Gonadotropin surge-induced up-regulation of the plasminogen activators (tissue plasminogen activator and urokinase plasminogen activator) and the urokinase plasminogen activator receptor within bovine periovulatory follicular and luteal tissue

This study examined the effect of the preovulatory gonadotropin surge on the temporal and spatial regulation of tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), and uPA receptor (uPAR) mRNA expression and tPA, uPA, and plasmin activity in bovine preovulatory follicles and new corpora lutea collected at approximately 0, 6, 12, 18, 24, and 48 h after a GnRH-induced gonadotropin surge. Messenger RNAs for tPA, uPA, and uPAR were increased in a temporally specific fashion within 24 h of the gonadotropin surge. Localization of tPA mRNA was primarily to the granulosal layer, whereas both uPA and uPAR mRNAs were detected in both the granulosal and thecal layers and adjacent ovarian stroma. Activity for tPA was increased in follicular fluid and the preovulatory follicle apex and base within 12 h after the gonadotropin surge. The increase in tPA activity in the follicle base was transient, whereas the increased activity in the apex was maintained through the 24 h time point. Activity for uPA increased in the follicle apex and base within 12 h of the gonadotropin surge and remained elevated. Plasmin activity in follicular fluid also increased within 12 h after the preovulatory gonadotropin surge and was greatest at 24 h. Our results indicate that mRNA expression and enzyme activity for both tPA and uPA are increased in a temporally and spatially specific manner in bovine preovulatory follicles after exposure to a gonadotropin surge. Increased plasminogen activator and plasmin activity may be a contributing factor in the mechanisms of follicular rupture in cattle.     

Tissue plasminogen activator and urokinase enhance the binding of plasminogen to thrombospondin

Thrombospondin (TSP) is a multifunctional platelet alpha-granule and extracellular matrix glycoprotein that binds specifically to plasminogen (Plg) via that protein's lysine-binding site and modulates activation by tissue activator (TPA). In this study we report that the plasminogen activators, TPA and urokinase, greatly influence the binding of Plg to TSP. Using an enzyme-linked immunosorbent assay and a TSP-Sepharose affinity bead-binding assay we have found that Plg-TSP complex formation was markedly enhanced (up to 5-fold) when catalytic concentrations of Plg activators were included in the reaction mixtures. The enhancement was dependent upon the generation of small amounts of active plasmin and was duplicated by pretreatment of the immobilized TSP with plasmin prior to addition of the Plg. The enhancement effect was associated with selective proteolysis of the immobilized TSP. Purified Lys-Plg (the plasmin modified form of native Glu-Plg) bound to TSP to a greater extent than Glu-Plg, and binding of both forms was augmented by Plg activators. The apparent KD values of complex formation were unchanged in the presence of Plg activators suggesting that the enhancement effect was due to the generation of additional binding sites. The increased amount of bound Plg was demonstrated to result in a similar increase in the amount of plasmin generated from the complexes by TPA. Plg activators did not influence binding of Plg to histidine-rich glycoprotein or of histidine-rich glycoprotein to TSP, demonstrating specificity. In addition when TSP was treated with other proteases (human thrombin or human leukocyte elastase) no augmentation of Plg binding was seen. Thus, the initial production of small amounts of plasmin from Plg immobilized on TSP in fibrin-free microenvironments could generate a positive feedback loop by enzymatically modifying both TSP and Plg, resulting in an increase in TSP-Plg complex formation leading to the localized production of substantially more plasmin.     

Regulation of urokinase plasminogen activator localization in keratinocytes by calcium ion and E-cadherin.

In keratinocyte culture, the cellular distribution of many adhesion markers and the organization of intercellular junctions are controlled by the calcium ion concentration of the medium. We show in the present study that urokinase plasminogen activator (uPA) localization in the human keratinocyte is similarly dependent upon calcium concentration. At 30 microM calcium, uPA is present throughout the cell, often with a perinuclear concentration. Upon calcium elevation to 1.0 mM, uPA is concentrated along the cell-cell borders, where it colocalizes (at the light microscope level) with E-cadherin. Blocking antibody to E-cadherin delays the calcium-induced redistribution of uPA, in a manner very similar to the previously observed delay in redistribution of several adhesion-related markers, including vinculin, desmoplakin, and beta 1 integrin. These data suggest a link between the redistribution of uPA to the cell-cell borders and the calcium-induced organization of intercellular junctions in the human keratinocyte. The presence of uPA along the intercellular borders suggests that this enzyme may be involved in regulation of epidermal adhesion through proteolysis.     

Kinetic analysis of the interactions between plasminogen activator inhibitor 1 and both urokinase and tissue plasminogen activator

Plasminogen activator inhibitor 1 (PAI-1) was purified from medium conditioned by cultured bovine aortic endothelial cells by successive chromatography on concanavalin A Sepharose, Sephacryl S-200, Blue B agarose, and Bio-Gel P-60. As shown previously for conditioned media (C. M. Hekman and D. J. Loskutoff (1985) J. Biol. Chem. 260, 11581-11587) the purified PAI-1 preparation contained latent inhibitory activity which could be stimulated 9.4-fold by sodium dodecyl sulfate and 45-fold by guanidine-HCl. The specific activity of the preparation following treatment with 0.1% sodium dodecyl sulfate was 2.5 X 10(3) IU/mg. The reaction between purified, guanidine-activated PAI-1 and both urokinase and tissue plasminogen activator (tPA) was studied. The second-order rate constants (pH 7.2, 35 degrees C) for the interaction between guanidine-activated PAI-1 and urokinase (UK), and one- and two-chain tPA are 1.6 X 10(8), 4.0 X 10(7), and 1.5 X 10(8) M-1 S-1, respectively. The presence of CNBr fibrinogen fragments had no affect on the rate constants of either one- or two-chain tPA. Steady-state kinetic analysis of the effect of PAI-1 on the rate of plasminogen activation revealed that the initial UK/PAI-1 interaction can be competed with plasminogen suggesting that the UK/PAI-1 interaction may involve a competitive type of inhibition. In contrast, the initial tPA/PAI-1 interaction can be competed only partially with plasminogen, suggesting that the tPA/PAI-1 interaction may involve a mixed type of inhibition. The results indicate that PAI-1 interacts more rapidly with UK and tPA than any PAI reported to date and suggest that PAI-1 is the primary physiological inhibitor of single-chain tPA. Moreover, the interaction of PAI-1 with tPA differs from its interaction with UK, and may involve two sites on the tPA molecule.     

Alterations in intracellular calcium homeostasis and platelet aggregation induced by ethanol

The in vitro effects of ethanol on intracellular Ca(2+) homeostasis and tyrosine phosphorylation have been investigated in human platelets in order to clarify the cellular mechanisms underlying its described anti-aggregant effects. Ethanol (1-50 mM) reduced, in a dose-dependent manner, the rate and amplitude of aggregation and attenuated the phosphotyrosine content both induced by 0.1U/ml of the physiological ligand, thrombin. Thrombin-induced Ca(2+) entry to the cytosol was significantly reduced, and capacitative Ca(2+) entry (CCE) significantly altered, by 50 mM ethanol, so that ethanol reduces CCE mediated by depletion of the 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ)-sensitive store but enhances CCE induced by the TBHQ-insensitive pool. In conclusion, we provide considerable evidence that ethanol reduces thrombin-induced aggregation, which is likely a result of a significant inhibition of Ca(2+) entry, as well as a reduction in the activity of protein tyrosine kinases.     

Effect of tetracaine on thrombocyte aggregation and mobilization of the intracellular calcium

Tetracaine (1 mM), a local anesthetics, lowers a degree of aggregation of human thrombocytes which is induced by thrombin (0.15 u/ml) and suppresses its appearance. Aggregation of thrombocytes induced by phorbol ester, TPA (10-8 M), an activator of protein kinase C, is inhibited completely by the mentioned doses of the anesthetics. In the presence of tetracaine the release of intracellular Ca is lower to some extent, but then it surpasses the control level. It is established that under the action of ionophore A23187 tetracaine exerts no effect on mobilization of intracellular Ca2+.     

Regulated localization confers multiple functions on the protease urokinase plasminogen activator

We have investigated the role of the plasminogen activation cascade in skeletal muscle differentiation. Migrating, undifferentiated myoblasts express urokinase plasminogen activator (uPA) and its cell surface receptor (uPAR). Consequently, uPA is localized predominantly to the cell surface. Preventing uPA from associating with its receptor with a noncatalytic form of uPA (NC-uPA) hinders migration of myoblasts and inhibits differentiation. When myoblasts reach confluence, cease migrating, and start to differentiate, uPAR gets downregulated, and uPA becomes redistributed from the cell surface to the extracellular space. The function of uPA at this stage was tested using the protease inhibitors aprotinin, α₂-antiplasmin, or plasminogen activator inhibitor-1 (PAI-1). Contrary to the role of cell-associated uPA, inhibition of soluble uPA/plasmin stimulates differentiation of myoblasts. Aprotinin can inhibit activation of latent TGFβ and stimulates differentiation, suggesting PAI-1 and α₂-antiplasmin also may stimulate differentiation via this mechanism. These data suggest that regulation of uPA localization allows a dual function for this protease in regulating cell migration and controlling cell differentiation. J. Cell. Physiol. 171:217–225, 1997. © 1997 Wiley-Liss, Inc.     

Mechanisms of cardiac fibrosis induced by urokinase plasminogen activator

Human hearts with end-stage failure and fibrosis have macrophage accumulation and elevated plasminogen activator activity. However, the mechanisms that link macrophage accumulation and plasminogen activator activity with cardiac fibrosis are unclear. We previously reported that mice with macrophage-targeted overexpression of urokinase plasminogen activator (SR-uPA+/o mice) develop cardiac macrophage accumulation by 5 weeks of age and cardiac fibrosis by 15 weeks. We used SR-uPA+/o mice to investigate mechanisms through which macrophage-expressed uPA causes cardiac macrophage accumulation and fibrosis. We hypothesized that: 1) macrophage accumulation and cardiac fibrosis in SR-uPA+/o mice are dependent on localization of uPA by the uPA receptor (uPAR); 2) activation of plasminogen by uPA and subsequent activation of transforming growth factor-beta1 (TGF-beta1) and matrix metalloproteinase (MMP)-2 and -9 by plasmin are critical pathways through which uPA-expressing macrophages accumulate in the heart and cause fibrosis; and 3) uPA-induced cardiac fibrosis can be attenuated by treatment with verapamil. To test these hypotheses, we bred the SR-uPA+/o transgene into mice deficient in either uPAR or plasminogen and measured cardiac macrophage accumulation and fibrosis. We also measured cardiac TGF-beta1 protein (total and active), Smad2 phosphorylation, and MMP activity after the onset of macrophage accumulation but before the onset of cardiac fibrosis. Finally, we treated mice with verapamil. Our studies revealed that plasminogen is necessary for uPA-induced cardiac fibrosis and macrophage accumulation but uPAR is not. We did not detect plasmin-mediated activation of TGF-beta1, MMP-2, or MMP-9 in hearts of SR-uPA+/o mice. However, verapamil treatment significantly attenuated both cardiac fibrosis and macrophage accumulation.     

Characterization of urokinase type plasminogen activator modified by phenylglyoxal

A chemical modification of single-chain urokinase-type plasminogen activator (scu-PA) with phenylglyoxal under mild conditions resulted in the scu-PA derivatives with various numbers of the modified Arg residues. The study of properties of the resulting derivatives demonstrated that the modification of 4-12 Arg residues did not cause any loss of the activator, fibrinolytic, and potential amidase activities of the activator. The scu-PA with four modified Arg residues was found to be the most stable derivative in human blood plasma; it causes a more efficient lysis of plasma clots than the native activator. Three of four modified Arg residues are supposed to be within the 178RRHRGGS184 cluster, which was localized in the superficial loop of the scu-PA globule and was shown to interact with the complementary series of negatively charged residues in the molecule of the main plasma inhibitor PAI-1. The neutralization of positively charged Arg residues in this cluster decreases the affinity of scu-PA and the double chain urokinase-type plasminogen activator for PAI-1, which results in an enhancement of the stability in plasma and the fibrinolytic efficiency of the activator. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.     

RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo

The invasive ability of tumor cells plays a key role in prostate cancer metastasis and is a major cause of treatment failure. Urokinase plasminogen activator-(uPA) and its receptor (uPAR)-mediated signaling have been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study was undertaken to investigate the biological roles of uPA and uPAR in prostate cancer cell invasion and survival, and the potential of uPA and uPAR as targets for prostate cancer therapy. uPA and uPAR expression correlates with the metastatic potential of prostate cancer cells. Thus, therapies designed to inhibit uPA and uPAR expression would be beneficial. LNCaP, DU145, and PC3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as demonstrated by their capacity to invade the extracellular matrix. In this study we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human uPA and uPAR. These small interfering RNA constructs significantly inhibited uPA and uPAR expression at both the mRNA and protein levels in the highly metastatic prostate cancer cell line PC3. Our data demonstrated that uPA-uPAR knockdown in PC3 cells resulted in a dramatic reduction of tumor cell invasion as indicated by a Matrigel invasion assay. Furthermore, simultaneous silencing of the genes for uPA and uPAR using a single plasmid construct expressing shRNAs for both uPA and uPAR significantly reduced cell viability and ultimately resulted in the induction of apoptotic cell death. RNA interference for uPA and uPAR also abrogated uPA-uPAR signaling to downstream target molecules such as ERK1/2 and Stat 3. In addition, our results demonstrated that intratumoral injection with the plasmid construct expressing shRNAs for uPA and uPAR almost completely inhibited established tumor growth and survival in an orthotopic mouse prostate cancer model. These findings uncovered evidence of a complex signaling network operating downstream of uPA-uPAR that actively advances tumor cell invasion, proliferation, and survival of prostate cancer cells. Thus, RNA interference-directed targeting of uPA and uPAR is a convenient and novel tool for studying the biological role of the uPA-uPAR system and raises the potential of its application for prostate cancer therapy.     

Secretion of macrophage urokinase plasminogen activator is dependent on proteoglycans.

The importance of proteoglycans for secretion of proteolytic enzymes was studied in the murine macrophage cell line J774. Untreated or 4beta-phorbol 12-myristate 13-acetate (PMA)-stimulated macrophages were treated with hexyl-beta-d-thioxyloside to interfere with the attachment of glycosaminoglycan chains to their respective protein cores. Activation of the J774 macrophages with PMA resulted in increased secretion of trypsin-like serine proteinase activity. This activity was completely inhibited by plasminogen activator inhibitor 1 and by amiloride, identifying the activity as urokinase plasminogen activator (uPA). Treatment of both the unstimulated or PMA-stimulated macrophages with xyloside resulted in decreased uPA activity and Western blotting analysis revealed an almost complete absence of secreted uPA protein after xyloside treatment of either control- or PMA-treated cells. Zymography analyses with gels containing both gelatin and plasminogen confirmed these findings. The xyloside treatment did not reduce the mRNA levels for uPA, indicating that the effect was at the post-translational level. Treatment of the macrophages with xylosides did also reduce the levels of secreted matrix metalloproteinase 9. Taken together, these findings indicate a role for proteoglycans in the secretion of uPA and MMP-9.     

Kinetics of the activation of plasminogen by natural and recombinant tissue-type plasminogen activator

The kinetics of the activation of plasminogen by tissue-type plasminogen activator were studied in the presence and the absence of CNBr-digested fibrinogen as a soluble cofactor. Michaelis-Menten kinetics applied and the kinetic parameters obtained were very similar to those previously reported for the activation in the presence of solid phase fibrin (Hoylaerts, M., Rijken, D. C., Lijnen, H. R., and Collen, D. (1982) J. Biol. Chem. 257, 2912-2919). The affinity of the enzyme for plasminogen dramatically increases in the presence of the soluble cofactor while the catalytic rate constant does not change significantly (KM drops from 83 to 0.18 microM and kcat increases from 0.07 to 0.28 s-1 for tissue-type plasminogen activator of melanoma origin). Fragments containing the lysine-binding sites of plasminogen compete with plasminogen for interaction with CNBr-digested fibrinogen. The dissociation constant of this interaction was found to be 4.5 microM for the high affinity lysine-binding site. No difference was found in the kinetic parameters for the activation of plasminogen by either tissue-type plasminogen activator of melanoma origin or by glycosylated forms of tissue-type plasminogen activator obtained by recombinant DNA technology. The present findings obtained in a homogenous liquid milieu support the previously proposed mechanism of the activation of plasminogen by tissue-type plasminogen activator in the presence of fibrin. This mechanism involves binding of both tissue-type plasminogen activator and plasminogen to fibrin.     

Effect of intracellular Na+ on platelet aggregation and Ca2+ mobilization

The effects of ionophores, which can carry alkali metal cations, on platelet aggregation were examined. At an alkaline extracellular pH, alkali metal cation/H+ exchanger nigericin accelerated aggregation in K+-enriched medium, whereas it rather inhibited aggregation in Na+-enriched medium, even though the intracellular pH was only slightly alkaline. The inhibitory effect of Na+ on platelet aggregation was more clearly shown with the alkali metal cation exchanger gramicidin D. The ionophore had no effect or a slightly accelerative effect on aggregation in K+-enriched medium, whereas it significantly inhibited aggregation induced by thrombin, ADP and platelet activating factor in Na+-enriched medium. Fluorescence studies on fura-2-labeled platelets revealed that in Na+-enriched medium gramicidin D inhibited agonist-induced Ca2+ mobilization both in the presence and absence of extracellular Ca2+. These results suggest that the intracellular Na+ inhibits platelet aggregation by inhibiting Ca2+ mobilization.     

Purification and characterization of human kidney plasminogen activator dissimilar to urokinase

The tissue activator was extracted with 2 M ammonium thiocyanate and purified by L-arginine methyl ester, concanavalin A and ion-exchange chromatographies, and Sephacryl S-200 gel filtration in buffers containing Triton X-100 and/or ammonium thiocyanate. The final preparations had specific activities of 25 000-40 000 IU/mg protein and were shown to be a single band with an apparent molecular weight of 54 00 by SDS-polyacrylamide gel electrophoresis with or without reducing agent. When subjected to isoelectric focusing, its major component had an isoelectric point of approx. 8.2 with minor components. (7.8-8.6). The purified tissue activator was a serine protease, dissimilar to urokinase in some respects including antigenicity, strong affinity to insoluble fibrin monomer and hydrolytic activities for synthetic substrates. The crude extract contained another plasminogen activator with antigen identity to urokinase, which constituted approx. 15% of the total activity in crude extract. These findings indicated that human kidney would produce at least two plasminogen activators, namely, the tissue activator as a major plasminogen activator and urokinase.     

Effect of calcium channel blocker (diltiazem) on platelet aggregation

Platelet aggregation with collagen, ADP and sodium arachidonate was significantly inhibited by 0.48 and 0.24 mg/ml of diltiazem but no significant effect occurred with 0.024 mg/ml of diltiazem. It is suggested that the antiplatelet property of diltiazem may be utilized in clinical setting and diltiazem may be tried synergistically with other antiplatelet drugs.