abruption (decidual hemorrhage) is known to promote decidual proteolysis and uterine contractions. This study investigates the separate and interactive effects of steroid hormones and thrombin on PAI-1, uPA, and tPA expression by term decidual cells (DCs). Term DCs were isolated by enzymatic digestion, purified, and depleted of leukocytes. Cells were treated with estradiol (10(-8) mol [E2]), medroxyprogesterone acetate (10(-7) mol [MPA]), both, or vehicle for 7 days. After 24-hour incubation with or without thrombin (0.1-2.5 U/mL), levels of PAI-1, uPA, and tPA in conditioned supernatant were measured by specific ELISA and Western blotting. Levels of PAI-1 and uPA mRNA were measured by quantitative RT-PCR. In the cultured term DCs, ELISA measurements indicated that basal output of PAI-1 was about 2 logs higher than that of either uPA or tPA (2.5 +/- 0.7 ng/mL per microg protein, 13.4 +/- 6.3 pg/mL per microg protein, and 25.4 +/- 10.8 pg/mL per microg protein, respectively). Although E2 alone did not affect PAI-1 output, MPA and E2+MPA significantly enhanced PAI-1 production (2.5 +/- 0.7 vs 8.2 +/- 2.0 ng/mL per microg protein for E2+MPA [3.3-fold]; P < .01). By contrast, uPA output was inhibited by exposure to MPA (13.4 +/- 6.3 vs 2.6 +/- 1.1 pg/mL per microg protein [0.2-fold]; P < .05), whereas tPA production was not affected by MPA. Thrombin did not significantly affect uPA and tPA production by term DCs. In contrast, in E2+MPA-treated term DCs, thrombin, a hemostatic proinflammatory cytokine, selectively increased PAI-1 output in a dose-dependent fashion, which could be blocked by the selective thrombin inhibitor, hirudin. Western blotting confirmed the effects of MPA and thrombin in elevating secreted levels of PAI-1. Unlike the increase in PAI-1 output elicited by thrombin, term DCs were unresponsive to either of the classic proinflammatory cytokines, TNFalpha or IL-1beta. Corresponding effects on PAI-1 mRNA levels were elicited by MPA and thrombin as seen for PAI-1 protein expression, suggesting that these up-regulatory effects are transcriptionally mediated. Progestin enhanced PAI-1 and inhibited uPA expression by term DCs, which may explain in part the pregnancy-prolonging properties of progesterone as a consequence of inhibited proteolytic activity at the maternal-fetal interface. Thrombin augmented PAI-1 expression in the absence of increased uPA or tPA expression by term DCs, suggesting that abruption-associated decidual proteolysis and preterm labor is mediated primarily by thrombin-enhanced matrix metalloproteinase expression rather than an indirect effect on the plasminogen activator/inhibitor system.

Stromal cells associated with early invasive foci in human mammary ductal carcinoma in situ coexpress urokinase and urokinase receptor

The transition from ductal carcinoma in situ (DCIS) of the breast to invasive ductal carcinoma is facilitated by proteolytic degradation of basement membrane. The transition can be identified as microinvasive foci in a small proportion of DCIS lesions. We have previously found that MMP-13 is frequently expressed in such foci. To establish whether plasmin-directed proteolysis is likely to be involved in early invasion, we have here studied the expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) in human DCIS lesions with and without microinvasion. uPA mRNA was detected in periductal stromal cells in all of 9 DCIS lesions with microinvasion and in 2 of 9 DCIS lesions without microinvasion by in situ hybridization. The uPA mRNA signal was seen in numerous stromal cells in microinvasive areas together with MMP-13 mRNA expressing cells. Double immunofluorescence analyses, using emission fingerprinting, showed that the uPA expressing stromal cells included both myofibroblasts and macrophages. The early invasive carcinoma cells were negative for uPA. uPAR immunoreactivity was focally upregulated in periductal stromal cells in all of the 9 DCIS lesions with microinvasion and in only 2 of the 9 DCIS lesions without microinvasion. uPAR was seen in both macrophages and myofibroblasts in microinvasive areas, and it was evident that uPA and uPAR colocalized in both fibroblast-like cells and macrophage-like cells. We conclude that periductal macrophages and myofibroblasts are strongly involved in the initial steps of breast cancer invasion by focally upregulating the expression of the plasminogen activation system and MMP-13.