Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The receptor for human urokinase-type plasminogen activator (u-PA) was purified from phorbol 12-myristate 13-acetate-stimulated U937 cells by temperature-induced phase separation of detergent extracts, followed by affinity chromatography with immobilized diisopropyl fluorophosphate-treated u-PA. The purified protein shows a single 55-60 kDa band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It is a heavily glycosylated protein, the deglycosylated polypeptide chain comprising only 35 kDa. The glycosylated protein contains N-acetyl-D-glucosamine and sialic acid, but no N-acetyl-D-galactosamine. Glycosylation is responsible for substantial heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines. The amino acid composition and the NH2-terminal amino acid sequence are reported. The protein has a high content of cysteine residues. The NH2-terminal sequence is not closely related to any known sequence. The identification of the purified and sequenced protein with the human u-PA receptor is based on the following findings: 1) the ability of the purified protein to bind u-PA and its amino-terminal fragment; 2) the identical electrophoretic mobilities observed for cross-linked conjugates, formed between either the purified protein or the u-PA receptor on intact U937 cells and the above ligands; 3) the identity of the apparent molecular weight of the purified protein to that predicted for the u-PA receptor in the same cross-linking studies; 4) the identical extent of glycosylation of the purified protein and of the u-PA receptor in crude membrane fractions, as detected after cross-linking; 5) the ability of antibodies raised against the purified protein to inhibit cellular binding of the amino-terminal fragment of u-PA.
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PMID:The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants. 215 52

The transplantation of PA-III rat prostate cancer cells onto rat skeleton produces osteoblastic metastases. Therefore w e studied the paracrine interactions between the PA-III cells and osteoblast-derived osteosarcoma cells (UMR 106 cells). A serine protease secreted by PA-III cells hydrolyzed IGF-binding protein-1 and IGF-binding protein-2 (IGFBP-1 and IGFBP-2) detected in the cell culture media (CM) of OMR 106 cells by western ligand blotting. The serine protease of PA-III cell CM was purified using a benzamidine affinity column. This protease was a protein of 45-50 kDa on polyacrylamide gel electrophoresis under non-reducing conditions but generated two protein bands under reducing conditions; a) one of 33-35 kDa possessing protease activity and b) another of 20-25 kDa which was proteinolytically inactive. Sequence analysis identified the amino acid sequence of the a-chain (20-25 kDa band) and of the b-chain (33-35 kDa band) of rat urokinase-type plasminogen activator molecule. Urokinase purified from PA-III cell CM hydrolyzed IGFBPs of UMR 106 cells and stimulated the proliferation of UMR 106 cells in serum-free cultures. Its protease activity was abolished by benzamidine and aprotinin. Its mitogenic activity for osteoblasts was inhibited by anti-IGF-I monoclonal antibody. Northern blot analysis documented the expression of the urokinase-type plasminogen activator gene in the mRNA extracted from PA-III cells. Urokinase expression was inhibited by dexamethasone. Therefore, we conclude that urokinase-type plasminogen activator stimulates osteoblasts via an IGF-I dependent mechanism. Hydrolysis of the IGFBOPs at the sites of PA-III cell-induced bone tumors account for an increased bioavailability of IGFs. This may facilitate the development and the growth of PA-III cell-induced bone tumor and can also mediate the subsequent local osteoblastic reaction.
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PMID:Urokinase-type plasminogen activator: a paracrine factor regulating the bioavailability of IGFs in PA-III cell-induced osteoblastic metastases. 768 89

The aim of the present study was to determine the role of transforming factor alpha (TGF alpha) and beta (TGF beta) in the regulation of prostaglandin (PG) secretion, and the relationships between PG and plasminogen activator (PA) activity in hen granulosa cells during ovarian follicular development. Cells from the first (F1), third (F3), and fifth and sixth (F5-6) largest preovulatory follicles were cultured for up to 21 h in the presence of TGF alpha (0.1-10 ng/ml) and/or TGF beta (4-20 ng/ml) or TGF alpha together with a cyclooxygenase inhibitor, indomethacin (0.05-0.5 microM). The release of PG into the incubation medium was determined by RIA. Cell-associated (PAc) and secreted (PAs) PA activities were measured by a fibrinolysis assay and characterized by zymography. Basal PGF secretion from F1, F3, and F5-6 cells was 2.2 +/- 0.3, 2.2 +/- 0.5, and 1.1 +/- 0.3 ng/micrograms DNA, respectively, and was higher than that of PGE. Basal total PA (PAc+PAs) activity from F1, F3, and F5-6 cells was 41 +/- 13,261 +/- 68, and 958 +/- 268 x 10(3) cpm/micrograms DNA, respectively. TGF alpha stimulated PG secretion and PA activity in a dose-dependent manner. The TGF alpha-induced PA activity was predominantly associated with a molecular mass of 30-35 kDa, corresponding to that of urokinase PA. The stimulation of PG secretion by TGF alpha was maximal in F3 and F1 granulosa cells whereas PA activity in the presence of TGF alpha was highest in cells from F5-6 follicles.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Avian granulosa cell prostaglandin secretion is regulated by transforming growth factor alpha and beta and does not control plasminogen activator activity during follicular development. 781 60

Granulosa cells from the first (F1), third (F3) and fifth and sixth (F5-6) preovulatory follicles and the small yellow follicles (SYFs; diameter 6-8 mm) were cultured for 21 h in the absence and presence of murine and human epidermal growth factors, fibroblast growth factor, transforming growth factors alpha and beta-I (TGF alpha, TGF beta), platelet-derived growth factor and insulin-like growth factor-I at concentrations of 0.1-100 ng/ml. Plasminogen activator (PA) activities in the cell (PAc) and in the medium (PAm) were measured by fibrinolysis and fibrin overlay methods. Basal PAc and PAm activities were highest in cell cultures from the less mature follicles (F5-6 and SYF) and decreased as the follicles matured (F3 > F1). PAc activity was greater than PAm activity, irrespective of the stage of follicular development. All growth factors examined at the 100 ng/ml level were effective in increasing PAc and PAm activities in cultures of granulosa cells from F1 follicles. However, only TGF alpha was able to increase PA activities at lower concentrations. The stimulation of the PA activities of granulosa cells from F3 follicles was inconsistent. None of the growth factors significantly increased PA activities in granulosa cells from F5-6 follicles and SYFs, as determined by fibrinolysis. The major PAc and PAm species (characterized by fibrin overlay) had a molecular mass of about 35 kDa, which is characteristic of the urokinase type. Both assay methods detected a stimulatory effect of the growth factors on PA activities in the granulosa cells from F1 follicles. However, an increase in PA activities in cells from F3 and F5-6 follicles and SYFs was indicated only after fibrin overlay analysis. Tritiated thymidine was incorporated into the DNA of granulosa cells at all stages of follicular development and was enhanced by all growth factors, although TGF alpha and TGF beta were the most effective and had a ranked order of activity: F3, F5-6 > F1, SYF. The present findings show that, of the growth factors examined, TGF alpha may be an effective regulator of PA activity in avian granulosa cells during follicular development, in addition to its observed mitogenic action.
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PMID:Influence of growth factors on the plasminogen activator activity of avian granulosa cells from follicles at different maturational stages of preovulatory development. 814 37

This study examined the influence of transforming growth factor-alpha (TGF alpha), TGF beta, and LH on progesterone (P4) secretion and plasminogen activator (PA) activity in cultured avian granulosa cells from the first (F1), third (F3), and fifth and sixth (F5-6) preovulatory follicles during a 21-h incubation period. PA activity in the cell (PAc) and the medium (PAm) fractions was measured by fibrinolysis and fibrin overlay methods. P4 was determined by RIA. Basal PAc and PAm activities were highest in cell cultures from the less mature (F5-6) follicles and decreased as follicles matured to the F1 stage of development. PAc activity was greater than PAm activity regardless of the stage of follicular maturation. TGF alpha (0.1-10 ng/ml) increased PA activity in cultures of granulosa cells from F1, F3, and F5-6 follicles in a concentration-dependent manner. TGF alpha-induced PAc and PAm activities were observed by 6 and 15 h of incubation, respectively, and increased rapidly between 15-21 h. LH (100 ng/ml) attenuated TGF alpha-induced PA activity by 15 h in cultures of granulosa cells from F1 and F3, but not F5-6, follicles. Basal PA activities were unaffected by the gonadotropin. TGF beta (2-100 ng/ml) stimulated PAc activity in a dose-dependent manner only in cultures of granulosa cells from F5-6 follicles and significantly enhanced TGF alpha-induced PAc and PAm activities in cell cultures from F3 and F5-6, but not F1, follicles. Basal and growth factor-induced PAc and PAm activities corresponded to a mol wt of about 35 kDa, a value consistent with that of the low mol wt uPA species. TGF alpha and TGF beta, alone or in combination, had no effect on basal P4 secretion at all stages of follicular development. TGF alpha, however, decreased LH-induced P4 secretion in F1 and F3 cultures. These results demonstrate a tightly controlled interaction of TGF alpha, TGF beta, and LH in regulating PA activity and P4 secretion during follicular development in the domestic hen.
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PMID:Interactions of transforming growth factor-alpha and -beta and luteinizing hormone in the regulation of plasminogen activator activity in avian granulosa cells during follicular development. 834 11

The occurrence of type-1 plasminogen activator inhibitor (PAI-1) in human cerebrospinal fluid (CSF) has not previously been reported. As a member of the serpin superfamily of serine protease inhibitors and an acute phase response component, PAI-1 has powerful potential roles in nervous system homeostasis. We have detected this serpin antigen using a polyclonal anti-PAI-1 antibody in normal human CSF. In Western blotting, PAI-1 in several CSF samples appears as a two-band antigen of Mr = 54 and 35 kDa, presumably the intact and proteolytic fragment, respectively. In vitro complex formation studies confirm that the 54 kDa form of PAI-1 interacts with 125I-urokinase after activation with SDS, but the 35 kDa form does not. Quantification of total PAI-1 antigen in 18 normal human CSF samples by ELISA reveals a mean value of 1.0 +/- 0.07 (SEM) micrograms/dL, indicating that a relatively low concentration of the inhibitor occurs in normal human CSF. This information should now allow comparison of PAI-1 levels and activity in various neurologic disorders.
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PMID:Plasminogen activator inhibitor 1, the primary regulator of fibrinolysis, in normal human cerebrospinal fluid. 845 10

The aim of the present study was to examine the regulatory role of transforming growth factor alpha (TGF alpha) on urokinase plasminogen activator (uPA) gene expression and protein levels in hen granulosa cells from different stages of ovarian follicular development in vitro. Granulosa cells from the first (F1), the second and third (F2-3), and the fourth, fifth, and sixth (F4-6) largest preovulatory follicles were cultured for 21 h in the absence and presence of TGF alpha (10 ng/ml). The uPA mRNA abundance and protein content were determined by Northern and Western blot analysis, respectively. Cell-associated and secreted PA activity was measured by a fibrinolysis assay and characterized by zymography. Hen granulosa cells produce a uPA with a molecular mass of about 35 kDa and a transcript size of approximately 2.5 kb. Basal uPA mRNA abundance, protein content, and activity were highest in granulosa cells from F4-6 follicles and decreased with follicular maturation. Granulosa cell uPA mRNA levels, protein content, and activity were increased in the presence of TGF alpha, reaching maximal levels in granulosa cells from less mature follicles, although the percentage of stimulation was higher in cells from late stages of follicular development. These findings clearly demonstrate specific expression of uPA in proliferatively active granulosa cells and responsiveness of uPA to TGF alpha at both transcriptional and translational levels. They support the concept that PA of the urokinase type plays an important role in extracellular matrix remodeling during TGF alpha-induced granulosa cell proliferation and ovarian follicular growth.
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PMID:Up-regulation of urokinase plasminogen activator messenger ribonucleic acid and protein in hen granulosa cells by transforming growth factor alpha in vitro during follicular development. 916 Jul 33

The specialized interaction between embryonic and maternal tissue is unique to mammalian development. This interaction begins with the invasion of the uterus by the first differentiated embryonic cells, the trophoblasts, and culminates in formation of the placenta. Because of their highly specialized behavior invasive cells must attach to the extracellular matrix proteins, secrete proteinases, capable of degrading matrix, and migrate through the degraded matrix; invasion is partially dependent on the proteinase activity of the cells. The objective, therefore, was to study a vitro system to examine the mechanism(s) of trophoblast cell invasion and its relationship to proteinases. Since little is known about the actual mechanism(s) involved. The mouse trophoblast cell lines established from placentas of different gestational ages were chosen to study their invasive properties in vitro. To begin to understand the biochemical basis of this behavior, the chromogenic assay and the substrate gel technique was used to analyze the cell associated and secreted plasminogen activators. All lines secrete and synthesize both urokinase-type (uPA) and tissue-type (tPA) plasminogen activators. The most invasive line SM9-2, derived from mid-gestation (day 9) placenta showed the highest enzymatic activity in the conditioned medium (CM), whereas in cell extract (CE) SM-10 line derived from late gestation placenta had the highest PAs activity. Four forms of secreted PAs in CM were of 79, 72, 43 and 35 kDa molecular weights, whereas in CE only 79 kDa molecular weight form of PA was detected using substrate SDS-PAGE gels. Additional observations from cells cultured on Marrigel Invasion Chambers also showed secretion of PAs by noninvading and invading cells in a biphasic pattern suggest the involvement of these enzymes in the extracellular proteolysis. The expression of matrix metalloproteinase gelatinase B (MMP-9) and tissue inhibitor of metalloproteinase (TIMP-1) were examined by RT-PCR in all the lines, however MMP-9 and TIMP-1 signals were strongly expressed in SM9-2 and SM-10 lines respectively. CM and CE were characterized by gelatin zymography, and the proteinases secreted by these cells in CM were confirmed to be metalloproteinases with approximate molecular masses between 52 to 92 kDa. Proteinases secreted by noninvading and invading cells cultured on Matrigel Invasion Chambers were not identical suggesting that specialized, temporally regulated metallopro-teinases are involved in trophoblast invasion. Trophoblast cell invasion in Matrigel Invasion Chambers was significantly inhibited in all the lines by using 1, 10-phenanthroline, an inhibitor of metalloproteinases. The results indicated that mouse trophoblast cells have matrix--degrading capabilities through metalloproteinase activity. Similar metalloproteinase activity has been reported to be necessary for human trophoblast invasion, suggesting a similar mechanism of implantation. Trophoblast culture system described here should be useful in studying some of the early events in human placentation.
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PMID:Mouse trophoblastic cell lines: II--Relationship between invasive potential and proteases. 962 4

The biochemical mechanism controlling nucleation of mineral crystals in developing bone, along with the growth and propagation of these crystals once formed, remains poorly understood. To define the nucleation mechanism, a proteomics analysis was begun on isolated biomineralization foci (BMF), sites of initial crystal nucleation in osteoblastic cell cultures and in primary bone. Comparative analyses of the protein profile for mineralized BMF with that for total osteoblast cultures revealed the latter were enriched in several proteins including BAG-75 and BSP, as well as fragments of each. When 12 protease inhibitors were added separately to UMR 106-01 osteoblastic cultures, only the serine protease inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) blocked cleavage of BAG-75 and BSP, and prevented mineral crystal nucleation within BMF. Consideration of the specificities of the inhibitors tested and the fact that AEBSF inhibition was not dependent upon inclusion of FBS in the culture media indicated that mineral nucleation does not require serine protease plasmin, thrombin, kallikrein, urokinase, C1s or furin. In contrast, SKI-1 (S1P or site-1) is a membrane-bound serine protease inhibitable by AEBSF. We show here for the first time that mineralizing UMR 106 cells express a 98-kDa active, soluble form of SKI-1 within BMF. In contrast, nonmineralizing UMR cells appear to differentially process SKI-1 into smaller immunoreactive fragments (<35 kDa). These findings suggest that SKI-1 plays a direct or indirect role in assembly of functional nucleation complexes containing BAG-75 and BSP and their fragments, thus facilitating initial mineral nucleation within these biomineralization foci.
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PMID:Potential role of proprotein convertase SKI-1 in the mineralization of primary bone. 1872 45