uPA mRNA Expression Research Articles

Pharmacological induction of heat shock protein exerts neuroprotective effects in experimental intracerebral hemorrhage

Heat shock proteins (HSPs) are reported to reduce inflammation and apoptosis in a variety of brain insults. Geranylgeranylacetone (GGA), developed as an antiulcer in Japan, has been known to induce HSP70 and to exert cytoprotective effects. In this study, we investigated whether GGA, as a specific HSP inducer, exerts therapeutic effects in experimentally induced intracerebral hemorrhage (ICH). ICH was induced with male Sprague–Dawley rats via the collagenase infusion. GGA (800 mg/kg) was administered via oral tube according to various schedules of treatment. The treatment with GGA, beginning before the induction of ICH and continuing until day 3, showed the reduction of brain water content and the increased level of HSP70 protein, as compared to the treatment with vehicle, although GGA started after the induction of ICH or administered as a single dose before ICH failed to up-regulate HSP70 and to reduce brain edema. The rats treated with GGA exhibited better functional recovery than those treated with vehicle. In the pre- and post- treatment group, inflammatory cells and cell death in the perihematomal regions were found to have been decreased. The treatment of GGA inhibited the mRNA expression of MMP-9, uPA, IL-6 and MIP-1, with concomitant increment of eNOS and phosphorylated STAT3 and Akt after ICH. We demonstrated that GGA induced a reduction in the brain edema along with marked inhibitory effects on inflammation and cell death after ICH.

Antimetastatic activity of insulin-like growth factor binding protein-3 in lung cancer is mediated by insulin-like growth factor–independent urokinase-type plasminogen activator inhibition

Insulin-like growth factor binding protein-3 (IGFBP-3), a major IGF-binding protein in human serum, regulates the growth of non-small cell lung cancer (NSCLC) cells through IGF-dependent and IGF-independent mechanisms. However, the role of IGFBP-3 in lung cancer metastasis is not well known. In the present study, we showed that noncytotoxic doses of adenoviral or recombinant IGFBP-3 significantly decreased the migration and invasion of H1299 and A549 NSCLC cells. Furthermore, treatment of human lung fibroblasts with recombinant IGFBP-3 suppressed their ability to stimulate the invasion of H1299 cells. Overexpression of IGFBP-3 markedly reduced lung metastasis of A549 cells in an experimental animal model system and prolonged the survival time of the animals. Urokinase-type plasminogen activator (uPA) inhibitor treatment or uPA small interfering RNA transfection of A549 and H1299 cells resulted in a significant decrease in invasion. Corresponding ELISA, Western blot, gelatin zymogram, and semiquantitative reverse transcription-PCR analyses revealed that IGFBP-3 reduced the expression of uPA mRNA through IGF-independent mechanisms. The specific role of uPA in anti-invasive activity of IGFBP-3 was further confirmed in NSCLC cells, in which uPA expression/activity was suppressed by the transfection with synthetic small interfering RNA or by the treatment with uPA inhibitor or induced by the infection with an adenoviral vector. IGFBP-3 also decreased the expression/activity of matrix metalloproteinase-2 through IGF-dependent but uPA-independent pathways. Taken together, our data suggest that IGFPB-3 effectively block uPA- and matrix metalloproteinase-2-stimulated invasion pathways, ultimately reducing lung cancer cell metastasis. Our findings indicate that IGFBP-3 may be a promising anti-invasive and antimetastatic therapeutic agent in lung cancer.

Smad4-dependent Regulation of Urokinase Plasminogen Activator Secretion and RNA Stability Associated with Invasiveness by Autocrine and Paracrine Transforming Growth Factor-β

Metastasis is a primary cause of mortality due to cancer. Early metastatic growth involves both a remodeling of the extracellular matrix surrounding tumors and invasion of tumors across the basement membrane. Up-regulation of extracellular matrix degrading proteases such as urokinase plasminogen activator (uPA) and matrix metalloproteinases has been reported to facilitate tumor cell invasion. Autocrine transforming growth factor-beta (TGF-beta) signaling may play an important role in cancer cell invasion and metastasis; however, the underlying mechanisms remain unclear. In the present study, we report that autocrine TGF-beta supports cancer cell invasion by maintaining uPA levels through protein secretion. Interestingly, treatment of paracrine/exogenous TGF-beta at higher concentrations than autocrine TGF-beta further enhanced uPA expression and cell invasion. The enhanced uPA expression by exogenous TGF-beta is a result of increased uPA mRNA expression due to RNA stabilization. We observed that both autocrine and paracrine TGF-beta-mediated regulation of uPA levels was lost upon depletion of Smad4 protein by RNA interference. Thus, through the Smad pathway, autocrine TGF-beta maintains uPA expression through facilitated protein secretion, thereby supporting tumor cell invasiveness, whereas exogenous TGF-beta further enhances uPA expression through mRNA stabilization leading to even greater invasiveness of the cancer cells.

Increased Expression of Urokinase-Type Plasminogen Activator mRNA Determines Adverse Prognosis in ErbB2-Positive Primary Breast Cancer

To evaluate and validate mRNA expression markers capable of identifying patients with ErbB2-positive breast cancer associated with distant metastasis and reduced survival. Expression of 60 genes involved in breast cancer biology was assessed by quantitative real-time PCR (qrt-PCR) in 317 primary breast cancer patients and correlated with clinical outcome data. Results were validated subsequently using two previously published and publicly available microarray data sets with different patient populations comprising 295 and 286 breast cancer samples, respectively. Of the 60 genes measured by qrt-PCR, urokinase-type plasminogen activator (uPA or PLAU) mRNA expression was the most significant marker associated with distant metastasis-free survival (MFS) by univariate Cox analysis in patients with ErbB2-positive tumors and an independent factor in multivariate analysis. Subsequent validation in two microarray data sets confirmed the prognostic value of uPA in ErbB2-positive tumors by both univariate and multivariate analysis. uPA mRNA expression was not significantly associated with MFS in ErbB2-negative tumors. Kaplan-Meier analysis showed in all three study populations that patients with ErbB2-positive/uPA-positive tumors exhibited significantly reduced MFS (hazard ratios [HR], 4.3; 95% CI, 1.6 to 11.8; HR, 2.7; 95% CI, 1.2 to 6.2; and, HR, 2.8; 95% CI, 1.1 to 7.1; all P < .02) as compared with the group with ErbB2-positive/uPA-negative tumors who exhibited similar outcome to those with ErbB2-negative tumors, irrespective of uPA status. After evaluation of 898 breast cancer patients, uPA mRNA expression emerged as a powerful prognostic indicator in ErbB2-positive tumors. These results were consistent among three independent study populations assayed by different techniques, including qrt-PCR and two microarray platforms.

C-phycocyanin transcriptionally regulates uPA mRNA through cAMP mediated PKA pathway in human fibroblast WI-38 cells

We have previously demonstrated the efficacy of c-phycocyanin in up-regulation of urokinase-type plasminogen activator (uPA) in bovine endothelial cell line. However, the mechanism of action and pathway elucidation in uPA regulation is unclear. In experiments reported here, we have investigated the mechanism of action of c-phycocyanin (c-pc) induced uPA gene modulation in human fibroblast (WI-38) cell line. ELISA test confirmed that c-pc increased the uPA antigen whereas PAI-1 antigen level was unaffected. Treatment of cells with c-pc significantly ( P < 0.05) enhanced the uPA mRNA level in a dose (50 μg/ml) and time dependent (up to 4 h) manner. This effect of c-pc was abolished by treatment with dichloro-1-β- d-ribofuranosyl benzamidazole (DRB) (10 μg/ml). Co-treatment of c-pc with 200 μg/ml cycloheximide (CHX), translation inhibitor, resulted in over accumulation of uPA mRNA. These results suggest that uPA induction by c-pc is transcriptionally regulated and does not require de novo protein synthesis. We also provide evidence that c-pc stimulates uPA gene through cAMP dependent pathway as adenylyl cyclase (AC) inhibitor, dideoxyadenosine (DDA) significantly inhibited the uPA mRNA expression and co-treatment with adenylyl cyclase analogue, dBcAMP recovered the effect of c-pc on gene activity. Furthermore, the present investigation provides evidence on the regulatory pathway involved in the c-pc stimulus. C-pc induced uPA expression was completely inhibited by PKA inhibitor (KT 5200), indicating the regulation is dependent on PKA pathway. Elimination of PKC pathway components by prolonged incubation with excess amount of phorbol 12-myristate 13-acetate (PMA) failed to abolish the c-pc effect on uPA expression indicating the regulation is independent of PKC pathway. Taken together, our data indicate that uPA gene regulation by c-pc is transcriptionally controlled through cAMP mediated PKA pathway.

Calcitonin inhibits invasion of breast cancer cells: Involvement of urokinase-type plasminogen activator (uPA) and uPA receptor

There is a growing body of evidence indicating that calcitonin (CT) and calcitonin receptor (CTR) are involved in the regulation of cell growth, differentiation, and survival and in tissue development. However, the precise functional role of CT/CTR in breast cancer is still unknown. It is well established that the urokinase plasminogen activator (uPA) system plays an important role in breast cancer invasion and metastasis. The goal of this study was to investigate the effects of CT on regulation of the uPA system and invasive capacity of breast cancer cells. In the highly invasive MDA-MB-231 cell line, 10(-8) M CT decreased both uPA and uPAR mRNA and protein expression which was associated with inhibition of the extracellular signal-regulated kinase (ERK) 1/2 pathway. Furthermore, two weeks of CT administration to nude mice inhibited the expression of uPA mRNA in primary tumors by 25% (P<0.05), as compared to control, untreated animals. CT also inhibited the invasiveness of MDA-MB-231 cells by 37% (10(-8) M CT, P<0.05), as determined by a Matrigel invasion assay. To the best of our knowledge, this is the first report describing a direct effect of CT on breast cancer cell invasion. Our data might suggest a close link between CT signaling, the uPA-mediated pathway, and breast cancer invasion.

LIM kinase 1 increases tumor metastasis of human breast cancer cells via regulation of the urokinase‐type plasminogen activator system

Mammalian LIM kinase 1 (LIMK1) phosphorylates and inactivates the actin-binding and -depolymerizing factor cofilin and induces actin cytoskeletal changes. LIMK1 is reported to play an important role in cell motility, but the mechanism of induction of cell motility and the role of LIMK1 in tumor growth, angiogenesis and invasion are poorly understood. Here we show that expression of LIMK1 in MDA-MB-435 human breast cancer cells enhanced cell proliferation and cell invasiveness and promoted in vitro angiogenesis. Since tumor metastasis requires degradation of the extracellular matrix by the serine protease urokinase type plasminogen activator (uPA), we examined the role of LIMK1 in the regulation of uPA/uPAR system. LIMK1 overexpression in breast cancer cells upregulated the uPA system, increased uPA promoter activity, induced uPA and uPAR mRNA and protein expression and induced uPA secretion. In contrast, cells transfected with the catalytically inactive LIMK mutant D460N-LIMK1 did not exhibit these phenotypic changes. Blocking antibodies against uPA and uPAR suppressed LIMK1-induced cell invasiveness. In addition, LIMK1 overexpression increased tumor growth in female athymic nude mice, promoted tumor angiogenesis and induced metastasis to livers and lungs, possibly by increasing uPA expression in the tumors. Finally, LIMK1 and uPAR were coordinately overexpressed in human breast tumors. These results suggested an important role for LIMK1 signaling in breast cancer tumor growth, angiogenesis and invasion and a regulatory connection between LIMK1 and the uPA system.

Correlative studies on uPA mRNA and uPAR mRNA expression with vascular endothelial growth factor, microvessel density, progression and survival time of patients with gastric cancer

To investigate the correlations between the expression of urokinase-type plasminogen activator (uPA) mRNA, uPA receptor (uPAR) mRNA and vascular endothelial growth factor (VEGF) protein and clinicopathologic features, microvessel density (MVD) and survival time. In situ hybridization and immuno-histochemistry techniques were used to study the expressions of uPA mRNA, uPAR mRNA, VEGF and CD34 protein in 105 gastric carcinoma specimens. Expressions of uPA mRNA, uPAR mRNA and VEGF protein were observed in 61 (58.1%) cases, 70 (66.7%) cases and 67 (63.8%) cases, respectively. The uPA mRNA and uPAR mRNA positive expression rates in infiltrating-type cases (73.7%, 75.4%), stage III-IV (72.1%, 75.4%), vessel invasion (63.2%, 69.9%), lymphatic metastasis (67.1%, 74.4%) and distant metastasis (88.1%, 85.7%) were significantly higher than those of the expanding-type (chi2 = 15.57, P = 0.001; chi2 = 6.91, P = 0.046), stage I-II (chi2 = 19.22, P = 0.001; chi2 = 16.75, P = 0.001), non-vessel invasion (chi2 = 11.92, P = 0.006; chi2 = 14.15, P = 0.002), non-lymphatic metastasis (chi2 = 28.41, P = 0.001; chi2 = 22.5, P = 0.005) and non-distant metastasis (chi2 = 12.32, P = 0.004; chi2 = 17.42, P = 0.002; chi2 = 11.25, P = 0.012; chi2 = 18.12, P = 0.002). The VEGF positive expression rates in infiltrating-type cases (75.4%), stage III-IV (88.5%), vessel invasion (82.9%), lymphatic metastasis (84.3%) and distant metastasis (95.2%) were significantly higher than those of the expanding-type (chi2 = 9.61, P = 0.021), stage I-II (chi2 = 16.66, P = 0.001), non-vessel invasion (chi2 = 29.38, P = 0.001), non-lymphatic metastasis (chi2 = 18.68, P = 0.005), and non-distant metastasis (chi2 = 22.72, P = 0.007; chi2 = 21.62, P = 0.004). The mean MVD in the specimens positive for the uPA mRNA, uPAR mRNA and VEGF protein was markedly higher than those with negative expression groups. Moreover, a positive relation between MVD and uPA mRNA (rs = 0.199, P = 0.042), uPAR mRNA (rs = 0.278, P = 0.035), and VEGF (rs = 0.398, P = 0.048) expressions was observed. The mean survival time in cases with positive uPA mRNA, uPAR mRNA and VEGF protein expression or MVD value > or = 54.9 was significantly shorter than those in cases with negative expression or MVD value < 54.9. uPA and uPAR expressions are correlated with enhanced VEGF-induced tumor angiogenesis and may play a role in invasion and nodal metastasis of gastric carcinoma, thereby serving as prognostic markers of gastric cancer.

The antithrombotic action of propylene glycol mannite sulfate (PGMS)

In order to reveal the antithrombotic mechanism of propylene glycol mannite sulfate (PGMS) on gene level and explore the correlation between PGMS and fibrinolytic system, the urokinase-type plasminogen activator (uPA) mRNA expression and uPA activity were detected in vitro and in vivo with semi-quantitative reverse transcription (RT)-PCR and chromogenic method. The results showed that PGMS was able to increase uPA activity in dosage-dependent manner in rat plasma, which resulted in the increase of rat fibrinolytic activity. The change of rat uPA mRNA expression with PGMS was similar to that of uPA activity. The results of experiments with cultured cells were similar to those results of experiments in vivo. The uPA mRNA expression and activities of cultured cells showed increases with PGMS in a concentration-dependent manner. All of these results indicated that PGMS may induce the expression of uPA mRNA. The increase of uPA mRNA expression may therefore reflect, in part, an increase in the stability of uPA mRNA and/or processing of nuclear uPA transcription, which, in turn, may increase the uPA activity in rat plasma and trigger the fibrinolytic system.

Prognostic significance of plasminogen system in advanced prostate cancer: A pilot study

4766 Background: Plasminogen system plays a significant role in cancer cell invasion and metastasis. Elevated tumor urokinase plasminogen activator (uPA), its receptor- uPAR, and inhibitor, plasminogen activator inhibitor (PAI-1) levels are associated with poor prognosis in several cancers (Manders et al. Cancer 2004, Duffy MJ. Clin Biochem 2004).We conducted two preliminary studies to evaluate its prognostic significance in advanced prostate cancer. Methods: In the first study fresh tissue from patients with local stage (n=9 prostatectomy specimens) and advanced stage (n=6 transurethral prostate resections) was collected. uPA and plasminogen (PLG) mRNA expression was evaluated by real-time polymerase chain reaction. Transcript abundance was normalized to 18s (control) and t-test used for detecting differences between means of both groups. In a second study plasma uPA, uPAR and PAI-1 levels were measured in advanced stage patients (pts) undergoing androgen deprivation (n=40) who were followed for overall and progression free survival (PFS). Mean values of all variables with and without progression were compared and univariate Cox proportional hazard ratios (HR) calculated. Results: uPA and PLG gene expression was found in all fresh tumor specimens (table). Elevated expression was observed in advanced stage specimens. In the second study 25/40 pts were found to have events (radiological progression) after a median follow up of 36.1 months, of these 17 have died. Prognostically only elevated uPAR levels were found to be statistically significant for PFS. Mean uPAR in pts with events versus without events was 11.04 vs 17.89 ng/ml (HR= 0.75, p= 0.028). Conclusions:This is the first analysis evaluating the role for the plasminogen system in advanced prostate cancer. Elevated uPAR was associated with 25% increase in PFS. We propose a prospective study of various plasminogen system proteases in advanced prostate cancer to better define its impact on survival. No significant financial relationships to disclose.

Protein synthesis and urokinase mRNA metabolism

Expression of the urokinase-type plasminogen activator (uPA) is under tight regulation by hormones, cytokines and growth factors under physiological conditions. Treatment of lung epithelial (Beas2B) cells with translation inhibitors induces uPA mRNA expression, as well as early response genes. To understand the specific expression and regulation of uPA mRNA, we treated Beas2B cells with cycloheximide (CycD), anisomycin, emitine and puromycin in a time-dependent manner and measured uPA mRNA expression by Northern blotting. All these agents induced uPA mRNA by two- to seven-fold within 3 h after treatment in Beas2B cells. CycD, emitine, puromycin and anisomycin also enhanced uPA mRNA half-life by three- to five-fold in Beas2B cells treated with DRB, an inhibitor of transcription. However, run-on-transcription experiments indicated that these agents failed to induce uPA mRNA transcription indicating that they augment uPA mRNA mainly due to increased stability. Using gel mobility shift, we identified an uPA mRNA binding protein (uPA mRNABp) that selectively binds to uPA mRNA [Gyetko MR, Todd III RF, Wilkinson CC, Sitrin RG: The urokinase receptor is required for human monocyte chemotaxis in vitro. J Clin Invest 93: 1380-1387, 1994]. Binding of both cytoplasmic and nuclear uPA mRNABp to uPA mRNA was abolished after treatment with translation inhibitors, which coincides with the maximal expression of uPA mRNA. We also found a similar decline in HuR and heterogeneous nuclear ribonucleoprotein C (hnRNPC) which are known to stabilize uPA mRNA both in the nuclear and cytosolic compartments. These results strongly suggest that increased uPA mRNA stability induced by translational inhibitors involves the interaction of uPA mRNA with a degrading protein factor rather than increased interaction of proteins that are known to stabilize uPA mRNA. These data also strongly suggests that down-regulation of the uPA-uPA mRNABp interaction by translational inhibitors rather than the translocation of uPA mRNABp contributes to increased uPA mRNA stability. This pathway may regulate uPA-mediated functions of the lung epithelium in the context of inflammation or neoplasia.

Significant overexpression of urokinase‐type plasminogen activator in pancreatic adenocarcinoma using real‐time quantitative reverse transcription polymerase chain reaction

Overexpression of urokinase-type plasminogen activator (uPA) has been shown to be strongly associated with an increased metastatic potential and poor prognosis in a variety of human malignancies. It was hypothesized that uPA would be overexpressed in highly metastatic pancreatic cancer. The aims of this study were to analyze uPA mRNA expression in pancreatic cancer and to correlate this to the expression of uPA protein and to the stage of the disease. Twenty-one pancreatic adenocarcinoma, six ampullary carcinoma and 10 benign mucinous cystadenoma samples, all with adjacent normal tissue, were collected. uPA mRNA was measured using real-time quantitative reverse transcription polymerase chain reaction. Localization of uPA within normal and pancreatic tumor sections was subsequently confirmed using immunohistochemistry. The median and range of the ratios of uPA mRNA measures between tumor tissue and non-involved pancreatic tissue was 17.1 (1.4-653.6) for pancreatic adenocarcinoma (P < 0.001), 3.9 (0.7-7.7) for ampullary carcinoma (P = 0.055) and 1.9 (0.6-5.9) for mucinous cystadenoma tissue (P = 0.052). uPA low tumors were associated with an exuberant stromal reaction, whereas uPA high tumors showed little stromal response. Immunohistochemistry confirmed that uPA protein was more prevalent in pancreatic adenocarcinoma tissue than in normal tissue and that it was membrane-bound. uPA mRNA expression was significantly associated with poorly differentiated pancreatic cancers (P < 0.05) and positively associated with tumor stage. These observations suggest that significant overexpression of uPA correlates closely to the rapid progression and invasiveness of pancreatic cancer and that uPA may provide a future therapeutic target for pancreatic cancer treatment.

Relationship between matrix metalloproteinase-2 mRNA expression and clinicopathological and urokinase-type plasminogen activator system parameters and prognosis in human gastric cancer

To investigate the relationship between matrix metalloproteinase-2 (MMP-2) mRNA expression and clinicopathologic and urokinase-type plasminogen activator (uPA) system parameter and prognosis in human gastric cancer. Expression of MMP-2 mRNA, uPA, and uPA-R mRNA in tumor tissues and > or =5 cm adjacent normal tissues from 67 cases of gastric cancer was studied using RT-PCR and Northern blot respectively. Survival analyses were done using the Kaplan-Meier method. The expression rates of MMP-2 mRNA, uPA and uPA-R mRNA in tumor tissues (31%, 41%, and 51%, respectively) were significantly higher than those in > or =5 cm adjacent tissues (19%, 11%, and 9%; chi(2) = 4.59, 43.58, and 53.24 respectively, P<0.05, 0.0001, and 0.0001, respectively). Expression of MMP-2 mRNA was significantly correlated with lymph node metastasis (metastasis: 61.9%, no metastasis: 39.1%, chi(2) = 7.61, P<0.05), Lauren's classification of diffuse/mixed types: 54.2%, intestinal type: 26.3%, chi(2) = 4.25, P<0.05, expression of uPA and uPA-R mRNA (uPA+: 55.1%, uPA-: 22.2% and uPA-R+: 54.9%, uPA-R-: 18.8%, chi(2) = 5.72 and 6.40 respectively, P<0.05). Kaplan-Meier survival analysis of MMP-2 mRNA expression did not show significant difference in all 67 cases, but revealed an association of the expression of MMP-2 mRNA, uPA, and uPA-R mRNA with worse prognosis (P = 0.0083, 0.0160, and 0.0094, respectively). MMP-2 may play an important role in the development of invasion and metastasis of gastric cancer.

Expressions of urokinase-type plasminogen activator, its receptor and plasminogen activator inhibitor-1 in gastric cancer cells and effects of Helicobacter pylori

ObjectiveDestruction of the extracellular matrix is essential for tumor invasion and metastasis. The relationship between Helicobacter pylori (H. pylori) infection and destruction of the extracellular matrix is not yet clear. Urokinase-type plasminogen activator (uPA) plays an important role in the destruction of the extracellular matrix and basement membrane. Urokinase-type plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1(PAI-1) appear to be associated with these processes. To clarify the role of H. pylori infection in the processes of destruction of the extracellular matrix and basement membrane in cancerous tissue, the effect of H. pylori on the expressions of uPA, uPAR and PAI-1 in cancer cells was investigated.Material and methodsGastric cancer cell lines (MKN45, KATO-III) were co-cultured with H. pylori standard strain (NCTC11637), cagA-negative strain and clinical isolated strain. The specific inductions of uPA, uPAR and PAI-1 mRNA were examined by reverse transcription-polymerase chain reaction (RT-PCR) amplification. The secreted uPA antigen was measured by enzyme-linked immunosorbent assay (ELISA). To evaluate the role of transcription factor NF-κB in uPA and uPAR gene transcription with H. pylori stimulation, the effect of NF-κB inhibitor MG132 on H. pylori-induced uPA and uPAR mRNA expression was examined.ResultsThe expressions of both uPA and uPAR mRNAs in the gastric cancer cell lines (MKN45 and KATO- III) were increased markedly (uPA mRNA; MKN45: 12-fold, KATO-III: 5-fold) (uPAR mRNA; MKN45: 3-fold, KATO-III: 3-fold) with H. pylori NCTC11637 strain stimulation, whereas the expression levels of uPA and uPAR mRNA did not increase with cagA-negative strain stimulation. These cancer cell lines slightly secreted uPA antigen into the culture medium, and the amount of uPA antigen increased dramatically by stimulation with H. pylori NCTC11637 and cagA-positive clinical isolated strains. These gastric cancer cell lines also slightly secreted PAI-1 antigen into the culture medium, and the amount of PAI-1 antigen was not affected by H. pylori NCTC11637 stimulation. H. pylori-induced uPA and uPAR mRNA expressions were strongly down-regulated by pretreatment with MG132 in both cell lines.ConclusionsThe results of this study indicated the possibility that cagA-positive H. pylori may play an important role not only in tissue remodeling, angiogenesis and wound healing but also in the process of degradation of the extracellular matrix breakdown, tumor invasion and metastasis by inducing uPA and uPAR complex in the gastric cancer cells.

Overexpression of Urinary Plasminogen Activator (uPA) Protein and mRNA in Thyroid Carcinogenesis

Urinary plasminogen activator (uPA), a protease, is one of the critical components of tumor invasion and metastasis. Its expression in thyroid carcinoma and potential role in thyroid tumorigenesis are unknown. The objective of this study was to determine whether uPA is differentially expressed in benign and malignant thyroid tumors. uPA expression was evaluated by immunohistochemistry (IHC) in 20 thyroid tumors (six classic papillary thyroid cancers (PTC) and three tall cell variants (TCV) and 11 adenomas). To validate IHC results, in situ hybridization was performed on both adenomas and cancer tissues to explore the expression of uPA at the mRNA level. The Fisher exact test was used to compare protein as well as mRNA expressions in adenomas and thyroid cancer. Intense granular cytoplasmic staining for uPA was observed in five of nine (56%) of thyroid cancers: 2/6 classic PTC (33%) and all tall cell variant PTC. Furthermore, uPA mRNA expression was found in the malignant thyroid epithelium but not in adjacent normal thyroid follicles or the stromal elements. None of the adenomas expressed uPA (P = .008). uPA staining was absent in histologically normal follicles adjacent to malignant thyroid follicles. uPA is expressed in thyroid carcinoma but not in benign adenomas or normal adjacent follicles. The selective expression of uPA in thyroid carcinoma provides evidence that uPA is useful in distinguishing benign and malignant thyroid neoplasms. More importantly, uPA may represent molecular target for therapeutic treatment of this malignancy.

Expression of urokinase plasminogen activator, its receptor and type-1 inhibitor in malignant and benign prostate tissue

The plasminogen activation (PA) cascade participates in degradation of extracellular matrix during cancer invasion. We have studied the expression of urokinase-type plasminogen activator (uPA) mRNA, uPA receptor (uPAR) mRNA and immunoreactivity, and type-1 plasminogen activator inhibitor (PAI-1) mRNA and immunoreactivity in 16 prostate adenocarcinomas and 9 benign prostate hyperplasias. uPA mRNA and uPAR mRNA expression were found in 9 and 8 of the adenocarcinomas, respectively, and in 7 and 6 of the benign hyperplasias, respectively. In both malignant and benign lesions, expression of these 2 mRNAs was predominantly seen in cells identified as macrophages, which in most of the carcinomas (approximately 90%) were located in the interstitial tissue between the tumor cell islands, while in most of the benign hyperplasias they were located in the lumen of the glands and were in only a few cases (approximately 30%) found in the interstitial tissue. uPAR immunoreactivity correlated with the mRNA expression and was, in addition, found in neutrophils. PAI-1 mRNA was detected in 13 of the 16 carcinomas and in 8 of the 9 benign hyperplasias, located in scattered fibroblast-like cells in both groups, in some vascular structures and in a few macrophages located in the interstitial tissue of both malignant and benign lesions. A similar expression pattern was found for PAI-1 immunoreactivity. In 8 of the 16 carcinomas, all 3 components were present, and in several areas colocalization was observed in stromal cells in close proximity to cancer cell islands. No immunoreactivity and/or mRNA expression of uPA, uPAR or PAI-1 was observed in cancer cells or in other epithelial cells in any of the cases.

Aggregated IgG Bind to Glomerular Epithelial Cells to Stimulate Urokinase Release through an Endocytosis-Independent Process

Background: In membranous nephropathy, the development of glomerular lesions is related to the formation of immune complexes at subepithelial sites. These deposits are associated with modifications in the fibrinolytic activity of glomerular cells leading to the appearance of fibrin degradation products in the deposits and the urine. A previous study has shown that immune complexes interact with glomerular epithelial cells (GEC) through the neonatal Fc receptor (FcRn). We therefore determined whether this binding could be responsible for a modification in the fibrinolytic activity of GEC. Methods: Endocytosis of heat-aggregated immunoglobulins (AgIgG) in cultured human GEC was studied by immunofluorescence and confocal microscopy. The release of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI) by GEC or whole glomeruli was assessed by ELISA, fibrin zymography and Northern blot. Results: Human GEC in culture bound AgIgG that possess characteristics similar to soluble immune complexes and internalized them by 10 min. This process was mediated by FcRn since chicken aggregated IgG (AgIgY), that do not bind FcRn, did not colocalize with AgIgG in GEC. AgIgG but not AgIgY induced a decrease of FcRn expression at the membrane and within the cells. The binding of AgIgG to GEC elicited a dose- and time-dependent increase in the release of uPA activity, as in the uPA protein and mRNA expression without modification in the release of PAI. This process was not abrogated by agents inhibiting endocytosis and/or transcytosis such as cytochalasin B, suggesting an endocytosis-independent uPA regulation. Conclusion: GEC response to AgIgG overload comprises at least two sequential steps: (1) a FcRn-mediated endocytosis; (2) an endocytosis-independent fibrinolytic imbalance leading to plasmin generation which could favor in vivo AgIgG clearance and matrix remodeling.

Inhibition of Tumor Invasion by Genomic Down-regulation of Matriptase through Suppression of Activation of Receptor-bound Pro-urokinase

Urokinase-type plasminogen activator (uPA) degrades the extracellular matrix and plays critical roles in tumor invasion and metastasis. Matriptase, a membrane-bound serine protease, was shown to activate uPA in a uPA receptor-free, solution-based study. We now investigate whether matriptase affects activation of receptor-bound uPA and contributes to the invasiveness of HRA human ovarian cancer cells in vitro and tumor behavior in nude mice. Here we show the following. 1) uPA expression was effectively stimulated by TGF-beta1 in HRA cells. 2) Antisense (AS)-matriptase transfection achieved a marked inhibition of receptor-bound pro-uPA activation without altering expression of uPA and uPA receptor mRNA and proteins, irrespective of whether cells were stimulated with TGF-beta1. 3) Tumor cell receptor-bound pro-uPA could be efficiently cleaved by matriptase to generate enzymatically active two-chain uPA. Thus, matriptase can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active uPA. 4) The AS-matriptase-treated cells had a decreased ability to invade an extracellular matrix layer, as compared with control cells. 5) When the AS-matriptase-treated cells were injected intraperitoneally into nude mice, the mice developed smaller tumors. Our data identify a novel role for matriptase for activation of receptor-bound uPA.

Transforming growth factor-beta1-dependent urokinase up-regulation and promotion of invasion are involved in Src-MAPK-dependent signaling in human ovarian cancer cells.

Urokinase-type plasminogen activator (uPA) has been implicated in tumor cell invasion and metastasis. We reported previously that transforming growth factor (TGF)-beta1 induces a dose- and time-dependent up-regulation of uPA mRNA and protein in highly invasive human ovarian cancer cell line HRA, leading to invasion. To further elucidate the mechanism of the invasive effect of TGF-beta1, we investigated which signaling pathway transduced by TGF-beta1 is responsible for this effect. Here, we show that 1) nontoxic concentrations of TGF-beta1 activated Src kinase; 2) TGF-beta1 rapidly phosphorylates ERK1/2 and Akt, but not p38; 3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment reduced TGF-beta1-induced phosphorylation of ERK1/2 and Akt by 85-90% compared with controls; 4) pharmacological inhibition of MAPK by PD98059 abrogated TGF-beta1-mediated Akt stimulation, whereas TGF-beta1-induced ERK1/2 stimulation was not inhibited by PI3K inhibitor LY294002 or AS-PI3K ODN transfection; 5) up-regulation of uPA mRNA in response to TGF-beta1 was almost totally blocked by PP2 and PD98059 and partially ( approximately 55%) by LY294002; 6) TGF-beta1-induced uPA mRNA up-regulation was inhibited by treatment with AS ODNs to c-Src or PI3K by 90 or 60%, respectively, compared with control ODN treatment; and 7) blockade of the release of the transcription factor NF-kappaB by pyrrolidinedithiocarbamate reduced the TGF-beta1-induced activation of the uPA gene by approximately 65%. In addition, curcumin, a blocker of the transcriptional factor AP-1, partially (35%) canceled this effect. Taken together, these data support a role for TGF-beta1 activation of two distinct pathways (Src-MAPK-PI3K-NF-kappaB-dependent and Src-MAPK-AP-1-dependent) for TGF-beta1-dependent uPA up-regulation and promotion of invasion.

Urokinase‐Type Plasminogen Activator and Insulin‐Like Growth Factor‐Binding Protein 3 mRNA Expression in Endometriotic Lesions and Eutopic Endometrium

The peritoneal fluid of women with endometriosis contains an increased insulin-like growth factor 1 (IGF-1) bioavailability, which is produced by limited hydrolysis of urokinase-type plasminogen activator (uPA) on IGF-binding protein 3 (IGFBP-3). Recently, IGF-1 was shown to inhibit apoptosis of endometrial-like cells in vitro, suggesting that a microenvironment of increased IGF-1 bioavailability can optimize the survival of endometrial cells grown ectopically. Here the expression of mRNA of IGFBP-3 and uPA in tissue biopsies from eutopic endometrium and endometriotic lesions obtained at laparoscopy from women with endometriosis have been analyzed, and it is documented that both IGFBP-3 and uPA mRNA expression are increased from 3- to 10-fold in endometriotic lesions versus eutopic endometrium. Consequently, the necessary components (uPA and IGFBP-3 expression) of endocrine/autocrine/paracrine enhancement of local IGF bioavailability mediated by uPA hydrolysis of the IGFBP-3 were present in endometriotic lesions. These data possibly explain the origin of the increased content of uPA activity, IGF-1 bioavailability, and NH(2)-truncated forms of IGFBP-3 in the peritoneal fluid of women with endometriosis.