Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The plasminogen activator urokinase promotes tumor invasion by converting plasminogen into plasmin, which degrades several extracellular matrix components. Urokinase can bind to a specific cell surface receptor, which leads to accelerated plasmin production. While there is good evidence indicating a role for this binding site in tumor invasion/metastasis, there is little information concerning the regulation of urokinase receptor expression in invasive cancer. To address this question a series of colon cancer cell lines, which demonstrate either a high or low ability to invade an extracellular matrix-coated porous filter, was characterized for receptor expression at the transcriptional and post-transcriptional levels. The invasive cell lines possessed 10-fold more receptors than their non-invasive counterparts as shown by cross-linking experiments and by Western blotting. Northern blotting indicated that this disparity in receptor number could be largely accounted for by a different amount of steady-state mRNA encoding the binding site. However, neither gene amplification nor enhanced mRNA stability could account for the augmented receptor protein observed for the invasive colon cancer cell types. In contrast, nuclear run-on experiments with representative cell lines revealed that the 10-fold difference in receptor display between the invasive-competent and invasive-deficient cells could be largely accounted for by differences in transcription rates. Transcription of the u-PAR gene in the receptor-deficient GEO cells, but not in the receptor-rich RKO cells, could be augmented by protein kinase C stimulation. These findings provide a clear rationale for studies to determine if the urokinase receptor promoter in invasive colon cancer is activated in cis or in trans.
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PMID:Transcriptional activation of the urokinase receptor gene in invasive colon cancer. 807 48

In LLC-PK1 cells, the urokinase-type plasminogen activator (uPA) gene is induced by two of the major signal transduction pathways, the protein kinase C (PKC) and the cAMP-dependent protein kinase (PKA) pathways. We have analyzed the chromatin structure of 26 kb of the uPA gene locus and have shown that PKA activation but not PKC activation induce major chromatin structural alterations in the uPA gene promoter. In uninduced cells, several DNase I hypersensitive (HS) sites were detected in the 5' and 3' flanking regions but not in the transcribed region. Two of the sites correspond to previously characterized regulatory sites: a cAMP responsive site at nucleotide position -3500 with respect to the initiation site, and the PEA3/AP1 site at -2100 that mediates PKC activation. After the activation of PKA but not PKC, a strong HS site was induced at -2600. Functional analysis of this region revealed cAMP responsive activity. Chromatin structural alterations again brought about specifically by PKA but not by PKC were were also detected in the upstream of the promoter by topoisomerase I cleavage site analysis, with two prominent sites appearing at -2800 and -3300. These results suggest that the strong cAMP induction of the uPA gene requires structural alterations that permit cooperative interactions between the multiple cAMP responsive sites.
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PMID:Activation of cAMP-dependent protein kinase alters the chromatin structure of the urokinase-type plasminogen activator gene promoter. 812 5

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

We have previously shown that the tumor promoter okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, transcriptionally induces the urokinase-type plasminogen activator (uPA) gene in LLC-PK1 cells. This induction occurs independently of the protein kinase C- and cAMP-dependent signaling pathways. Here we show that a sequence located 2.0 kilobases upstream of the uPA gene, which resembles an AP-1-recognition sequence, mediates the action of OA. DNA-protein interaction studies, together with mRNA and protein analyses, indicate that c-Jun, but not c-Fos, is involved in OA-dependent uPA gene induction. The appearance of high levels of uPA mRNA and DNA binding activity of c-Jun to the AP-1-like site correspond to the appearance of c-Jun accumulation, suggesting that c-Jun accumulation is a critical event in OA-dependent uPA gene induction. c-Jun protein levels increase significantly between 100 and 160 min following OA treatment, whereas c-Jun translation increases only slightly in this time frame, suggesting that post-translation mechanisms are also involved in c-Jun accumulation. Pulse-chase analyses shows that OA specifically stabilizes c-Jun. We discuss our results with respect to the possibility that protein phosphatase 2A maintains c-Jun in its down-regulated state in LLC-PK1 cells.
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PMID:Okadaic acid-dependent induction of the urokinase-type plasminogen activator gene associated with stabilization and autoregulation of c-Jun. 830 Jun 23

Fibroblast growth factors (FGFs) play a role in biological processes such as cell growth and development, angiogenesis, and wound healing. Several genes have been shown to be induced by FGFs, but the underlying mechanisms have not been elucidated. We investigated the effect of FGF-2 (basic FGF) on the urokinase-type plasminogen activator (uPA) gene in NIH 3T3 fibroblasts. We found that the uPA gene is transcriptionally induced by FGF-2 as well as by 12-O-tetradecanoylphorbol-13 -acetate involving a PEA3/AP1 element located 2.4 kb upstream of the transcription initiation site; neither induction requires ongoing protein synthesis. Unlike 12-O-tetradecanoylphorbol-13-acetate induction, FGF-2 induction was not impaired by protein kinase C down-regulation. Analyses of various signaling molecules by Western blotting, extracellular signal-regulated kinase (ERK) activity assays, and transient transfection assays (cotransfection of a uPA-reporter gene construct with expression vectors for wild-type or dominant negative type of these molecules or for ERK-specific protein phosphatase MKP-1) showed that a Ras/Raf-1/MEK/ERK-2/JunD pathway is induced by FGF-2 and 12-O-tetradecanoylphorbol-13-acetate, leading to the activation of the uPA gene.
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PMID:Elucidation of a signaling pathway induced by FGF-2 leading to uPA gene expression in NIH 3T3 fibroblasts. 854 15

Previous studies have shown that high glucose levels and diabetes induce an elevation in protein kinase C (PKC) activity in vascular cells and tissues susceptible to diabetic complications. In addition, PKC activation has been shown to modulate vascular cell growth, permeability, and gene expression, processes thought to be involved in the development of vascular complications. Using two in vivo model systems, we have identified a novel inhibitor of diabetic vascular dysfunction, LY290181. LY290181 prevented glucose-induced increases in blood flow and permeability in rat granulation tissue and corresponding vascular changes in the retina, sciatic nerve, and aorta of diabetic rats. Tested for its ability to inhibit PKC-regulated processes, LY290181 inhibited phorbol ester-stimulated plasminogen activator activity in a dose-dependent manner in bovine retinal endothelial cells and in human dermal fibroblasts. In addition, LY290181 inhibited phorbol ester-stimulated activation of the porcine urokinase plasminogen activator (uPA) promoter (-4600/+398) linked to the chloramphenicol acetyltransferase (CAT) reporter gene (p4660CAT). More detailed analysis of the uPA promoter revealed that LY290181 inhibited phorbol ester-stimulated activation of the uPA phorbol response element (-2458/-2349) located upstream of the thymidine kinase promoter (puPATKCAT). LY290181 appears to inhibit uPA promoter activation by blocking phorbol ester-stimulated binding of nuclear proteins to the uPA PEA3/12-0-tetradecanoylphorbol 13-acetate responsive element (TRE). These results suggest that LY290181 may inhibit diabetes-induced vascular dysfunction by inhibiting transcription factor binding to specific PKC-regulated genes involved in vascular function.
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PMID:LY290181, an inhibitor of diabetes-induced vascular dysfunction, blocks protein kinase C-stimulated transcriptional activation through inhibition of transcription factor binding to a phorbol response element. 862 Oct 17

Both in cell culture and in vivo, keratinocytes that are migrating in response to a wound express enhanced levels of both urokinase-type plasminogen activator (uPA) and the uPA cell surface receptor (uPA-R). To explore the mechanism of this up-regulation, keratinocyte cultures were treated proir to wounding with a variety of metabolic and growth factor inhibitors in order to evaluate their effect on uPA and uPA-R expression. Actinomycin D and cycloheximide inhibited the up-regulation of both uPA and uPA-R, as determined by immunohistochemistry, indicating that RNA and protein syntheses are required for their induction in migrating keratinocytes. Neither removal of protein growth factors from the medium nor addition of inhibitory antibodies to a number of growth factors depressed uPA or uPA-R induction; these findings suggest that a variety of exogenous or endogenous growth factors [i.e., basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), amphiregulin, and tumor necrosis factor-alpha (TNF-alpha) do not have a critical role in the induction of uPA or uPA-R. In contrast, when protein kinase C (PKC) was either down-regulated with bryostatin 5 or inhibited with Ro31-8220 or staurosporine, the expression of both uPA and uPA-R was greatly decreased in migrating keratinocytes. Furthermore, pharmacologic activation of PKC enhanced uPA levels in non-wounded cultures. These data suggest that the enhanced expression of uPA and uPA-R in migrating keratinocytes is mediated by selective activation of PKC in these cells, perhaps secondary to alterations in the cytoskeleton induced by wounding. To test the requirement for uPA during keratinocyte migration in vitro, the extent of migration was quantified in the presence and absence of a variety of inhibitors in the wounded culture model. Migration was not altered by actinomycin D, cycloheximide, any of the above growth factor inhibitors, anti-uPA antibodies, a variety of inhibitors of uPA or plasmin enzymatic activity, or exogenous uPA. The independence of keratinocyte migration in vitro from uPA was further suggested by experiments which combined the phagokinetic assay of migration and the zymographic assay for pericellular uPA activity; no relationship was observed between pericellular uPA activity and the motility of individual cells.
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PMID:Protein kinase C mediates up-regulation of urokinase and its receptor in the migrating keratinocytes of wounded cultures, but urokinase is not required for movement across a substratum in vitro. 865 4

To investigate the regulation of promoters containing classical phorbol ester response sequences (PEA-3/12-O-tetradecanoylphorbol-13-acetate response element motifs) by protein kinase C (PKC) isozymes, co-transfections were performed in human dermal fibroblasts with a plasmid containing either the human collagenase promoter or the porcine urokinase plasminogen activator (uPA) promoter linked to the chloramphenicol acetyltransferase gene and a plasmid expressing an individual PKC isozyme. Using this experimental design, seven PKC isozymes were analyzed for their ability to trans-activate the collagenase and uPA promoters. Our results demonstrate that only PKC delta, epsilon, and eta trans-activated the collagenase promoter and that binding of Ap-1 family members to the collagenase 12-O-tetradecanoylphorbol-13-acetate response element (TRE) was not responsible for the isozyme-specific trans-activation. In contrast, the uPA promoter was stimulated by all of the PKC isozymes examined (PKC alpha, betaII, gamma, delta, epsilon, zeta, and eta). These results indicate that PKC isozymes differentially regulate promoters containing PEA-3/TRE motifs and suggest that individual isozymes play unique roles within the cell.
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PMID:Protein kinase C isozymes differentially regulate promoters containing PEA-3/12-O-tetradecanoylphorbol-13-acetate response element motifs. 870 56

In order to reach the sites of inflammation, lymphocytes leave the bloodstream and migrate into peripheral tissues, in a process involving integrin-mediated adhesion to the vascular endothelium, followed by transmigration across the endothelial barrier and through the underlying interstitial matrix. We have investigated the role of the plasminogen activator/plasmin system in normal T cell migration. Receptors for urokinase plasminogen activator (uPAR) were not expressed in resting T lymphocytes, but could be efficiently induced at the mRNA and protein level by coclustering of the antigen receptor complex and beta1 or beta2 integrins, through a signalling pathway involving both protein kinase C activation and an increase in intracellular cyclic AMP. Catalytic activation of plasminogen by uPAR-expressing T cells promoted their migration through an extracellular matrix in vitro. Plasmin-induced invasion was inhibited by plasmin-and urokinase inhibitors and by anti-uPAR antibodies. Finally, cytofluorimetric and immunohistochemical analysis of primary human tumor specimens showed the presence of uPAR positive infiltrating T cells in vivo. Collectively, these findings suggest that plasminogen activation may play a role in lymphocyte migration in vivo, and that integrin-dependent expression of membrane-associated endopeptidases could represent an additional step in the regulated process of leukocyte transmigration.
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PMID:Integrin-dependent induction of functional urokinase receptors in primary T lymphocytes. 878 76

Plasminogen activation by urokinase-type plasminogen activator (uPA) is implicated in tumor invasion and metastasis by the breakdown of extracellular matrix. We have recently demonstrated the inhibitory effect of cAMP on uPA gene transcription in RC-K8 human lymphoma cells (Biochim Biophys Acta 1268: 293-9, 1995). Prostacyclin produced by endothelial cells is shown to increase cellular cAMP levels by activating adenylate cyclase. We, therefore, examined the effect of a stable analogue of prostacyclin, Beraprost, on uPA production in RC-K8 cells. uPA activity gradually increased in the conditioned medium with time. Beraprost (0.1 nM-1.0 microM) inhibited uPA accumulation in a dose-dependent manner without affecting cell viability. Fibrinzymography demonstrated that high and low molecular forms of uPA were present in the conditioned medium and that after Beraprost-treatment all forms of uPA decreased and no PA/PA inhibitor complex was present. Northern blot analysis revealed that after exposure to Beraprost, uPA mRNA levels increased transiently and then rapidly decreased to below control levels. Treatment with Beraprost resulted in a rapid activation of cellular cyclic AMP-dependent protein kinase (PKA). Beraprost completely negated uPA gene expression induced by phorbol myristate acetate, an activator of protein kinase C (PKC). These results suggest that Beraprost inhibits uPA production by suppressing uPA gene expression through the PKA pathway and that PKA-mediated signals are dominant in uPA gene expression as compared to those medicated by PKC. This inhibition of uPA expression by a prostacyclin analogue may be an important fact to explain the mechanism of anti-metastatic effects of prostacyclin.
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PMID:Inhibitory effect of a synthetic prostacyclin analogue, beraprost, on urokinase-type plasminogen activator expression in RC-K8 human lymphoma cells. 882 88


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