Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UNIPROT:P00750 (PLA)
16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The HT-1080 human fibrosarcoma cell line exhibited a plasminogen-dependent ability to inactivate recombinant anaphylatoxin C5a or zymosan-activated serum. The inactivation was obtained at physiological levels of both plasminogen (2 microM) and C5a (1-5 nM). Inactivated C5a and zymosan-activated serum were no longer able to induce chemotaxis and degranulation of neutrophils. Inactivation of C5a paralleled the emergence of plasmin activity, assayed by cleavage of the synthetic substrate H-D-valyl-L-leucyl-L-lysine-p-nitroanilide (S-2251). Both C5a inactivation and S-2251 cleavage were inhibited by the plasmin inhibitor alpha 2-antiplasmin, the urokinase inhibitor amiloride, and by anti-urokinase antibodies. In a cell-free system, inactivation of C5a was shown to depend on the simultaneous presence of urokinase and plasminogen and was inhibited by alpha 2-antiplasmin and by anti-urokinase antibodies. SDS-polyacrylamide electrophoresis demonstrated the cleavage of C5a by the plasminogen activation system and inhibition of the cleavage by amiloride. Amino acid sequencing of the band corresponding to the C5a degradation product revealed that C5a was cleaved at positions Lys14-His15 and Arg40-Ile41; cleavage at position Arg40-Ile41 seemed to be responsible for the loss of activity. Since neoplastic cells extensively produce and exhibit plasminogen activator activity, the present observations suggest that plasminogen activation may, by inactivation of C5a, reduce the anti-tumor immune response and support the immunological escape phenomenon of tumors.
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PMID:Inactivation of human anaphylatoxin C5a and C5a des-Arg through cleavage by the plasminogen activator activity of a human fibrosarcoma cell line. 792 54

The production of proteolytic enzymes by osteoblasts is considered important for initiating osteoclastic bone resorption. Using the established cell line NY as an example of osteoblast-like cells, the effect of intracellular cyclic AMP (cAMP) and protein kinase C (PKC) on plasminogen activator secretion and its specific binding to the cells were investigated. HT-1080 cells were used as the control. NY cells predominantly secrete single-chain urokinase-type plasminogen activator (scu-PA) and some two-chain u-PA. Both scu-PA and u-PA were present in the cell surface and cell lysate of NY cells, and their distribution in HT-1080 cells was quite similar to that of NY cells. Exposing cells to phorbol myristate acetate (PMA) or dibutyryl cyclic AMP (db cAMP) enhanced the secretion of scu-PA and two-chain u-PA, whereas 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) decreased scu-PA secretion, indicating that it is enhanced by protein kinase C (PKC) as well as by cAMP in NY cells. On the other hand, in HT-1080 cells, PMA decreased the level of two-chain u-PA secretion into the conditioned medium. The binding assay of 125I-DFP-u-PA to NY cells revealed the presence of a single class of binding sites with a Kd of 2.23 nM and Bmax of 0.82 x 10(6) binding sites/cell. PMA however, altered neither the Kd nor the Bmax. Dibutyryl cAMP increased the Bmax 1.9 fold. Thus, NY cells secrete u-PA and express specific binding sites on the cell surface, which are modulated by cAMP and PKC.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of scu-PA secretion and u-PA receptor expression in osteoblast-like cells. 816 59

The importance of cell-associated plasminogen activation in tumor invasion and metastasis is becoming increasingly evident. To clarify the modulators of cell-associated plasminogen activation in malignant states, we have recently established an assay system utilizing endogenous plasminogen activators on the cell surface. In the present study using the assay system, we found that the conditioned medium from phorbol 12-myristate 13-acetate (PMA)-stimulated human lymphoid cell lines, HUT 78 and Raji, strongly enhanced plasminogen activator (PA) activity on the surface of human malignant tumor cell lines (WI-38 VAI3 2RA, A431, A549 and HT-1080). The enhancing effect was inhibited by the addition of actinomycin D. By gel filtration, the active substances in PMA-stimulated HUT 78- and Raji-conditioned media were eluted in similar fractions corresponding to molecular weights of 60 to 80 kDa. The active substance was heat-labile. The enhanced PA activities were completely inhibited by anti-urokinase-type plasminogen activator (uPA) IgG. Moreover, the active substance was found to increase in cell-bound uPA antigen. These findings suggest that a population of activated lymphocytes produces a plasminogen activator modulator that induces uPA on the surface of malignant tumor cells.
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PMID:Phorbol-ester-stimulated human lymphoid cell lines produce a plasminogen activator modulator inducing cell-bound urokinase-type plasminogen activator in malignant tumor cell lines. 856 14

The importance of cell-associated plasminogen activation in the extracellular matrix degradation processes is becoming increasingly evident. To elucidate the modulators of net plasminogen activation on the cell surface, we have recently established an assay system. Using this system, we examined the effects of several candidate modulators on cell surface plasminogen activator in the human fibrosarcoma cell line HT-1080 and the SV40-transformed human lung fibroblast cell line WI-38 VA 13 2RA. Although the majority of the candidates had no effect or a selective effect on either cell line, only retinoic acid markedly enhanced cell surface plasminogen activator activity in both HT-1080 and WI-38 VA13 2RA cells in a time-dependent manner. The effect of retinoic acid was neutralized by actinomycin D. The enhanced activity was inhibited by anti-uPA IgG and by pretreatment with phosphatidylinositol-specific phospholipase C. These findings suggest that retinoic acid increases the amount of receptor-bound uPA via de novo synthesis, and that it plays an important role in modulating cell-associated plasminogen activation.
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PMID:Retinoic acid enhances plasminogen activation on the cell surface. 857 37

The human tissue-type plasminogen activator gene (t-PA) is induced by the phorbol ester, phorbol 12-myristate 13-acetate (PMA), in HeLa cells. Previous studies in transfected HeLa cells identified two cis-acting regulatory elements within the t-PA gene promoter responsible for both constitutive and PMA-inducible expression. One element differs from the consensus cAMP response element (CRE) by a single nucleotide substitution (referred to in this report as t-PACRE) and another which bears similarity to the AP-2 recognition sequence. In HT-1080 fibrosarcoma cells, t-PA mRNA levels are expressed at higher constitutive levels and are suppressed by PMA. Nuclear run-on transcription experiments indicate that PMA-mediated suppression of t-PA in these cells is associated with a decrease in t-PA gene template activity. We designed experiments to determine whether nuclear t-PACRE or AP-2-like binding proteins were differentially expressed in HeLa and HT-1080 cells and, accordingly, if these could be correlated with the opposite effect of PMA on t-PA expression. Band shift analyses indicated that the migration profiles of HeLa and HT-1080 nuclear proteins interacting with the AP-2-like site were indistinguishable; however, those produced with the t-PACRE binding site were qualitatively and quantitatively distinct. The distribution of t-PACRE binding proteins in these cells was investigated in a supershift assay using specific antibodies against members of the fos/jun and CRE-binding protein (CREB)/activating transcription factor (ATF) families. In HT-1080 cells, CREB-1 was the most prominent t-PACRE-binding activity detected and was greatly increased in cells treated with PMA. In contrast, CREB-1 activity was absent in HeLa cells, but antibodies specific for ATF-2 produced a marked supershifted complex which was unaffected by PMA treatment. Since CREB-1 can repress transcription of other target genes (including c-jun) via association with identical cis-acting CRE-like sequences, we suggest that the mechanism for the transcriptional down-regulation of t-PA by PMA in HT-1080 cells requires CREB-1 binding to the t-PACRE while ATF-2, by associating with the same site, plays a role in PMA-mediated induction of t-PA in HeLa cells.
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PMID:Differential binding of cAMP-responsive-element (CRE)-binding protein-1 and activating transcription factor-2 to a CRE-like element in the human tissue-type plasminogen activator (t-PA) gene promoter correlates with opposite regulation of t-PA by phorbol ester in HT-1080 and HeLa cells. 864 95

Vitamin D and its derivatives (deltanoids) are potent regulators of cell proliferation and differentiation. Targeted production of proteolytic enzymes like serine proteases and metalloproteinases is an important part of the invasive process of cancer cells. Treatment with 1 alpha25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] decreases the invasive properties of breast carcinoma cells. Here we have analyzed the effects of 1alpha,25(OH)2D3 and its synthetic analogues on the secretion and cell surface association of the components of the plasminogen activator (PA) system and on the secretion of certain matrix metalloproteinases (MMPs) and their inhibitors in MDA-MB-231 breast carcinoma cells. Deltanoids were able to decrease the secretion of urokinase PA and tissue-type PA activity in a dose-dependent manner and to increase PA inhibitor 1 secretion, leading to reduced total PA activity. CB1093 was the most potent analogue, effective at concentrations several logarithms lower than 1alpha,25(OH)2D3. Transient transfection of different urokinase PA promoter reporter constructs to HT-1080 fibrosarcoma indicator cells indicated that vitamin D-responsive sequences were located between nucleotides -2350 and -1870 in the 5' region of the promoter. Treatment of MDA-MB-231 cells with 1alpha,25(OH)2D3 or other deltanoids also resulted in decreased MMP-9 levels in association with increased tissue inhibitor of MMP 1 activity. Membrane-type 1-MMP expression or proteolytic processing were not appreciably affected by deltanoids. Vitamin D and its analogues caused a decrease in Matrigel invasion assays of MDA-MB-231 cells. Cancer cell invasion is associated with coordinated secretion of proteolytic enzymes and their inhibitors. Vitamin D and its derivatives can evidently influence invasive processes by two means: (a) decreasing the expression and activity of cell invasion-associated serine proteases and metalloproteinases; and (b) inducing their inhibitors.
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PMID:1alpha,25-dihydroxyvitamin D3 and its analogues down-regulate cell invasion-associated proteases in cultured malignant cells. 1077 39

The tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 2 (PAI-2) genes are differentially regulated by 12-phorbol 13-myristate acetate (PMA) in HT-1080 fibrosarcoma cells. PMA transcriptionally down-regulates the t-PA gene in HT-1080 cells, while the PAI-2 gene is simultaneously induced by this agonist. The t-PA and PAI-2 gene promoters harbour a cAMP-response element (CRE) which influences the expression of both genes. We have compared the binding activity of nuclear factors that recognise these CRE sites. We show that CREB (CRE binding protein) recognises each CRE and that the degree of constitutive Ser119-phosphorylated t-PA CRE-bound CREB was greater than for PAI-2 CRE bound CREB. Stable transfection of HT-1080 cells with a plasmid containing a CREB that could not be phosphorylated on Ser119 (pCI-CREB(ala119)) did not influence PMA-mediated suppression of t-PA mRNA, but markedly impaired PMA-mediated induction of PAI-2 mRNA. Our results demonstrate that the Ser119 residue of CREB plays a crucial role in PMA-mediated induction of PAI-2 gene expression, whereas PMA-mediated suppression of t-PA in HT-1080 cells requires a different process.
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PMID:Overexpression of a dominant negative CREB protein in HT-1080 cells selectively disrupts plasminogen activator inhibitor type 2 but not tissue-type plasminogen activator gene expression. 1101 26

Previous studies have shown that the adhesion protein, vitronectin, directs the localization of urokinase-type plasminogen activator (uPA) to areas of cell-substrate adhesion, where uPA is thought to regulate cell migration as well as pericellular proteolysis. In the present study, HT-1080 cell lines expressing either wild-type vitronectin or vitronectin containing a single amino-acid substitution in the integrin binding domain were used to assess whether ligation of the alphavbeta5 integrin was required for uPA localization to focal adhesions. The synthesis of wild-type vitronectin by HT-1080 cells adherent to either collagen or fibronectin resulted in the redistribution of both the alphavbeta5 integrin as well as uPA to focal adhesion structures. In contrast, cells synthesizing mutant vitronectin, containing the amino-acid substitution in the integrin binding domain, were unable to direct the redistribution of either alphavbeta5 or uPA to focal adhesions. Recombinant forms of wild-type and mutant vitronectin were prepared in a baculovirus system and compared for their ability to direct the redistribution of vitronectin integrin receptors as well as uPA on human skin fibroblasts. In the absence of vitronectin, fibroblast cells adherent to fibronectin assemble focal adhesions which contain the beta1 integrin but do not contain uPA. Addition of recombinant wild-type, but not mutant, vitronectin to fibroblasts adherent to fibronectin resulted in the redistribution of alphavbeta3, alphavbeta5, and uPA into focal adhesions. However, when cells were plated directly onto antibodies directed against either the alphavbeta3 or alphavbeta5 integrins, uPA was not localized on the cell surface. These data indicate that ligation of vitronectin integrin receptors is necessary but not sufficient for the localization of uPA to areas of cell matrix adhesion, and suggest that vitronectin may promote cell migration by recruiting vitronectin integrin receptors and components of the plasminogen activator system to areas of cell matrix contact.
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PMID:Localization of urokinase type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors. 1105 58

The human tissue-type plasminogen activator (t-PA) gene is regulated in a cell-type dependent manner. The t-PA gene is transcriptionally induced by the phorbol ester PMA in HeLa cells, but suppressed by PMA in HT-1080 cells. A cAMP responsive element (tPACRE) and a Sp-1 site located within the proximal t-PA gene promoter are functionally important in both cell systems. HeLa and HT-1080 cells contain a different repertoire of factors that associate with the tPACRE. In HT-1080 cells, CREB and c-Jun are the two major t-PACRE binding proteins identified, while activating transcription factor 2 (ATF-2) is a predominant t-PACRE binding protein in HeLa cells. To determine whether alteration in the distribution of tPACRE binding proteins would influence the differential regulation of the t-PA gene in these cells, the tPACRE binding profiles in these two cell systems were manipulated by over expressing ATF-2 in HT-1080 cells and CREB in HeLa cells. Supershift experiments confirmed that the overexpression of these factors resulted in binding to the tPACRE site. However, the presence of ATF-2 in HT-1080 cells did not affect either constitutive or PMA-mediated suppression of the endogenous t-PA gene. In contrast, enforced tPACRE-binding activity of CREB in HeLa cells significantly reduced the magnitude of PMA-mediated induction of t-PA mRNA in HeLa cells. These results indicate that the introduction of CREB into HeLa cells disrupts the regulation of the t-PA gene.
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PMID:Ectopic expression of the cAMP-responsive element binding protein inhibits phorbol ester-mediated induction of tissue-type plasminogen activator gene expression. 1117 65

Objective. Endothelial tissue-type plasminogen activator (t-PA) release is a pivotal response to protect the circulation from occluding thrombosis. We have shown that the t-PA gene is epigenetically regulated and greatly induced by the histone deacetylase (HDAC) inhibitor valproic acid (VPA). We now investigated involvement of known t-PA promoter regulatory elements and evaluated dependence of potential interacting transcription factors/cofactors. Methods. A reporter vector with an insert, separately mutated at either the t-PA promoter CRE or GC box II or GC box III elements, was transfected into HT-1080 and HUVECs and challenged with VPA. HUVECs were targeted with siRNA against histone acetyl transferases (HAT) and selected transcription factors from the Sp/KLF family. Results. An intact VPA-response was observed with CRE mutated constructs, whereas mutation of GC boxes II and III reduced the magnitude of the induction by 54 and 79% in HT-1080 and 49 and 50% in HUVECs, respectively. An attenuated induction of t-PA mRNA was observed after Sp2, Sp4, and KLF5 depletion. KLF2 and p300 (HAT) were identified as positive regulators of basal t-PA expression and Sp4 and KLF9 as repressors. Conclusion. VPA-induced t-PA expression is dependent on the proximal GC boxes in the t-PA promoter and may involve interactions with Sp2, Sp4, and KLF5.
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PMID:Dependence of Proximal GC Boxes and Binding Transcription Factors in the Regulation of Basal and Valproic Acid-Induced Expression of t-PA. 2696 81


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