Form Of uPA Research Articles

Inhibition of Human Urokinase-Type Plasminogen Activator (uPA) Enzyme Activity and Receptor Binding by DNA Aptamers as Potential Therapeutics through Binding to the Different Forms of uPA.

Urokinase-type plasminogen activator is widely discussed as a marker for cancer prognosis and diagnosis and as a target for cancer therapies. Together with its receptor, uPA plays an important role in tumorigenesis, tumor progression and metastasis. In the present study, systematic evolution of ligands by exponential enrichment (SELEX) was used to select single-stranded DNA aptamers targeting different forms of human uPA. Selected aptamers allowed the distinction between HMW-uPA and LMW-uPA, and therefore, presumably, have different binding regions. Here, uPAapt-02-FR showed highly affine binding with a KD of 0.7 nM for HMW-uPA and 21 nM for LMW-uPA and was also able to bind to pro-uPA with a KD of 14 nM. Furthermore, no cross-reactivity to mouse uPA or tissue-type plasminogen activator (tPA) was measured, demonstrating high specificity. Suppression of the catalytic activity of uPA and inhibition of uPAR-binding could be demonstrated through binding with different aptamers and several of their truncated variants. Since RNA aptamers are already known to inhibit uPA-uPAR binding and other pathological functions of the uPA system, these aptamers represent a novel, promising tool not only for detection of uPA but also for interfering with the pathological functions of the uPA system by additionally inhibiting uPA activity.

Abstract A71: TMPRSS4 proteolytically activates pro-uPA to induce cancer cell invasion

Abstract The invasive nature of tumor cells is critical for cancer metastasis. Urokinase-type plasminogen activator (uPA), which is usually derived from stromal cells, is a well-known serine protease involved in invasion and metastasis. uPA is produced as an inactive single-chain protein (known as pro-uPA or sc-uPA) that is processed into the active disulfide-linked two-chain form of uPA by a proteolytic event. TMPRSS4 is a novel type II transmembrane serine protease that is highly expressed on the cell surface in pancreatic, thyroid, colon, and other cancer tissues. Previously, we demonstrated that TMPRSS4 mediates colorectal tumor cell invasion, migration, and metastasis and increased TMPRSS4 expression is associated with colorectal cancer progression. We also demonstrated that TMPRSS4 upregulates uPA gene expression through c-Jun N-terminal kinase signaling activation to induce cancer cell invasion. However, it remains unknown how proteolytic activity of TMPRSS4 contributes to invasion. Here, we report that TMPRSS4 directly converted inactive pro-uPA into the active form through its proteolytic activity. Analysis of conditioned medium from cells overexpressing TMPRSS4 demonstrated that the active TMPRSS4 protease domain is released from the cells and is associated with the plasma membrane. Furthermore, TMPRSS4 could increase pro-uPA-mediated invasion in a serine proteolytic activity-dependent manner. These observations suggest that TMPRSS4 is an upstream regulator of invasion through the regulation of uPA. This study provides valuable insights into the proteolytic function of TMPRSS4 as well as mechanisms for the control of invasion. Citation Format: Hye-Jin Min, Semi Kim. TMPRSS4 proteolytically activates pro-uPA to induce cancer cell invasion. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A71. doi:10.1158/1538-7445.CHTME14-A71

Urokinase plasminogen activator augments cell proliferation and neointima formation in injured arteries via proteolytic mechanisms

Urokinase plasminogen activator (uPA) has been implicated in the healing responses of injured arteries, but the importance of its various properties that influence smooth muscle cell (SMC) proliferation and migration in vivo is unclear. We used three recombinant (r-) forms of uPA, which differ markedly in their proteolytic activities and abilities to bind to the uPA receptor (uPAR), to determine, which property most influences the healing responses of balloon catheter injured rat carotid arteries. After injury, uPA and uPAR expression increased markedly throughout the period when medial SMCs were rapidly proliferating and migrating to form the neointima. Perivascular application of uPA neutralizing antibodies immediately after injury attenuated the healing response, significantly reducing neointima size and neointimal SMC numbers. Perivascular application of r-uPAwt (wild type uPA) or r-uPA/GDF (r-uPA with multiple mutations in its growth factor-like domain) doubled the size of the neointima. Four days after injury these two uPAs nearly doubled neointimal and medial SMC numbers in the vessels, and induced greater reductions in lumen size than injury alone. Proteolytically inactive r-uPA/H/Q (containing glutamine rather than histidine-204 in its catalytic site) did not affect neointima or lumen size. Also, in contrast to the actions of proteolytically active uPAs, tissue plasminogen activator (tPA) did not affect the rate of neointima development. We conclude that uPA is an important factor regulating the healing responses of balloon catheter injured arteries, and its proteolytic property, which cannot be mimicked by tPA, greatly influences SMC proliferation and early neointima formation.

Chemotactic Effect of Urokinase Plasminogen Activator: a Major Role for Mechanisms Independent of Its Proteolytic or Growth Factor Domains

Urokinase type plasminogen activator (uPA) converts plasminogen to plasmin and is highly chemotactic for many cell types. We examined, using recombinant wild type and mutated forms of uPA, the extent to which its proteolytic properties, its growth-like domain (GFD) and/or interactions with the specific receptor (uPAR) contribute to the chemotactic activity towards vascular smooth muscle cells (SMC). Recombinant wild type uPA (r-uPA) stimulated cell migration nearly 5,8-fold, inactive r-uPA, with a mutation in the catalitic domain (r-uPA(H/Q)), 3-fold, uPA without growth factor like domain (r-uPA(GFD)), 2.6-fold, and a form containing both mutations (r-uPA(H/Q, GFD), 3.3-fold. All recombinant forms of uPA, wild type and those with mutations were equally and highly effective (IC5∼20 nM) in displacing 125I-r-uPA bound to SMC. These results indicate that additional mechanisms, not dependent on uPA's proteolytic activity or the binding ability of its GFD to uPAR, are the major contributors to its chemotactic action on SMC

164 Conformational differences between free and complexed forms of uPA and PAI-1, as probed by proteolytic susceptibility

164 Conformational differences between free and complexed forms of uPA and PAI-1, as probed by proteolytic susceptibility

Quantitative assessment of interaction of urokinase-type plasminogen activator and its receptor (CD87) by use of a solid-phase uPA-ligand binding assay

Summary Urokinase-type plasminogen activator (uPA) is a serine protease which has been implicated in numerous physiological and pathological processes, e.g. tissue remodelling, embryogenesis, fibrinolysis, and tumour spread. uPA protease binds to a specific high-affinity receptor (uPAR; CD87) on normal and tumour cells. This binding is mediated by the growth factor domain of uPA and thus independent of its proteolytic activity. An ELISA-type, solid-phase microtitre plate assay is presented, designed for the quantitation of such uPA molecules capable of binding to the receptor uPAR. This solid-phase uPA-ligand binding assay makes use of the specific, high-affinity interaction of uPA with uPAR. This assay format is different from the common uPA-ELISA which measures uPA antigen but not uPAR-reactivity. Recombinant soluble uPAR (CHO-uPAR), attached to the well of a microtitre plate, serves as the capture molecule for uPA. uPA-containing samples are added to allow binding of uPA to immobilized uPAR. Receptor-bound uPA is then detected by reaction of uPA with biotinylated monoclonal antibody no. 377 (American Diagnostica, Greenwich, CT, USA) directed to the kringle domain of uPA followed by avidin-peroxidase. The solid-phase uPA-ligand binding assay detects various forms of uPA: pro-uPA, HMW-uPA, ATF, GFD but does not react with the low molecular weight form of uPA (LMW-uPA) lacking the uPAR-reactive domain. uPA molecules in which the uPAR-binding domain has been impaired by proteolysis and uPA/uPAR complexes are also excluded from detection. The high sensitivity of the solid-phase uPA-ligand binding assay (lower limit 2 pM = 0.1 ng uPA/ml) allowed to measure reactive uPA (and fractions thereof) in the supernatants of cultured ovarian cancer cells, in extracts of ovarian and breast cancer tissues, in placenta tissue extracts and in malignant ascites. The solid-phase uPA-ligand binding assay was also used to screen the receptor binding reactivity of recombinant human uPA-polypeptides synthesized by yeast cells and that of synthetic uPA-peptides. The uPAR-blocking capability of uPA-peptides uPA14-32, uPA14-32/H29A, and uPA14-32/N32A, determined by the solid-phase uPA-ligand binding assay, was confirmed by flow cytofluorometric analysis employing fluorescent pro-uPA (FITC-pro-uPA) and the uPAR-rich promyeloid cell line U937.

Inhibitory Effect of a Synthetic Prostacyclin Analogue, Beraprost, on Urokinase-type Plasminogen Activator Expression in RC-K8 Human Lymphoma Cells

Plasminogen activation by urokinase-type plasminogen activator (uPA) is implicated in tumor invasion and metastasis by the breakdown of extracellular matrix. We have recently demonstrated the inhibitory effect of cAMP on uPA gene transcription in RC-K8 human lymphoma cells (Biochim Biophys Acta 1268: 293-9, 1995). Prostacyclin produced by endothelial cells is shown to increase cellular cAMP levels by activating adenylate cyclase. We, therefore, examined the effect of a stable analogue of prostacyclin, Beraprost, on uPA production in RC-K8 cells. uPA activity gradually increased in the conditioned medium with time. Beraprost (0.1 nM-1.0 microM) inhibited uPA accumulation in a dose-dependent manner without affecting cell viability. Fibrinzymography demonstrated that high and low molecular forms of uPA were present in the conditioned medium and that after Beraprost-treatment all forms of uPA decreased and no PA/PA inhibitor complex was present. Northern blot analysis revealed that after exposure to Beraprost, uPA mRNA levels increased transiently and then rapidly decreased to below control levels. Treatment with Beraprost resulted in a rapid activation of cellular cyclic AMP-dependent protein kinase (PKA). Beraprost completely negated uPA gene expression induced by phorbol myristate acetate, an activator of protein kinase C (PKC). These results suggest that Beraprost inhibits uPA production by suppressing uPA gene expression through the PKA pathway and that PKA-mediated signals are dominant in uPA gene expression as compared to those medicated by PKC. This inhibition of uPA expression by a prostacyclin analogue may be an important fact to explain the mechanism of anti-metastatic effects of prostacyclin.

Phosphorylation of a surface receptor bound urokinase-type plasminogen activator in a human metastatic carcinomatous cell line

The 32P-labeled urokinase (uPA) bound to surface receptors of Detroit 562 cells was immunoprecipitated by anti-uPA antibody. Amino acid analysis showed that tyrosines and serines were the acceptors. Inhibition of protein kinases greatly reduced the 32P incorporation, suggesting that the respective cellular src gene product and protein kinase C were involved in the phosphorylations. Proteins purified on chromatographic columns contained two forms of uPA, a high (HMW) and a low (LMW) molecular weight. Tyrosine-phosphorylation occurs in the HMW and A-chain. Such modifications might modulate the extracellular activities of uPA.

A Monoclonal Antibody that Recognizes the Receptor Binding Region of Human Urokinase Plasminogen Activator

An anti-urokinase monoclonal antibody 5B4 (MAB 5B4) was obtained by fusing the murine myeloma cell line X63-Ag8.653 with the spleen cells from a female BALB/c mouse immunized with high-molecular-weight urokinase (HMW-uPA). MAB 5B4 is an IgG1 that binds selectively to the single-chain form of uPA (sc-uPA), to HMW-uPA and to the 17,000 Mr aminoterminal fragment of the A-chain (ATF) but not to the low-molecular-weight urokinase (LMW-uPA) nor to the reduced form of HMW-uPA. This strongly suggests that MAB 5B4 recognizes a conformational determinant on the A-chain. The antibody has an affinity constant for uPA-Sepharose of 1.42 X 10(7) M-1, calculated from equilibrium binding data, and can be used for one step purification of HMW-uPA by immunoaffinity chromatography. MAB 5B4 and the previously obtained antibody 105IF10 recognize the A-chain: the epitopes, however, are distinct as shown by double-antibody-sandwich enzyme immunoassay. Finally MAB 5B4 inhibits the binding of ATF to the uPA receptor of different human cells, whereas 105IF10 does not. Thus this antibody represents a potentially, useful tool for the study of uPA receptor physiology.