Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bovine vascular smooth muscle cells (SMC) express the urokinase-type plasminogen activator receptor (u-PAR) claimed to be important in cell invasion. Receptor numbers and affinity are regulated by thrombin and several other mitogens involved in SMC proliferation. We investigated the effects of these mitogens on u-PAR mRNA levels. On continuous thrombin stimulation the u-PAR message in SMC was 10 +/- 2.3-fold elevated reaching a maximum between 6 and 9 hours and declining to control values within 48 hours. Thrombin present for 30 minutes on the cell surface produced similar effects. Stimulation with the thrombin receptor activation peptide S-F-L-L-R-N representing the NH2-terminus of the tethered ligand also increased u-PAR mRNA levels with an identical time course. D-Phe-Pro-Arg-chloromethyl ketone (PPACK) active site blocked thrombin and the catalytically inactive thrombin mutant S205A did not affect u-PAR mRNA levels. Thrombin stimulation also resulted in a 2 +/- 0.2-fold transient increase in thrombin receptor mRNA preceding the rise in u-PAR message. Transforming growth factor beta 1 (TGF beta 1) and platelet-derived growth factor (PDGF) showed similar time courses for the elevation of u-PAR mRNA levels with a maximal 5.5 +/- 0.9 and 12 +/- 2.5-fold increase, respectively. Basic fibroblast growth factor (bFGF) and phorbol myristate acetate (PMA) showed a more prolonged effect increasing u-PAR mRNA levels 8 +/- 2.0-fold and 12.3 +/- 2.5-fold, respectively, within 6 hours but remaining 5 to 10-fold elevated at 48 hours. In order to decide if the u-PAR mRNA increase was due to message stabilization or a consequence of transcriptional activation we used the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB) during the stimulation experiments. u-PAR mRNA levels on TGF beta 1 stimulation of SMC decayed after the addition of DRB indicating that enhancement of transcriptional activity was involved in the induction. In contrast, the time course of u-PAR mRNA elevation on thrombin, bFGF, and PMA stimulation was not significantly altered in the presence of DRB suggesting that in these latter cases u-PAR mRNA message accumulation was at least in part due to mRNA stabilization. Increased transcriptional activity, mRNA stabilization and expression of u-PAR protein on the SMC surface in response to growth factors may facilitate enhanced cell surface protease activity, cell migration, and development of atheromatous lesions.
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PMID:Effect of thrombin, the thrombin receptor activation peptide, and other mitogens on vascular smooth muscle cell urokinase receptor mRNA levels. 794 25

The Escherichia coli transcription factor NusA and the bacteriophage lambda antiterminator Q proteins were expressed as inducible glutathione S-transferase (GST) fusion proteins. The fusion proteins were purified under nondenaturing conditions by affinity chromatography on glutathione agarose. Thrombin cleavage of the glutathione agarose-bound fusion proteins yielded homogeneously pure NusAN+15 (5 mg/g cells) and almost homogeneously pure QN+13 protein (0.7 mg/g cells), where N+x indicates the presence of x additional amino acids at the N-terminus of the protein. The purified NusAN+15 exhibited the same activities as wildtype NusA in enhancement of transcriptional pausing, enhancement of termination at Rho-independent terminators, and enhancement of Q-mediated antitermination in vitro. The QN+13 protein exhibited both anti-pausing and antitermination activities in Q-mediated transcription antitermination. However, the antitermination activity of QN+13 was lost gradually during storage if the thrombin used for cleavage of the GST fusion protein was not removed. This was due to cleavage by thrombin after Arg22 within the Q protein itself, at a noncanonical thrombin cleavage site, so the truncated protein (QN+22) lacked the first 22 amino acids at the N-terminus of Q. The expression vectors described here can be used to rapidly produce large quantities of these proteins, and the truncated Q protein can be used to evaluate the requirement for the N-terminus of Q in antitermination, anti-pausing, interactions with the DNA template (qut site), and interaction with RNA polymerase itself.
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PMID:Expression and functional characterization of Escherichia coli NusA and lambda Q as glutathione S-transferase fusion proteins. 853 55

The traditional view on the role of serine proteases in tumor biology has changed with the recent discovery of a family of protease-activated receptors (PARs). In this study we explored the expression and functional role of the thrombin receptor PAR-1 in human colon cancer cells. Reverse transcriptase-polymerase chain reaction analysis showed that PAR-1 mRNAs are present in 11 of 14 human colon cancer cell lines tested but not in normal human colonic epithelial cells. This is in line with the immunolocalization of PAR-1 in human colon tumors and its absence in normal human colonic mucosa. The functional significance of the aberrant expression of PAR-1 in colon cancer cells was then investigated. We found that 1) a prompt increase in intracellular calcium concentration was observed on thrombin (10 nmol/L) or PAR-1 agonist AP1 (100 micro mol/L) challenge of HT29 cells; 2) HT29 quiescent cells treated with thrombin (0.01 to 20 nmol/L) or AP1 (1 to 300 micro mol/L) exhibited dramatic mitogenic responses (3.5-fold increase in cell number). Proliferative effects of thrombin or AP1 were also observed in other colon cancer cell lines expressing PAR-1. This effect was reversed by the MEK inhibitor PD98059 in consonance with the ability of thrombin or AP1 to induce phosphorylation of p42/p44 extracellular-regulated protein kinases. 3) PAR-1 activation by thrombin or AP1 led to a two-fold increase in cell motility of wounded HT29-D4. Our results demonstrate for the first time the aberrant expression of the functional thrombin receptor PAR-1 in colon cancers and its important involvement in cell proliferation and motility. Thrombin should now be considered as a growth factor for human colon cancer.
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PMID:Aberrant expression and activation of the thrombin receptor protease-activated receptor-1 induces cell proliferation and motility in human colon cancer cells. 1270 33

Thrombin is a serine protease involved in many physiological functions and its receptor. the protease-activated receptor-1 (PAR-1), has a wide tissue distribution. We hypothesized that PAR-1 is expressed in gastric epithelial cells and that thrombin can modulate defence mechanisms through PAR-1. The rat gastric epithelial cell line (RMG1) and gastric biopsy specimens from gastritis patients were used in the study. Reverse transcriptase polymerase chain reaction analysis showed that the thrombin receptors PAR-1, PAR3 and PAR-4 are expressed by RGM1 gastric epithelial cell line. Immunohistochemical and electron microspcopic studies also showed PAR-1 expression in human gastric epithelial cells. Thrombin stimulated the secretion of mucin and prostaglandin E2 (PGE2) formation in RGM1 cells in a dose-dependent manner. PAR-1 agonist also stimulated PGE2 formation. In addition, thrombin significantly increases the expression of the PGE2 receptors EP2-R and EP4-R in RGM1 cells. In conclusion, the results of the present study showed for the first time that gastric epithelial cells express thrombin receptors and that these receptors may play a protective role in the gastric mucosa.
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PMID:Expression and cytoprotective effect of protease-activated receptor-1 in gastric epithelial cells. 1273 39

Thrombin, its main inhibitor (protease nexin-1) and its related receptors (protease-activated receptors, PAR-1,-2, -3, -4) were studied in rat hippocampus following administration of trimethyltin (TMT), a neurotoxin inducing neuronal degeneration and reactive gliosis. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry revealed that while expression of prothrombin and protease nexin-1 did not change significantly in TMT-treated hippocampi, PARs (in particular PAR-1 and to a lesser extent PAR-2 and PAR-3) were upregulated in reactive astrocytes, suggesting their involvement in neurodegeneration and in the consequent response of the nervous tissue.
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PMID:Trimethyltin-induced differential expression of PAR subtypes in reactive astrocytes of the rat hippocampus. 1499 20