The regulation of tissue plasminogen activator activity by human fibroblasts

Hoal, Eileen G.; Wilson, EL; Dowdle, Eugene B.

doi:10.1016/0092-8674(83)90158-7

Cited by 61 publications

(31 citation statements)

References 21 publications

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“…In a thoughtful presentation of their observations, Markus et al (1984) (Pennica et al, 1983), we feel that it is improbable that the 100,000 Mr species is a precursor of the lower molecular weight forms. We feel that it is more likely that it represents an enzymatically active complex formed by the combination of the enzyme with a specific binding molecule similar to the one that we have previously described on fibroblasts (Hoal et al, 1983) or that Levin has described as a 40,000 dalton inhibitor that binds to tissue plasminogen activator and converts it to an inactive or 'latent' species (Levin, 1983 (Hoal et al, 1983) and that enzyme is removed by this mechanism. Studies are currently under way to exanine these two alternatives.…”

Section: Resultsmentioning

confidence: 99%

Characterization of seven human melanoma cell lines: Melanogenesis and secretion of plasminogen activators

Helden¹,

Wilson²,

Dowdle³

1986

Br J Cancer

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Summary Permanent cell lines (UCT-Mel 1 through 7) were established from biopsies of metastatic tissue taken from seven patients with malignant melanoma. Cells from these lines were all aneuploid and all grew as non-contact-inhibited, adherent monolayers. All of the lines, with the remarkable exception of UCT-Mel 6, formed tumours in nude mice, expressed the melanoma M-18 antigen and synthesized plasminogen activators exclusively of the tissue-type. UCT-Mel 6 cells were non tumourigenic, they lacked the M-18 antigen and they synthesized plasminogen activators exclusively of the urokinase type. UCT-Mel I and UCT-Mel 2 formed pigment in vitro and both of these lines showed an increase in pigment content and tyrosinase synthesis with increasing cell density. The rate of plasminogen activator released by UCT-Mel 1 and UCT-Mel 3 declined strikingly as the cells became confluent.Assuming (a) that proteolytic activity is required for cell migration in vivo; (b) that tyrosinase synthesis reflects expression of the differentiated phenotype and (c) that melanoma cells retain some of the characteristics of neural crest cells, we suggest that the effects of confluence and close cell-cell contact provide a useful experimental counterpart for the study of normal neural crest all behaviour that is characterized by an inverse relationship between migration and a protease secretion on the one hand and pigmentation on the other.Human melanoma cells cultured in vitro provide a useful system for examining processes that are relevant both to this particular class of tumours and, at the same time, to cellular biological phenomena of a more general nature.Melanogenesis, for example, is an obvious and readily quantifiable marker of cellular differentiation that can be used to study factors that govern expression of the differentiated phenotype in pigmenting cell lines. Furthermore, as derivatives of the embryonic neural crest, melanoma cells share a common origin with a variety of ectomesenchymal structures, cells of the autonomic nervous system, calcitonin-producing cells, cells of the carotid body and sensory neurones in spinal ganglia and cranial nerves. It is now clear that, in choosing one of these diverse alternative destinies, pluripotential neural crest 'stem-cells' are influenced by environmental cues that come from neighbouring embryonic structures (Le Douarin, 1982 serum (State Vaccine Laboratories, Cape Town), 300 g penicillin ml-1, 200, g streptomycin sulphate ml-1 and 10 yg tylocine ml-1. Cultures were maintained at 37°C in a humid atmosphere containing 5% CO2 in air.Culture media were changed twice weekly. Confluent adherent cultures were passaged by detachment with 0.25% trypsin and 0.02% EDTA in Tris-buffered saline (TBS: 0.137 M NaCl, 5 mM KCl, 0.7 mM Na Phosphate, 25 mM TrisHCl; pH7.4) and re-seeding at -2x 105 cells/ 35 mm dish.Cell lines were tested for mycoplasma contamination (Chen, 1977) and found to be negative.Growth curves were constructed by seeding cells at a low density (usually 105 cells/35mm dish) and fe...

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Section: Resultsmentioning

confidence: 99%

Characterization of seven human melanoma cell lines: Melanogenesis and secretion of plasminogen activators

Helden¹,

Wilson²,

Dowdle³

1986

Br J Cancer

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“…In addition to removal of fibrin from the vascular bed, the Plg activatorplasmin system is important in other physiological and patho-logical processes (30), such as inflammation (33), neoplasia (34), and embryogenesis (35,36,37). Through a complex system of controls including the level of tissue production of TPA (38), localized degradation and inactivation of TPA (39,40), inactivation of plasmin by circulating inhibitors (21), and microenvironmental influences on the kinetics of Plg activation, Plg activation may be a general mechanism for generating localized proteolysis in a microenvironment (30). TSP may play an important role in such non-fibrin mediated Plg activator systems.…”

Section: Discussionmentioning

confidence: 99%

Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator.

Silverstein¹,

Leung²,

Harpel³

et al. 1984

J. Clin. Invest.

168

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A bstract. Thrombospondin (TSP), a multifunctional a-granule glycoprotein of platelets, binds fibrinogen, fibronectin, heparin, and histidine-rich glycoprotein and thus may play an important role in regulating thrombotic influences at vessel surfaces. In this study we have demonstrated that purified human platelet TSP formed a complex with purified human plasminogen (Plg). Complex formation was detected by rocket immunoelectrophoresis of mixtures of the purified radiolabeled proteins. Significant complex formation of fluid-phase Plg with adsorbed TSP was also demonstrated by enzyme-linked immunosorbent assay (ELISA). The complex formation was specific, saturable, and inhibited by excess fluid-phase TSP, with an apparent KD of -35 nM. In both ELISA and rocket immunoelectrophoresis systems, complex formation was inhibited by 10 mM eamino-n-caproic acid, implying that there is a role for the lysine binding sites of Plg in mediating the interaction. TSP also formed a complex with plasmin as detected by ELISA but did not directly inhibit plasmin activity measured with a synthetic fluorometric substrate or with a '251-fibrin plate assay. TSP, when incubated with Plg before addition to '25I-fibrin plates significantly inhibited the generation of plasmin activity by tissue plasminogen activator (TPA) in a manner that was calcium dependent. A kinetic study of Plg activation by TPA in the presence of TSP demonstrated that Michaelis-Menten kinetics were followed and that TSP acted as a noncompetitive inhibitor. These studies support the hypothesis that TSP,

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“…Kadouri and Bohak (11) suggested a negative feedback-type control for t-PA expression in human lung fibroblasts but did not identify the mechanism by which expression was regulated. Although t-PA has been reported to undergo receptor-mediated binding to a variety of cultured cells (12)(13)(14)(15), the biological significance of this phenomenon remains uncertain. Reports on the internalization and lysosomal degradation of t-PA have been largely confined to studies on hepatocytes, which have a known clearance function (for reviews, see Refs.…”

mentioning

confidence: 99%

Low Density Lipoprotein Receptor-related Protein Modulates the Expression of Tissue-type Plasminogen Activator in Human Colon Fibroblasts

Hardy

Feder

Wolfe

et al. 1997

Journal of Biological Chemistry

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Human colon fibroblasts (HCF) produce tissue-type plasminogen activator (t-PA) in culture, but after 24 -48 h, t-PA ceases to accumulate in the medium. Here, we report negative feedback regulation of t-PA expression, exerted by t-PA or complexes of t-PA with its physiological inhibitor, plasminogen activator inhibitor type 1 (PAI-1). Inhibition of t-PA expression could be induced by addition of exogenous t-PA or t-PA⅐PAI-1 complexes and reversed by monoclonal antibody directed against the active site of t-PA. Analysis of metabolically radiolabeled protein and cellular mRNA showed that both t-PA protein and mRNA levels declined considerably after 24 h. When 125 I-labeled t-PA or t-PA⅐PAI-1 complexes were incubated with HCF, monensin-inhibitable endocytosis and catabolism were observed. The low density lipoprotein receptor-related protein (LRP) was found to be expressed by HCF and to mediate these events. Addition of the 39-kDa receptor-associated protein (RAP), an antagonist for ligand interactions with LRP, removed the block to t-PA expression and restored its accumulation in the medium. Moreover, RAP completely prevented the degradation of exogenous 125 I-labeled t-PA by HCF, suggesting that LRP is the endocytic receptor for t-PA in these cells. These results demonstrate that cellular modulation of t-PA expression in HCF involves LRP receptor-mediated clearance of t-PA. This LRP receptor-mediated event results in down-regulation of t-PA expression at the mRNA level.Fibrinolysis is a complex process that requires precise regulation in vivo to ensure that it is neither deficient nor excessive. The plasminogen activator system is critical for thrombolysis, as well as other physiological processes including cell migration and tissue remodeling (1, 2). Human tissue-type plasminogen activator (t-PA) 1 (M r 68,000), a key serine protease in this system, is of particular pharmacological interest because of its value in the treatment of thromboembolic disorders. Regulation of the plasminogen activator system involves modulation by cofactors and inhibitors as well as controlled synthesis of the key components. It is well documented that t-PA expression is influenced by a variety of exogenous factors such as hormones, growth factors, and cytokines (3-6). Regulation by these factors appears to be receptor-mediated and is imposed at the transcriptional level (7-10). However, information on the role of t-PA in the local regulation of its own biosynthesis, release, and clearance is limited. Kadouri and Bohak (11) suggested a negative feedback-type control for t-PA expression in human lung fibroblasts but did not identify the mechanism by which expression was regulated. Although t-PA has been reported to undergo receptor-mediated binding to a variety of cultured cells (12-15), the biological significance of this phenomenon remains uncertain. Reports on the internalization and lysosomal degradation of t-PA have been largely confined to studies on hepatocytes, which have a known clearance function (for reviews, see Refs. 16,17)....

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The regulation of tissue plasminogen activator activity by human fibroblasts

Cited by 61 publications

References 21 publications

Characterization of seven human melanoma cell lines: Melanogenesis and secretion of plasminogen activators

Characterization of seven human melanoma cell lines: Melanogenesis and secretion of plasminogen activators

Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator.

Low Density Lipoprotein Receptor-related Protein Modulates the Expression of Tissue-type Plasminogen Activator in Human Colon Fibroblasts

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