Plasminogen activators in human breast cancer cell lines: hormonal regulation and properties

To understand the hormonal regulation of plasminogen activators (PAs) in human breast cancer, we have examined the hormonal regulation and properties of PAs in four human breast cancer cell lines that differ markedly in their estrogen receptor (ER) content: MCF-7 cells contain high levels of ER (approx 7 pmol/mg DNA) and their PA activity was increased 3-4-fold by physiological concentrations of estradiol; T47-D and ZR-75-1 cells contain lower levels of ER (0.9 and 2.1 pmol/mg DNA respectively) and their PA activity was also increased 3-4-fold by estradiol. In contrast, MDA-MB-231 cells, which do not contain ER, showed a high level of PA activity that was not modulated by estradiol. SDS-PAGE followed by zymography indicated that MCF-7 cells secreted tissue-type PA (t-PA), T47-D and ZR-75-1 cells secreted urokinase-type PA (u-PA), and MDA-MB-231 cells secreted both types of PAs. The types of PAs secreted by these cell lines did not change upon treatment with estradiol. Dose-response curves for the stimulation of MCF-7 PA activity by different estrogens showed an excellent correlation between affinities of the estrogens for ER and their potency in stimulating PA activity. With a clonal subline of MCF-7 cells, MCF-L, a soluble inhibitor of both t-PA and u-PA was secreted. Incubation of purified t-PA or u-PA with the serum-free conditioned medium from MCF-L cells resulted in a shift in the mobility of t-PA and u-PA in SDS-polyacrylamide gels to forms increased in molecular mass by about 50,000-70,000. The shifts in molecular mass could be prevented by the presence of the competitive inhibitor p-aminobenzamidine, indicating that the active sites of the PAs were involved in the formation of these complexes. Furthermore, co-cultivation, of RT4-D rat neuroblastoma cells, which exhibit high levels of t-PA activity, with MCF-L cells resulted in a marked decrease in the PA activity of the RT4-D cells. Our results were consistent with the following conclusions: t-PA, u-PA or both were secreted by human breast cancer cells. In the ER-containing cell lines, depending upon the specific cell line, t-PA or u-PA was stimulated by estrogens. The unstimulated levels of PA activity and the magnitude of PA stimulation by estrogens were not closely related to ER content.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of a tissue-type plasminogen activator from porcine urine

Porcine urine, unlike human urine, does not contain detectable amounts of urokinase-type plasminogen activator (u-PA). The plasminogen activator present in porcine urine is of tissue-type (t-PA) as identified by the following criteria. (1) Porcine urine PA exhibits an Mr of 65,000 similar to the Mr of human t-PA (64-70,000) but distinct from the Mr of human u-PA (55,000). (2) Antibodies against human t-PA bind and inhibit crude and purified porcine urine PA, while human u-PA-specific antibodies do not react with porcine urine PA. (3) Plasminogen activation by porcine urine PA is markedly stimulated in the presence of fibrinogen fragments. (4) Porcine urine PA activity is not affected by concentration of amiloride substantially suppressing human u-PA activity.     

Construction and expression of a hybrid plasminogen activator gene with sequences from non-protease region of tissue-type plasminogen activator (t-PA) and protease region of urokinase (u-PA)

There are two physiological plasminogen activators (PAs), tissue-type PA (t-PA) and urokinase (u-PA) which possess distinct immunological and biochemical characteristics. Using genetic engineering techniques a hybrid t:u-PA cDNA, comprised of amino acid (aa) sequences corresponding to the non-protease region (aa 1-261) of t-PA and the protease region (aa 132-411) of u-PA, was constructed. The t:u-PA gene after insertion into the SV40 expression vector was expressed in monkey Cos-1 cells. The 66-67 kDa t:u-PA was produced in an enzymatically active form. The fibrinolytic activity of the t:u-PA could be quenched by anti-urokinase as well as by anti-t-PA sera. Like urokinase, the t:u-PA showed a high intrinsic plasminogen activation. This activity, as in the case of t-PA, was stimulated by fibrin. The u-PA, on the other hand, stimulated plasminogen activation marginally in the presence of fibrin. Both the t:u-PA and t-PA showed binding affinity for fibrin clot. This study strongly suggests the autonomous nature of the structural domains in PA and also demonstrates the feasibility of shuffling these domains without loss of their functional activities.     

Production of proteases type plasminogen activator and their inhibitor in cornea

Corneal epithelial cells secrete tissue plasminogen activator (t-PA), urokinase type plasminogen activator (u-PA) and their inhibitor (PAI), whereas these cell types in other tissues are known to secrete only u-PA hitherto. Endothelial cells in the cornea produce mostly u-PA and only small amounts of t-PA and PAI which remain confined in the cellular compartment contrary to the situation in the vascular endothelial cells where they are liberated into the circulation in the order PAI greater than t-PA greater than U-PA. These unique features of activator/inhibitor secretion and production may play an important role in the remodeling of the corneal matrix.     

Extracellular matrix produced by cultured corneal and aortic endothelial cells contains active tissue-type and urokinase-type plasminogen activators

Incubation of plasminogen with the subendothelial extracellular matrix (ECM) synthesized by cultured bovine corneal and aortic endothelial cells resulted in generation of fibrinolytic activity, indicated by proteolysis of ¹²⁵I-fibrin in a time-and dose-dependent manner. Both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) were identified in the ECM by fibrin zymography, immunoblotting, and inhibition of plasminogen activation by anti-u-and anti-t-antibodies. Most of the ECM-resident plasminogen activator (PA) activity did not originate from intracellular PA release occurring when the endothelial cells were lyzed and the ECM exposed, since a comparable amount of PA was associated with the ECM when the cells were lyzed with Triton X-100 or removed intact by treatment with 2 M urea. Active u-PA and t-PA were released from ECM by treatment with heparanase (endo-β-D-), indicating that some of the ECM-resident PA activity is sequestered by heparan sulfate side chains. These results indicate that both u-PA and t-PA produced by endothelial cells are firmly sequestered in an active form by the subendothelial ECM. It is suggested that ECM-resident plasminogen activators participate in sequential matrix degradation during cell invasion and tumor metastasis. PA activity may also function in release of ECM-bound growth factors (i.e., basic fibroblast growth factor) and activation of proenzymes (i.e., prothrombin), resulting in modulation of the ECM growth-promoting and thrombogenic properties. © 1993 Wiley-Liss, Inc.     

Enhanced production of tissue-type plasminogen activator by estradiol in a novel type variant of human breast cancer MCF-7 cell line

ES-1 cells, which showed a higher sensitivity to the cytocidal action of estradiol were isolated from a human breast cancer MCF-7 cell line. Growth of ES-1 cells was inhibited by a dose of 17-beta estradiol that stimulated the growth of the parental MCF-7 cells. Proteins secreted from MCF-7 and ES-1 cells when cultured with 17-beta estradiol were compared by sodium dodecyl sulfate-containing polyacrylamide gel electrophoresis (SDS-PAGE). Addition of estradiol to culture medium enhanced secretion of a protein of molecular mass of 52 kDa in media for both MCF-7 and ES-1 cell lines, but the secretion of a second 67 kDa protein was enhanced about 10-fold only in ES-1 cells. The analysis by SDS-PAGE of culture medium immunoprecipitated with anti-tissue-type plasminogen activator (t-PA) antibody demonstrated that the band of 67 kDa protein specifically secreted from estradiol-treated ES-1 cells contained t-PA. Zymography assays, quantitative immunoreactive assays, and Northern analysis showed about 5-fold specific increase by estradiol of t-PA with molecular mass of 65-70 kDa in ES-1 but not in its parental MCF-7 cells. Cellular level of the plasminogen activity was also specifically enhanced in ES-1 cells by estradiol, but only a slightly in MCF-7 cells. By contrast, another urokinase-type PA (u-PA) with molecular weight of 55 kDa showed very low level activity in both MCF-7 and ES-1 cell lines in the presence of estradiol. Formation of t-PA mRNA was specifically enhanced in ES-1 cells when ES-1 cells were treated for more than 12 h with 10(-8) M 17-beta estradiol. Estradiol did not elongate the lifetime of t-PA mRNA in ES-1 cells. A unique phenotype of ES-1 cells in response to estradiol is discussed in relation to activating expression of the t-PA gene.     

Urokinase-type plasminogen activator and its inhibitor secreted by cultured human monocyte-macrophages

Human blood monocytes in culture differentiate to macrophagelike cells within 1 week. Coinciding with this morphological transition the cells started releasing increasing amounts of the serine proteinase plasminogen activator (PA; Mr 56,000) of the urokinase (u-PA) type and the proteinase inhibitor alpha-2-macroglobulin (alpha 2M). Unlike the cell-associated PA activity, which was also readily detected in fresh monocytes, the activity secreted into the serum-free culture medium could be measured only after treatment of the samples with sodium dodecyl sulphate. Heat or acid treatment of the medium was not sufficient to reveal the PA activity, suggesting that, apart from alpha 2M, another PA-inhibiting substance was present in the culture medium. The inhibitor (Mr 65,000) was found to be synthesized by macrophages and specifically inhibited u-PA activity but not tissue-type PA (t-PA) or plasmin activity. Dexamethasone decreased the secretion of PA by differentiated macrophages without affecting the production of alpha 2M or the PA inhibitor. Dexamethasone also inhibited the morphological differentiation of the cells when added to the monocyte-phase cells.     

Endotoxin induction of plasminogen activator and plasminogen activator inhibitor type 1 mRNA in rat tissues in vivo

The tissue-specific distribution of tissue-type and urokinase-type plasminogen activator (t-PA and u-PA) and their inhibitor type 1 (PAI-1) was analyzed at mRNA level in five major rat organ tissues. t-PA mRNA was detected in lung, kidney, heart, and liver. u-PA mRNA was detected in kidney and lung. Presence of PA mRNA correlated with the detection of PA activity in extracts of these tissues. PAI-1 mRNA was detected predominantly in heart and lung. Although PAI activity could not be measured directly in tissue extracts, the presence of PAI-1 mRNA correlated with the occurrence of PA.PAI complex in fibrin autography of tissue extracts. Endotoxin injection caused a very large increase in plasma PAI activity. This increase correlated with a marked increase in PAI-1 mRNA in nearly all tissues studied. The increase in PAI-1 mRNA is most pronounced in lung and liver. Endotoxin injection also caused an increased level of t-PA mRNA in heart and kidney, and an increased u-PA mRNA level in kidney. mRNA analysis of freshly isolated and separated subfractionated liver cells showed that the marked increase in PAI-1 mRNA in the liver after endotoxin injection may be due mainly to a strong increase of PAI-1 mRNA in the liver endothelial cells.     

Concentration of plasminogen activators and inhibitor in the human endometrium at different phases of the menstrual cycle.

The concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA) and plasminogen activator inhibitor (PAI-1) have been determined in endometrial curettings obtained from 46 subfertile women during proliferative, early or late secretory phases of the menstrual cycle. t-PA activity and antigen concentrations was significantly higher (P < 0.001) in late secretory endometrium than in proliferative or early secretory endometrium. Higher concentrations of PAI-1 antigen (P < 0.05) were also noted in late secretory phase than in proliferative and early secretory endometrium. However, u-PA concentration was not significantly different and no PAI activity could be demonstrated in the menstrual phases studied. Zymography studies confirmed the presence of both t-PA and u-PA in the endometrium. Ovarian hormonal patterns may therefore influence the activity of plasminogen activators especially of t-PA in the endometrium during various phases of the menstrual cycle.     

Isolation and interrelationships of the multiple molecular tissue-type and urokinase-type plasminogen activator forms produced by cultured human umbilical vein endothelial cells

Primary and early subcultures (1st- to 3rd passage) of human umbilical vein endothelial cells produce tissue-type plasminogen activator (t-PA) antigen, consisting only of a major Mr 110,000 t-PA form. Later subcultures (greater than 4th passage) produce increasing amounts of t-PA antigen, consisting of a major Mr 110,000 and a minor Mr 68,000 form as well as increasing amounts of urokinase-type plasminogen activator (u-PA) antigen, consisting of a minor Mr 95,000 and major Mr 54,000 form. All of the major plasminogen activator forms were purified to homogeneity from 72 h serum-free conditioned media (3 liters, 1-1.8 x 10(9) cells) by a combination of immunoaffinity and gel filtration chromatography. Typically, 4th to 6th passage cultures produced/secreted t-PA-type proteins consisting of an inactive Mr 110,000 (220 IU/mg) and active Mr 68,000 (76,500 IU/mg) form representing about 39 and 8%, respectively, of the total starting sodium dodecyl sulfate stable t-PA activity, and u-PA-type proteins consisting of an inactive Mr 95,000 (700 IU/mg) and active Mr 54,000 (81,000 IU/mg) form representing about 9 and 38%, respectively, of the total starting sodium dodecyl sulfate stable u-PA activity. The isolated Mr 68,000 t-PA and Mr 54,000 u-PA proteins, exist only as two-chain forms in the absence of aprotinin and as mixtures of single- and two-chain proteins in the presence of aprotinin. Treatment with nucleophilic agents completely dissociated the Mr 110,000 t-PA and Mr 95,000 u-PA proteins into their respective Mr 68,000 t-PA and Mr 54,000 u-PA activity forms and a common Mr 46,000 protein, confirming the enzyme-inhibitor complex nature of these inactive plasminogen activator forms.     

Co-localization of urokinase and its receptor on established human umbilical vein endothelial cell.

Vascular endothelial cells possess antithrombotic properties, which are determined by the balance between plasminogen activators (PAs) and PA inhibitors (PAls). A cell line, TKM-33, has been established and cloned from human umbilical vein endothelial cells, was previously reported to produce a large amount of urokinase-type PA (u-PA) and small amounts of tissue-type plasminogen activator (t-PA) and PA inhibitor-1 (PAI-1). Moreover, TKM-33 expressed the u-PA receptor (u-PAR) which plays an important role in the localization of fibrinolytic activity on cell surface. In the present study, we investigated the localization of u-PA, t-PA, PAI-1 and u-PAR in TKM-33 by using immunofluorescence staining technique. The endothelial cells were strongly stained with anti-PAI-1, anti-u-PA and anti-u-PAR IgGs, and slightly with anti-t-PA IgG. The double immunofluorescence staining with mouse anti-u-PA IgG and rabbit anti-u-PAR IgG followed by rhodamine-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit IgG showed the co-localization of u-PA and u-PAR on the same section of endothelial cells. Although u-PA antigen also existed in the cytoplasm of endothelial cells, u-PAR antigen did not. The treatment of endothelial cells with phorbol-myristate-acetate (PMA) upregulated the expression of u-PA and u-PAR antigens. In this stimulation, u-PAR antigen was detected not only on the surface of the cells but also in the cytoplasm. Thus, the binding of u-PA to u-PAR was confirmed by double immunofluorescence staining.     

Human tissue-type plasminogen activator gene located near chromosomal breakpoint in myeloproliferative disorder.

Plasminogen activators (PA) convert the inactive proenzyme plasminogen into plasmin, which is involved in the process of fibrinolysis, tissue remodeling, and cell migration. There are two distinct forms of PA: urokinase (u-PA) and tissue-type plasminogen activator (t-PA). t-PA has higher affinity for fibrin and is the main form involved in thrombolysis. By in situ chromosomal hybridization and Southern blot analysis of somatic cell hybrid DNA, we have assigned the human t-PA gene to chromosome 8, bands 8p12----q11.2. We have detected a common EcoRI restriction fragment length polymorphism within the t-PA gene that thus provides a precisely localized highly informative marker for genetic linkage studies. The t-PA gene localization coincides with a translocation breakpoint observed in myeloproliferative disorders. Whereas leukemic cells usually secrete both types of PA, a correlation exists between acute myeloid leukemic cells that release only t-PA and failure to respond to chemotherapy.     

Human endothelial cells produce a plasminogen activator inhibitor and a tissue-type plasminogen activator-inhibitor complex

Serum-free conditioned media and cell extracts from cultured human umbilical vein endothelial cells were analyzed for plasminogen activator by SDS-polyacrylamide gel electrophoresis and enzymography on fibrin-indicator gels. Active bands of free and complexed tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA) were identified by the incorporation of specific antibodies against, respectively, t-PA or u-PA in the indicator gel. The endothelial cells predominantly released a high-molecular-weight t-PA (95000–135000). This t-PA form was converted to M_r-72000 t-PA by 1.5 M NH₄OH/39 mM SDS. A component with high affinity for both t-PA and u-PA could be demonstrated in serum-free conditioned medium and endothelial cell extract. The complex between this component and M_r-72000 t-PA comigrated with high-molecular-weight t-PA. From the increase in M_r of t-PA or u-PA upon complex formation, the M_r of the endothelial cell component was estimated to be 50000–70000. The reaction between t-PA or u-PA and the plasminogen activator-binding component was blocked by 5 mM p-aminobenzamidine, while the complexes, once formed, could be cleaved by 1.5 M NH₄OH/39 mM SDS. These observations indicated that the active center of plasminogen activator was involed in the complex formation. It was further noted that serum-free conditioned medium of endothelial cell extract inhibited plasminogen activator activity when assayed by the fibrin-plate method. Evidence is provided that the plasminogen activator-binding component was different from a number of the known plasma serine proteinase inhibitors, the placenta inhibitor and the fibroblast surface protein, proteinase-nexin. We conclude that cultured endothelial cells produce a rapid inhibitor of u-PA and t-PA as well as a t-PA-inhibitor complex.     

Cytokines regulate plasminogen activation system in astrocytoma cells.

We report here that human astrocytoma cell line U373-MG is able to express genes of the following components of plasminogen activation system: PA1-1, PN-1, u-PA and t-PA. Treatment of these cells with IL-1beta results in accumulation of PA1-1, PN-1 and u-PA mRNAs, whereas t-PA mRNA remains unaffected. IFNy preferentially enhances PN-1 and PA1-1, EGF enhances PA1-1, u-PA and t-PA expression. Simultaneous addition of anti-inflammatory cytokines IL-4, IL-13 and IL-10 has little effect on the tested components, except induction of u-PA mRNA wich was further enhanced by IL-4. We have confirmed interesting time-dependent regulation of plasminogen activation system by EGF/IFNgamma. Cells stimulated with EGF/IFNgamma show at first increased proteolytic activity but after 24 h inhibition of proteolysis with PA1-1 would prevail. To understand the cooperative effect of EGF and IFNgamma in PA1-1 induction the kinetics of activation of STAT1 was studied. It was found that although EGF alone does not activate STAT1, the STAT1 binding activity in the cells treated with the mixture of EGF/IFNgamma was considerably prolonged. Our results indicate the importance of inflammatory cytokines and EGF in gene regulation of plasminogen activation system in astrocytoma cells.     

Kinetics of inhibition of tissue-type and urokinase-type plasminogen activator by plasminogen-activator inhibitor type 1 and type 2

Highly purified plasminogen-activator inhibitors of type 1 (PAI-1) and type 2 (PAI-2), low-Mr form, were compared with respect to their kinetics of inhibition of tissue-type (t-PA) and urokinase-type plasminogen activator (u-PA). The time course of inhibition of plasminogen activator was studied under second-order or pseudo-first-order conditions. Residual enzyme activity was measured by the initial rate of hydrolysis of a chromogenic t-PA or u-PA substrate or by an immunosorbent assay for t-PA activity. PAI-1 rapidly reacted with single-chain t-PA as well as with two-chain forms of t-PA and u-PA. The second-order rate constant k for inhibition of single-chain t-PA (5.5 x 10(6) M-1 s-1) was about three times lower than k for inhibition of the two-chain activators. PAI-2 reacted slowly with single-chain t-PA, k = 4.6 x 10(3) M-1 s-1. The association rate was 26 times higher with two-chain t-PA and 435 times higher with two-chain u-PA. The k values for inhibition of single-chain t-PA, two-chain t-PA and two-chain u-PA were respectively, 1200, 150 and 8.5 times higher with PAI-1 than with PAI-2. The removal of the epidermal growth factor domain and the kringle domain from two-chain u-PA did not affect the kinetics of inhibition of the enzyme, suggesting that the C-terminal proteinase part of u-PA (B chain) is responsible for both the primary and the secondary interactions with PAI-1 and PAI-2. The k values for inhibition of single-chain t-PA and endogenous t-PA in plasma by PAI-1 or PAI-2 were identical indicating that t-PA in blood consists mainly in its single-chain form.     

Vascular origin determines plasminogen activator expression in human endothelial cells. Renal endothelial cells produce large amounts of single chain urokinase type plasminogen activator

Positioned at the boundary between intra- and extravascular compartments, endothelial cells may influence many processes through their production of plasminogen activators (PA). Available data have shown that tissue-type plasminogen activator (t-PA) is the major form produced by human endothelial cells. We have compared the molecular forms of PA produced by human endothelial cells from different microvascular and large vessel sources including two different sites within the circulation of the kidney. Using combined immunoactivity assays specific for u-PA and t-PA activity and antigen, we found that both human renal microvascular and renal artery endothelial cells produced high levels of u-PA antigen (60.48 ng/10(5) cells/24 h and 50.42 ng/10(5) cells/24 h, respectively) and corresponding levels of u-PA activity after activation with plasmin. Activity was not evident before plasmin activation, showing that the u-PA produced is almost exclusively as single chain form U-PA. In contrast, human omental microvascular endothelial cells and human umbilical vein endothelial cells produced exclusively t-PA (8.80 ng/10(5) cells/24 h and 2.17 ng/10(5) cells/24 h, respectively). Neither endothelial cell type from human kidney produced plasminogen activator inhibitor, as determined by reverse fibrin autography and titration assays. Agents including phorbol ester, thrombin, and dexamethasone were shown to regulate the renal endothelial cell production and mRNA expression of both u-PA and t-PA. Among the macro- and microvascular endothelial cells tested, only those from the renal circulation produced high levels of single chain form U-PA, suggesting the vascular bed of origin determines the expression of plasminogen activators.     

Tissue plasminogen activator is released into cultured medium by cultured human uveal melanocytes

Melanoma cells produce tissue plasminogen activator (t-PA) that plays an important role in tumor invasion and metastasis. The production of t-PA by normal human uveal melanocytes has not been reported previously. In order to explore this possibility, we studied the production of t-PA by cultured human uveal melanocytes and compared that with the production by cultured human uveal melanoma cells and epidermal melanocytes. Human adult uveal melanocytes were isolated and cultured from donor eyes. The cells were cultured in serum-free medium for 48 h and the conditioned medium then collected for the plasminogen activator (PA) activity assay. Free PA activity was tested in an amidolytic assay using a t-PA standard curve. PA type was identified by fibrinography and antihuman t-PA and urokinase plasminogen activator (u-PA) blocking antibodies. Free PA activity was found in the conditioned medium of normal melanocytes and melanoma cells. The predominant PA activity was t-PA. Normal uveal melanocytes produced more t-PA (3.23 +/- 0.73 IU/105 cells/24 h) than that of epidermal melanocytes (1.25 IU/105 cells/24 h) but much less than uveal melanoma cells (11.0 +/- 3.39 IU/105 cells/24 h). Western blot analysis revealed that most t-PA in conditioned media were one-chain t-PA with molecular weight of 69 kDa. Our study indicates that uveal melanocytes may contribute to the free t-PA activity previously found in aqueous humor and choroidal eye cup superfusions. Therefore, this function of uveal melanocytes may play a role in intraocular matrix remodeling, fibrinolysis and aqueous humor outflow.