scholarly journals Transcriptional regulation of the rat tissue type plasminogen activator gene: localization of DNA elements and nuclear factors mediating constitutive and cyclic AMP-induced expression.

1993 ◽  
Vol 13 (1) ◽  
pp. 266-275 ◽  
Author(s):  
M Ohlsson ◽  
G Leonardsson ◽  
X C Jia ◽  
P Feng ◽  
T Ny
Keyword(s):  
Cyclic Amp ◽  
Plasminogen Activator ◽  
Granulosa Cells ◽  
Constitutive Expression ◽  
Cell Types ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Nuclear Extracts ◽  
Nuclear Factors ◽  
Type Plasminogen Activator

We have characterized tissue type plasminogen activator (tPA) promoter elements and nuclear factors required for follicle-stimulating hormone (FSH)-induced transcription of the rat tPA gene in granulosa cells and constitutive expression of the gene in the rat neuroblastoma cell line B103. Run-on transcription analysis of isolated nuclei revealed that B103 cells transcribe the tPA gene at a high and constitutive level, while FSH was found to induce tPA gene transcription in a rapid and transient manner in granulosa cells. The maximal FSH-induced transcription rate was obtained after 20 min and was similar in the absence or presence of the protein synthesis inhibitor cycloheximide. However, in the presence of cycloheximide, tPA transcription was not turned off but continued at a high rate for several hours. This phenomenon may at least partly explain the earlier finding that tPA mRNA is superinduced by FSH in the presence of cycloheximide. DNase I footprinting analysis of the first 621 bp of the tPA promoter revealed a total of six regions that interact with nuclear factors from B103 and granulosa cells. Deletion of the promoter region from positions -269 to -621, a region that includes the two most-upstream footprints, had no effect on constitutive or FSH-induced transcription in transient expression experiments. Nuclear extracts from both granulosa cells and B103 cells showed strong binding to a consensus cyclic AMP-responsive element (CRE) at positions -178 to -185 and a neighboring binding site for nuclear factor 1 (NF1) at positions -145 to -158. The factors binding to these two regions were identified as members of the CRE-binding protein and NF1 families of transcription factors, respectively. Footprints were also obtained over two GC boxes at positions -64 to -71 and -41 to -49. These footprints were more pronounced with nuclear extracts from B103 cells than with extracts from untreated or FSH-treated granulosa cells, but gel shift assays indicate that similar amounts of two distinct factors bind to the two GC boxes in both cell types. Transfection experiments using promoter constructs with inactivated promoter elements indicate that both the CRE and NF1 sites contribute to the FSH responsiveness of the rat tPA gene in granulosa cells, while only the NF1 site is important for constitutive expression in B103 cells. The two GC boxes were found to be necessary both for constitutive expression in B103 cells and for FSH-induced expression in granulosa cells, and inactivation of both GC boxes essentially eliminated the tPA promoter activity in both cell types.

Transcriptional regulation of the rat tissue type plasminogen activator gene: localization of DNA elements and nuclear factors mediating constitutive and cyclic AMP-induced expression

1993 ◽  
Vol 13 (1) ◽  
pp. 266-275
Author(s):  
M Ohlsson ◽  
G Leonardsson ◽  
X C Jia ◽  
P Feng ◽  
T Ny
Keyword(s):  
Cyclic Amp ◽  
Plasminogen Activator ◽  
Granulosa Cells ◽  
Constitutive Expression ◽  
Cell Types ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Nuclear Extracts ◽  
Nuclear Factors ◽  
Type Plasminogen Activator

We have characterized tissue type plasminogen activator (tPA) promoter elements and nuclear factors required for follicle-stimulating hormone (FSH)-induced transcription of the rat tPA gene in granulosa cells and constitutive expression of the gene in the rat neuroblastoma cell line B103. Run-on transcription analysis of isolated nuclei revealed that B103 cells transcribe the tPA gene at a high and constitutive level, while FSH was found to induce tPA gene transcription in a rapid and transient manner in granulosa cells. The maximal FSH-induced transcription rate was obtained after 20 min and was similar in the absence or presence of the protein synthesis inhibitor cycloheximide. However, in the presence of cycloheximide, tPA transcription was not turned off but continued at a high rate for several hours. This phenomenon may at least partly explain the earlier finding that tPA mRNA is superinduced by FSH in the presence of cycloheximide. DNase I footprinting analysis of the first 621 bp of the tPA promoter revealed a total of six regions that interact with nuclear factors from B103 and granulosa cells. Deletion of the promoter region from positions -269 to -621, a region that includes the two most-upstream footprints, had no effect on constitutive or FSH-induced transcription in transient expression experiments. Nuclear extracts from both granulosa cells and B103 cells showed strong binding to a consensus cyclic AMP-responsive element (CRE) at positions -178 to -185 and a neighboring binding site for nuclear factor 1 (NF1) at positions -145 to -158. The factors binding to these two regions were identified as members of the CRE-binding protein and NF1 families of transcription factors, respectively. Footprints were also obtained over two GC boxes at positions -64 to -71 and -41 to -49. These footprints were more pronounced with nuclear extracts from B103 cells than with extracts from untreated or FSH-treated granulosa cells, but gel shift assays indicate that similar amounts of two distinct factors bind to the two GC boxes in both cell types. Transfection experiments using promoter constructs with inactivated promoter elements indicate that both the CRE and NF1 sites contribute to the FSH responsiveness of the rat tPA gene in granulosa cells, while only the NF1 site is important for constitutive expression in B103 cells. The two GC boxes were found to be necessary both for constitutive expression in B103 cells and for FSH-induced expression in granulosa cells, and inactivation of both GC boxes essentially eliminated the tPA promoter activity in both cell types.

Elevated tissue type plasminogen activator in human granulosa cells correlates with fertilizing capacity

1988 ◽  
Vol 3 (5) ◽  
pp. 597-599 ◽  
Author(s):  
Josef Deutinger ◽  
Johannes C. Kirchheimer ◽  
Alexander Reinthaller ◽  
Günter Christ ◽  
Gerhart Tatra ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Granulosa Cells ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Human Granulosa Cells ◽  
Type Plasminogen Activator

scholarly journals Human tissue-type plasminogen activator

2014 ◽  
Vol 112 (08) ◽  
pp. 243-254 ◽  
Author(s):  
Egbert K. O. Kruithof ◽  
Sylvie Dunoyer-Geindre
Keyword(s):  
Plasminogen Activator ◽  
Gene Transcription ◽  
Current Knowledge ◽  
Plasminogen Activator Inhibitor ◽  
Cell Types ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Central Nervous System Activity ◽  
Its Gene

SummaryTissue-type plasminogen activator (t-PA ) plays an important role in the removal of intravascular fibrin deposits and has several physiological roles and pathological activities in the brain. Its production by many other cell types suggests that t-PA has additional functions outside the vascular and central nervous system. Activity of t-PA is regulated at the level of its gene transcription, its mRNA stability and translation, its storage and regulated release, its interaction with cofactors that enhance its activity, its inhibition by inhibitors such as plasminogen activator inhibitor type 1 or neuroserpin, and its removal by clearance receptors. Gene transcription of t-PA is modulated by a large number of hormones, growth factors, cytokines or drugs and t-PA gene responses may be tissue-specific. The aim of this review is to summarise current knowledge on t-PA function and regulation of its pericellular activity, with an emphasis on regulation of its gene expression.

scholarly journals Evidence for carbohydrate-independent endocytosis of tissue-type plasminogen activator by liver cells

1992 ◽  
Vol 285 (3) ◽  
pp. 799-804 ◽  
Author(s):  
E Stang ◽  
N Roos ◽  
M Schlüter ◽  
T Berg ◽  
J Krause
Keyword(s):  
Plasminogen Activator ◽  
Liver Cell ◽  
Hepatic Clearance ◽  
Mannose Receptor ◽  
Cell Types ◽  
Tissue Type ◽  
Side Chains ◽  
Tissue Type Plasminogen Activator ◽  
Hepatic Cells ◽  
Type Plasminogen Activator

In the liver, tissue-type plasminogen activator (t-PA) is endocytosed by hepatic parenchymal (PC), endothelial (EC) and Kupffer (KC) cells. Although the endocytosis is receptor-mediated, it remains a matter of discussion which receptors are involved in this catabolic process. To evaluate the role of a protein-specific receptor, as well as the possible involvement of the galactose receptor on PC and the mannose receptor on EC, we have employed different glycosylation variants of t-PA in biochemical and immunocytochemical studies. Partial or total removal of carbohydrate side-chains by endoglycosidases did not prevent clearance and hepatic endocytosis of t-PA by either of the liver cell types. Blockade of the galactose and mannose receptors by co-application of a large excess of the glycoprotein ovalbumin remained without effect on the binding and uptake of t-PA by hepatic cells. However, the contribution of different liver cell types to the hepatic clearance of t-PA was to a certain extent dependent on the type of oligosaccharide chains removed. The mannose receptor on EC is partially responsible for the clearance of t-PA by this cell type, whereas the galactose receptor does not seem to be involved in this process. The results obtained in this study further demonstrate that the major portion of the hepatic catabolism of t-PA is independent of its carbohydrate side-chains.

scholarly journals Butyrate stimulates tissue-type plasminogen-activator synthesis in cultured human endothelial cells

1987 ◽  
Vol 247 (3) ◽  
pp. 605-612 ◽  
Author(s):  
T Kooistra ◽  
J van den Berg ◽  
A Töns ◽  
G Platenburg ◽  
D C Rijken ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cyclic Amp ◽  
Plasminogen Activator ◽  
Tissue Type ◽  
Mrna Levels ◽  
Dibutyryl Cyclic Amp ◽  
Human Endothelial Cells ◽  
Tissue Type Plasminogen Activator ◽  
Cell Extracts ◽  
Type Plasminogen Activator

Incubation of cultured human endothelial cells with 5 mM-dibutyryl cyclic AMP led to an approx. 2-fold increase in tissue-type plasminogen-activator (t-PA) production over a 24 h incubation period. The stimulating effect of dibutyryl cyclic AMP could be explained by the slow liberation of butyrate, as the effect could be reproduced by addition of free butyrate to the medium, but not by addition of 8-bromo cyclic AMP or forskolin, agents known to raise intracellular cyclic AMP levels. With butyrate, an accelerated accumulation of t-PA antigen in the conditioned medium (CM) was observed after a lag period of about 6 h. Increasing amounts of butyrate caused an increasingly stimulatory effect, reaching a plateau at 5 mM-butyrate. The relative enhancement of t-PA production in the presence of 5 mM-butyrate varied among different endothelial cell cultures from 6- to 25-fold in 24 h CM. Such an increase in t-PA production was observed with both arterial and venous endothelial cells. The butyrate-induced increases in t-PA production were accompanied by increased t-PA mRNA levels. Analysis of radiolabelled CM and cell extracts by SDS/polyacrylamide-gel electrophoresis indicated that the potent action of butyrate is probably restricted to a small number of proteins. The accumulation of plasminogen activator inhibitor type 1 (PAI-1) in CM from butyrate-treated cells varied only moderately. In our study of the relationship between structure and stimulatory activity, we found that a straight-chain C4 monocarboxylate structure with a methyl group at one end and a carboxy moiety at the other seems to be required for the optimal induction of t-PA in cultured endothelial cells.

scholarly journals Tissue-Type Plasminogen Activator and Tenecteplase-Mediated Increase in Blood Brain Barrier Permeability Involves Cell Intrinsic Complement

2020 ◽  
Vol 11 ◽  
Author(s):  
Charithani B. Keragala ◽  
Trent M. Woodruff ◽  
Zikou Liu ◽  
Be'eri Niego ◽  
Heidi Ho ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Therapeutic Potential ◽  
Cell Types ◽  
Receptor Activation ◽  
Tissue Type ◽  
C5a Receptor ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Bbb Permeability

Background: Tissue-type plasminogen activator (t-PA) has been the mainstay of therapeutic thrombolysis for patients with acute ischaemic stroke (AIS). However, t-PA can cause devastating intracerebral hemorrhage. t-PA can also influence the CNS in part by modulation of BBB permeability. Complement activation also occurs after AIS and has also been reported to increase BBB permeability. The complement components, C3 and C5, can also be activated by t-PA via plasmin formation and cell intrinsic complement may be involved in this process. Tenecteplase (TNK-tPA) is a t-PA variant with a longer plasma half-life, yet the ability of TNK-tPA to modulate the BBB and complement is less clear.Aim: To evaluate the effect of C5 and C5a-receptor 1 (C5aR1) inhibitors on t-PA- and TNK-tPA-mediated opening of the BBB.Methods: We used an in vitro model of the BBB where human brain endothelial cells and human astrocytes were co-cultured on the opposite sides of a porous membrane assembled in transwell inserts. The luminal (endothelial) compartment was stimulated with t-PA or TNK-tPA together with plasminogen, in the presence of PMX205 (a non-competitive C5aR1 antagonist), Avacopan (a competitive C5aR1 antagonist) or Eculizumab (a humanized monoclonal inhibitor of human C5). BBB permeability was assessed 5 and 24 h later. Immunofluorescence was also used to detect changes in C5 and C5aR1 expression in endothelial cells and astrocytes.Results: PMX205, but not Avacopan or Eculizumab, blocked t-PA-mediated increase in BBB permeability at both the 5 and 24 h time points. PMX205 also blocked TNK-tPA-mediated increase in BBB permeability. Immunofluorescence analysis revealed intracellular staining of C5 in both cell types. C5aR1 expression was also detected on the cell surfaces and also located intracellularly in both cell types.Conclusion: t-PA and TNK-tPA-mediated increase in BBB permeability involves C5aR1 receptor activation from cell-derived C5a. Selective inhibitors of C5aR1 may have therapeutic potential in AIS.

A Monoclonal Antibody that Does Not Recognize Tissue-Type Plasminogen Activator Bound to Its Naturally Occurring Inhibitor

1986 ◽  
Vol 56 (03) ◽  
pp. 328-332 ◽  
Author(s):  
Raymond R Schleef ◽  
Nancy V Wagner ◽  
Manjula Sinha ◽  
David J Loskutoff
Keyword(s):  
Monoclonal Antibody ◽  
Plasminogen Activator ◽  
Solid Phase ◽  
Plasminogen Activator Inhibitor ◽  
Cell Types ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Naturally Occurring ◽  
Type Plasminogen Activator ◽  
Activator Inhibitor

SummaryHybridoma clones specific for tissue-type plasminogen activator (tPA) were screened in solid-phase radioimmunoassays for their reactivity toward free tPA and tPA complexed to the endothelial cell-derived, (3-migrating plasminogen activator inhibitor (β-PAI). Two monoclonal antibodies (MABs) were identified with quite distinct properties. The first, MAB LI72D1, bound to free tPA but did not recognize tPA complexed to the β-PAI, whereas the second, MAB HI72C1, bound both to free tPA and to tPA in complex with β-PAI. These properties were maintained when the MABs were immobilized to plastic micro- titer wells, thus permitting the development of immunoradiometric assays (IRMAs) to quantitate free tPA and total tPA antigen in various samples. The IRMAs were employed to analyze the tPA in media conditioned by several human cell types. The results indicate that in some cases, tPA may be present entirely as a free and active enzyme, while in others it apparently exists entirely in complex with β-PAI. Interestingly, some cells appear to contain both forms of tPA.

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