Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.68 (tissue plasminogen activator)
 11,311 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasmin-mediated extracellular proteolysis has been implicated in the degradation of bone in normal and pathological conditions. Normal and malignant osteoblasts can produce both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). We have used the osteosarcoma cell line MG63 to address the question of whether the enhanced bone turnover in osteosarcomas is mediated by t-PA or by u-PAA and to study the effect of the cytokine interleukin-1 alpha (IL-1 alpha), known to influence bone degradation, on the plasminogen activator production and extracellular matrix degradation in malignant osteoblastic cells. Furthermore, the effect of IL-1 alpha on the synthesis of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) was analyzed. u-PA production by MG63 was high (approximately 180 ng/10(6) cells/24 h). Also t-PA and PAI-1 production was observed. u-PA production was rapidly increased in MG63 by IL-1 alpha (10 ng/ml), whereas an effect on t-PA production was only found after a prolonged incubation and hardly any effect of IL-1 alpha on PAI-1 production was observed. mRNA analysis revealed similar effects. u-PA receptor (u-PAR) mRNA was detectable in MG63 cells and could be increased by IL-1 alpha after 24 h. In MG63, u-PA-mediated extracellular matrix degradation was detectable, and IL-1 alpha increased the u-PA-mediated matrix degradation (approximately 2-fold). Under control conditions in MG63, only MMP-2, TIMP-1, and TIMP-2 mRNA could be observed. After the addition of IL-1 alpha, a very rapid increase in MMP-1 and MMP-3 mRNA could be observed as well as a moderate increase in TIMP-1 mRNA. The presence of MMP-2 was demonstrated by gelatin zymography. These results show that IL-1 alpha can stimulate u-PA production and can regulate extracellular proteolytic activity mainly via u-PA induction in the MG63 osteosarcoma cell line. Furthermore, IL-1 alpha has a strong stimulating effect on the production of MMP-1 and MMP-3. These findings suggest that u-PA and possibly MMP-1 and MMP-3 play an important role in the process of bone turnover in osteosarcomas.
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PMID:Regulation of plasminogen activation, matrix metalloproteinases and urokinase-type plasminogen activator-mediated extracellular matrix degradation in human osteosarcoma cell line MG63 by interleukin-1 alpha. 750 10

Human omental microvascular endothelial (HOME) cells seeded on Matrigel begin to migrate within 1 h, forming honeycomb-like structures and capillary-like networks within 18 h. Cross-sections of the capillary networks show them to be tube-like structures. Northern blot analysis showed that tissue-type plasminogen activator (t-PA) mRNA synthesis increased from the initial state at 0 h after seeding on Matrigel, reaching a steady state after 4 h. This elevated cellular t-PA mRNA level decreased markedly at 24 h. In contrast, the cellular plasminogen activator inhibitor-1 (PAI-1) mRNA level demonstrated biphasic curves during the 24 h after seeding on Matrigel: the PAI-1 mRNA level was increased eightfold initially at 4 h over that at 0 h, then declined, and again secondarily increased to greater than tenfold at 18 h. Cellular levels of both 72 kD type IV collagenase and tissue inhibitor of metalloproteinase (TIMP-2) mRNA were increased only a slightly within 2-4 h. These elevated mRNA levels were maintained for 18 h, while the TIMP-1 mRNA level increased up to 18 h, reaching around three times the level at 0 h. However, on collagen-coated dishes, cellular levels of t-PA, PAI-1, 72 kD type IV collagenase, TIMP-1, and TIMP-2 mRNA were not greatly changed during incubation for 24 h. On Matrigel, the cellular t-PA mRNA level at 18 h after seeding was greatly increased when treated with specific anti-transforming growth factor-beta (TGF-beta) antibody. In contrast, both PAI-1 and TIMP-1 mRNA levels at 18 h were reduced in the presence of anti-TGF-beta antibody. Development of the capillary network on Matrigel was inhibited in the presence of anti-t-PA antibody. Epidermal growth factor (EGF) enhanced t-PA gene expression and TGF-beta inhibited its expression in HOME cells cultured on collagen-coated dishes. On the other hand, TGF-beta enhanced cellular expression of the PAI-1 gene. The formation of a capillary network by HOME cells on Matrigel appears to be balanced by angiogenic EGF and anti-angiogenic TGF-beta through modulation of PA activity.
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PMID:Expression of tissue-type plasminogen activator and its inhibitor couples with development of capillary network by human microvascular endothelial cells on Matrigel. 782 31

The cellular events causing pathological extracellular matrix (ECM) accumulation in vivo are not well understood. Prolonged serial passage of several cell types in culture leads to increased production of extracellular matrix (ECM) proteins, but the mechanism for these putative fibrotic changes is not known. Here, human fetal glomerular mesangial cells were subjected to serial passage (P) in culture and the expression of ECM proteins, proteases and protease inhibitors was comprehensively evaluated. From P11 through P14, a series of phenotypic changes occurred. Steady-state expression of mRNA for alpha 1 chains of type III and type IV (but not type I) collagen, and for laminin beta 1 and gamma 1, increased 2- to 8-fold, while expression of mRNA for interstitial collagenase (MMP-1) and gelatinase A (MMP-2) virtually ceased. Expression of tissue-type plasminogen activator (tPA) mRNA also decreased markedly. Expression of mRNA for the tissue inhibitor of metalloproteinases (TIMP)-1, and of the smaller of two mRNA species for the PA inhibitor PAI-1, ceased by P14. There was a switch in expression of the two species of TIMP-2 mRNA: whereas the ratio of signal intensity comparing the 3.5 kb mRNA species to the 1.0 kb species was 5:1 up to P11, it was reversed (1:5) at P14 and later. Serial passage also led to changes in protein expression, with increased type IV collagen and laminin, but decreased interstitial collagenase and gelatinase A. The cells showed a progressive increase in staining for type IV collagen. These findings define the appearance of a matrix-accumulating phenotype in later-passage mesangial cells. Matrix expansion in vivo has been associated with increased transforming growth factor (TGF)-beta synthesis; the cells were found to show at least 5-fold increased expression of TGF-beta 1 mRNA from P8 to P16. However, treatment of P9 or P10 cells with graded doses of TGF-beta 1 increased expression of both collagen IV and gelatinase A mRNA and did not alter the ratio of signal intensity for TIMP-2 mRNA species. Thus, assumption of a matrix-accumulating phenotype by these cultured fetal glomerular mesangial cells is not accelerated by exogenous TGF-beta. These data describe an in vitro model of mesangial cell matrix turnover in which matrix accumulation could result from a concerted increase in ECM synthesis and decrease in ECM degradation.
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PMID:Increased expression of extracellular matrix proteins and decreased expression of matrix proteases after serial passage of glomerular mesangial cells. 892 13

We report on the effect of prolonged hyperglycaemic (11 and 30 mM D-glucose) culture conditions on human mesangial cell matrix metalloproteinases (MMPs), plasminogen activators and their inhibitors. The results indicate that hyperglycaemic conditions modulate the potential proteolytic activity of the enzymes secreted by confluent cultures of these cells. Gelatinase A (MMP-2) activity was always higher in cultures maintained under hyperglycaemic than under normoglycaemic conditions (4 mM D-glucose). In contrast, gelatinase B (MMP-9) activity was decreased under the same conditions. Matrilysin (MMP-7) activity was decreased by up to 100% under hyperglycaemic conditions. Reverse transcriptase-PCR and Western-blotting analyses indicate that in all cases both the transcripts and the protein level were correlated with enzymic activity. One tissue inhibitor of metalloproteinases, TIMP-2, was barely detectable under hyperglycaemic conditions (30 mM D-glucose). In contrast, TIMP-1 increased during the initial 2 weeks of culture in hyperglycaemic conditions and remained elevated to the end of the experiment (4 weeks). Under normoglycaemic conditions TIMP-1 decreased after 2 weeks of culture. Hyperglycaemic conditions also decreased markedly the activity of tissue plasminogen activator (t-PA). This seemed to be due to increased synthesis of its inhibitor, plasminogen activator inhibitor 1, under these conditions rather than to decreased expression of the t-PA enzyme.
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PMID:Modulation of neutral protease expression in human mesangial cells by hyperglycaemic culture. 900 62

Cultured fibroblasts from patients with systemic sclerosis (SSc) and normal individuals were examined for gene expression of types I and III collagen, decorin, matrix metalloproteinases (MMP) MMP-1, MMP-2, and MMP-3, tissue inhibitors of metalloproteinases (TIMP) TIMP-1 and TIMP-2, urokinase- and tissue-type plasminogen activators (u-PA and t-PA). Fibroblasts from patients with early stage SSC (less than 1 year duration of disease) exhibited higher levels of types I and III procollagen, decorin, MMP-1, MMP-3, TIMP-1, and PAs than those from normal individuals. The gene expression of procollagen alpha 1(I) and TIMP-1 mRNAs were increased, but those of decorin, MMP-1, MMP-2, and MMP-3 were decreased, in fibroblasts from SSc patients with mid-stage SSc (2 to 4 years duration) as compared with those from normal individuals. In contrast, no significant difference in gene expression was found between fibroblasts from normal individuals and from patients with late-stage SSc (more than 6 years duration). These results suggest that gene expression of collagen, decorin, and degrading factors is dynamically modulated during fibrillogenesis. The responses of procollagen alpha 1(I) mRNA to IL-1 and TGF-beta were lower in fibroblasts from SSc patients with early and mid-stage disease, but not in those from patients with-late stage disease, than in control fibroblasts, which indicates that these cytokines may be involved in the earlier phases of fibrosis in SSc.
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PMID:Gene expression of types I and III collagen, decorin, matrix metalloproteinases and tissue inhibitors of metalloproteinases in skin fibroblasts from patients with systemic sclerosis. 937 15

Al-trans retinoic acid (RA) enhanced human, S-type, SK-N-SH neuroblastoma cell invasion of reconstituted basement membrane in vitro but did not induce terminal differentiation of this cell line. In contrast to basal invasion, which was urokinase (uPA)- and plasmin-dependent, RA-enhanced invasion was dependent on tissue-type plasminogen activator (t-PA) and plasmin activity. Neither basal nor RA-enhanced invasion involved TIMP-2 inhibitable metalloproteinases. Enhanced invasion was associated with the induction of t-PA expression, increased expression of the putative t-PA receptor amphoterin, increased association of t-PA with cell membranes and increased net membrane-associated PA activity. Enhanced invasion was not associated with significant changes in the expression of uPA or its membrane receptor UPAR; plasminogen activator inhibitors PAI-1 and PAI-2; metalloproteinases MMP-1, MMP-2, MMP-3, MMP-9 and membrane type MMP1; or tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2. RA stimulated the association of t-PA with the external cell membrane surface, which could be inhibited by heparin sulphate but not by mannose sugars or chelators of divalent cations, consistent with a role for amphoterin. Our data indicate that RA can promote the malignant behavior of S-type neuroblastoma cells refractory to RA-mediated terminal differentiation by enhancing their basement membrane invasive capacity. We suggest that this results from the action of a novel, RA-regulated mechanism involving stimulation of t-PA expression and its association with the cell membrane leading to increased PA-dependent matrix degradation.
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PMID:Retinoic acid-enhanced invasion through reconstituted basement membrane by human SK-N-SH neuroblastoma cells involves membrane-associated tissue-type plasminogen activator. 939 56

Primary varicose veins are functionally characterized by venous back-flow and blood stagnation in the upright position. Dilatation and tortuosity provide evidence for progressive venous wall remodelling, with disturbance of smooth muscle cell/extracellular matrix organization. Affected areas are not uniformly distributed, some areas being hypertrophic, whereas others are atrophic or unaffected. In 12 varicose veins and ten control veins, the proteolytic enzyme/inhibitor balance which may participate in the remodelling of the venous wall was investigated. For this purpose, the presence and enzymatic activity of matrix metalloproteinases (MMP-2, MMP-9), tissue inhibitors of MMPs (TIMP-1, TIMP-2), urokinase-type (uPA) and tissue-type (tPA) plasminogen activators (PAs), and plasminogen activator inhibitor-1 (PAI-1) were quantified by western blot and gelatin or plasminogen-casein zymography. In addition, MMP-2, TIMP-1, TIMP-2, and PAI-1 levels were measured by ELISA. A high TIMP-1 level and a low MMP-2 level/activity were found in varicose veins (p<0.005), resulting in a three-fold increase in the TIMP-1/MMP-2 ratio in varicose versus control veins. Levels of PAs (uPA and tPA) as well as PAI-1 were both lower in varicose veins (p<0.005), with minimal change in the PAI/PA ratio. These results demonstrate that varicose veins are characterized by a higher than normal TIMP/MMP ratio, which may facilitate extracellular matrix accumulation in the diseased venous wall.
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PMID:Increased TIMP/MMP ratio in varicose veins: a possible explanation for extracellular matrix accumulation. 1095 7

The aim of this study was to determine the expression of proteinases and inhibitors from the matrix metalloproteinase (MMP) (MMPs 1, 2, 3, 9, tissue inhibitors of metalloproteinases (TIMPs) 1, 2) and plasminogen activator ((PA) urokinase (uPA), tissue type (tPA), uPAR, plasminogen activator inhibitors (PAIs) 1, 2) systems in colorectal cancer pathology by gelatin zymography, enzyme-linked immunosorbent assays (ELISAs) and quenched fluorescent substrate hydrolysis. The levels of all studied MMPs, uPA, uPAR, TIMP-1 and PAIs were significantly greater in tumour tissues than normal tissues. However, tPA and TIMP-2 were greater in normal colon (P<0.05, Mann-Whitney) e.g. PAI-1: tumour, median 14.9 (range 0.2-80.2) ng/mg total protein; normal, 2.1 (0.1-65.0). Tumour levels of several factors, in particular MMP-1 and PAI-1, correlated with pathology, i.e. Dukes' stage, differentiation, lymphatic or vascular invasion and tumour depth. The interactions between proteinase systems in colorectal cancer are complex and the balance between active proteinases and their inhibitors is important for extracellular matrix (ECM) degradation/remodelling at each stage of the metastatic cascade.
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PMID:The plasminogen activator and matrix metalloproteinase systems in colorectal cancer: relationship to tumour pathology. 1270 68

The degradation of basement membranes by tumor cells involves secretion and activation of proteinases, such as matrix metalloproteinases (MMPs) and the plasminogen activation system (uPA, tPA, PAI-1), and results from an imbalance between their inhibitors and activators, controlled by various growth factors or cytokines. Among them, the TGF-beta family is one of the most intriguing because it has been reported either to decrease or promote cancer progression. In the present paper, we studied the effect of TGF-beta1 in a mouse melanoma model. In vivo, TGF-beta1 inhibited tumor growth after subcutaneous injection of B16F1 cells in syngenic mice. In vitro, TGF-beta1 did not alter B16F1 cell proliferation, but strongly decreased their migration through Matrigel-coated membranes. The protease production was analyzed by zymography, Western blot, or RT-PCR. MMP-2 and TIMP-2 expression were not altered by TGF-beta1. In contrast, TGF-beta1 triggered a large decrease of uPA and tPA, as well as a decrease of uPA and uPAR mRNAs. By Western blot and RT-PCR analyses, TGF-beta1 was shown to induce a strong increase of PAI-1 synthesis. Collectively, these results suggest that TGF-beta1 may inhibit melanoma tumor growth by specifically decreasing plasmin activity of tumor cells and play a protective role during the earliest stages of tumor progression.
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PMID:Transforming growth factor-beta1 inhibits tumor growth in a mouse melanoma model by down-regulating the plasminogen activation system. 1459 3

Uncontrolled activation of matrix metalloproteinases (MMPs) can result in tissue injury and inflammation, yet little is known about the activation of MMPs during orthotopic liver transplantation (OLT). OLT is associated with increased fibrinolytic activity due to elevated plasmin generation. The serine-protease plasmin not only causes degradation of fibrin clots but is also thought, amongst others, to play a role in the activation of some matrix metalloproteinases. We therefore studied the evolution of MMP-2 and -9 plasma concentrations during OLT and the effect of serine-protease inhibition by aprotinin on the level and activation of these MMPs. In a group of 24 patients who participated in a randomized, double-blind, placebo-controlled study we determined serial MMP-2 and MMP-9 plasma levels during transplantation using ELISA (total MMP), activity assays (activatable MMP) and zymography. In addition, the MMP-inhibitors TIMP-1 and TIMP-2 were assessed by ELISA. The putative regulating factors tumor necrosis factor alpha (TNF-alpha) and tissue-type plasminogen activator (t-PA) were assessed as well. Patients were administered high-dose aprotinin, regular-dose aprotinin or placebo during surgery. Plasma TIMP-1, TIMP-2 and MMP-2 level gradually decreased during transplantation. Approximately two-thirds of total MMP-2 appeared to be in its activatable proMMP form. No release of MMP-2 from the graft could be detected. In contrast, plasma levels of MMP-9 increased sharply during the anhepatic and postreperfusion periods. Peak MMP-9 levels of about eight times above baseline were found at 30 minutes after reperfusion. Most MMP-9 appeared to be in its active/inhibitor-complexed form. No significant differences were observed between the three treatment groups. However, in patients with more severe ischemia/reperfusion (I/R) injury the MMP-9 concentration, particularly of the active/inhibitor-complexed form, remained high at 120 minutes postreperfusion compared to patients with no or mild I/R injury. The decrease in plasma levels of MMP-2, TIMP-1 and TIMP-2 during OLT occurred irrespective of the severity of the I/R injury. There was a significant correlation between MMP-9 and t-PA levels, but not with TNF-alpha. In conclusion, OLT is associated with a sharp increase of MMP-9 during the anhepatic and postreperfusion periods, which coincided with the changes in t-PA. MMP-2, TIMP-1 and TIMP-2 gradually decreased during OLT. The composition of these MMPs was not altered by the use of aprotinin, suggesting that serine-protease/plasmin-independent pathways are responsible for MMP regulation during OLT. In addition, only MMP-9 seems to be involved in I/R injury during human liver transplantation.
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PMID:Plasma MMP-2 and MMP-9 and their inhibitors TIMP-1 and TIMP-2 during human orthotopic liver transplantation. The effect of aprotinin and the relation to ischemia/reperfusion injury. 1498 26


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