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)

Activation of either muscarinic cholinergic or thrombin receptors increases phosphoinositide turnover, Ca2+ mobilization, and redistribution of protein kinase C and induces rapid transient increases in c-fos mRNA and c-jun mRNA in 1321N1 cells. To determine whether the increases in c-fos and c-jun mRNA induced by carbachol and thrombin are sufficient to stimulate AP-1-mediated transactivation, 1321N1 cells were transfected with a reporter carrying two copies of the tetradecanoyl phorbol acetate response element and the firefly luciferase gene. Thrombin was significantly more effective than carbachol at stimulating AP-1-mediated transactivation. To identify the factors underlying the difference in AP-1 activity induced by carbachol and thrombin, members of the fos and jun families which encode components of AP-1 were examined. Carbachol and thrombin have similar effects on expression of c-fos, fosB, fra-2, junB, and junD, both acutely and over a 24-h time course. However, whereas carbachol leads only to transient induction of c-jun (maximal at 0.5 h), thrombin induces a biphasic increase in c-jun mRNA--an initial peak at 0.5 h and a second, more-prolonged increase at 12 h. Thrombin but not carbachol also induces a late increase in fra-1 mRNA, which peaks at 12 h. The secondary increase in c-jun mRNA is associated with marked increases in c-Jun protein levels and AP-1 DNA-binding activity. The late induction of c-jun and fra-1 mRNA can be prevented by adding the antagonist hirudin 30 min after thrombin, which results in loss of thrombin-stimulated increases in c-Jun protein, AP-1 DNA-binding activity, and AP-1-mediated transactivation. These findings suggest that rapid and transient conduction of c-fos and c-jun mRNA is insufficient to induce prominent changes in gene transcription, while the sustained increase in c-jun mRNA and perhaps the late induction of fra-1 mRNA are required for generation of AP-1 DNA-binding activity and transactivation through AP-1.
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PMID:Biphasic increase in c-jun mRNA is required for induction of AP-1-mediated gene transcription: differential effects of muscarinic and thrombin receptor activation. 132 61

A functional cellular assay system was developed for the detection of substances modulating the activity of G protein-coupled receptors, linked to the phospholipase C second messenger system. The human adenocarcinoma cell line A549 was transformed with the Photinus pyralis luciferase gene under the control of the ICAM-1 gene 5'regulatory region and, subsequently, stably transfected with the human neurokinin 2 (NK2) receptor gene. The ICAM-1 promoter is known to be inducible via the phospholipase C signal transduction pathway. In this NK2 receptor test cell line, expression of luciferase was inducible by neurokinin A and other NK2-specific agonists. The order of potency of the three neurokinins substance P, neurokinin A and neuromedin K was consistent with published data and results from ligand binding studies performed with the same NK2 test cell line. The agonistic effect of neurokinin A could be inhibited in a dose-dependent manner by simultaneous addition of NK2-specific antagonists or protein kinase C-inhibitors. Similarly, a stable test cell line expressing the human serotonin 2 receptor was established. Agonist-induced luciferase expression in this cell line was abolished in the presence of 5-HT2-specific antagonists. These cellular assay systems can be employed for the identification of competitive, non-competitive and allosteric modulators of the NK2 and the 5-HT2 receptor, and they represent prototypes for analogous test cell lines for other phospholipase C-coupled receptors.
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PMID:Establishment of a cellular assay system for G protein-linked receptors: coupling of human NK2 and 5-HT2 receptors to phospholipase C activates a luciferase reporter gene. 752

We have studied effects of ferric transferrin (FeTF), ferric lactoferrin (FeLF), ferric complexes of pyridoxal- or salicylaldehyde-isonicotinoyl hydrazone, (Fe-PIH, Fe-SIH), and ferric ammonium citrate (FAC) on expression of protein kinase C (PKC) mRNA transcripts in a variety of cultured cell lines. FeTF supported an increase of PKC-beta mRNA transcripts in T-lymphoblastoid (CCRF-CEM; Jurkat), B-lymphoblastoid (Daudi; Raji), promyelocyte (HL-60), erythroleukemia (K562), and monocyte (U937) cell lines. By contrast, FeLF, Fe-PIH, and Fe-SIH did not support an increase of PKC-beta mRNA transcripts in any of these cell lines. Furthermore, FAC supported an increase of PKC-beta mRNA transcripts in HL-60, K562, and U937 cells only. Preincubation of cells with desferrioxamine (DF), a cell-permeable iron chelator, abolished the increments of PKC-beta mRNA observed in response to FeTF or FAC. In contrast to results with PKC-beta, neither FeTF nor FAC caused an increase of PKC-alpha transcripts in any cell line. To locate iron-responsive DNA regulatory elements of the PKC-beta gene, we prepared genetic constructs containing various portions of the human PKC-beta 5'-flanking DNA linked to the firefly luciferase gene. Constructs were cotransfected with the neomycin resistance plasmid, Pwl-neo, into HRE H9 cells, and stable transfectants were selected in G418. Treatment with FeTF of transfectants bearing chimeric gene constructs with 2,200 bp of the PKC-beta 5'-flanking region increased luciferase activity and mRNA transcripts 2.5-fold. This increase was blocked by DF. Neither luciferase activity nor mRNA increased with FeTF in stable transfectants bearing constructs with 342 bp or 587 bp of the PKC-beta 5'-flanking region. These data provide direct confirmation that iron is involved in regulation of PKC-beta but not PKC-alpha gene expression in many cell lines. The form in which iron is presented to these cell lines appears to affect its availability for this function, and cells vary in their capabilities to use nontransferrin iron to support PKC-beta gene expression. Finally, transcriptional upregulation of PKC-beta by FeTF is mediated by DNA sequences located between -2200 bp and -587 bp in the 5'-flanking region of the human PKC-beta gene.
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PMID:Regulation of protein kinase C (PKC) expression by iron: effect of different iron compounds on PKC-beta and PKC-alpha gene expression and role of the 5'-flanking region of the PKC-beta gene in the response to ferric transferrin. 794 5

Signal transduction pathways evoked by interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) to stimulate expression of other cytokines in mesenchymal cells are not clearly understood. Stimulation of the murine bone marrow stromal cell line +/(+)-1.LDA 11 with IL-1 (500 U/ml) in combination with TNF-alpha (500 U/ml) (IL-1 plus TNF-alpha) induced expression of c-jun mRNA as well as granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA. We investigated the possibility that arachidonic acid metabolites, acting through protein kinase C (PKC) and perhaps also through the PKC-responsive transcription factor c-jun/AP-1, may be responsible for regulating GM-CSF transcription in these stromal cells. Expression of GM-CSF mRNA was preceded by IL-1 plus TNF-alpha induced arachidonate release (assayed using the 3H-derivative). Pretreatment of cells with the phospholipase A2 inhibitor quinacrine (20 microM) inhibited accumulation of both c-jun and GM-CSF mRNA but had no influence on expression of other genes induced by IL-1 and TNF-alpha, including leukemia inhibitory factor (LIF). In addition, quinacrine partially blocked IL-1 plus TNF-alpha induced 3H-arachidonic acid release from prelabeled stromal cells. Furthermore, exogenous arachidonate (10 to 50 microM) induced expression of c-jun. To investigate the role of arachidonate in GM-CSF transcription, we used a reporter vector consisting of the murine GM-CSF promoter linked to firefly luciferase. Transfection efficiency was monitored by assessing expression of a constitutively active gene, RSV-beta galactosidase. In this system, quinacrine significantly inhibited IL-1 plus TNF-alpha induced GM-CSF transcription assayed with the reporter construct. Exogenous arachidonic acid alone (10 microM) increased activity of GM-CSF reporter vector 1.5-fold over control. These results are consistent with the hypothesis that arachidonate metabolites are involved in the signaling pathway that leads to IL-1 plus TNF-alpha induced GM-CSF gene expression. Thus, transcriptional activation of GM-CSF gene is mediated, in part, by the arachidonate cascade.
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PMID:Regulation of interleukin-1 and tumor necrosis factor-alpha induced granulocyte-macrophage colony-stimulating factor gene expression: potential involvement of arachidonic acid metabolism. 828 65

The protein kinase inhibitor 2-aminopurine (2-AP) greatly stimulated expression in human promonocytes-macrophages of plasmid constructs carrying various reporter genes (chloramphenicol acetyltransferase, lacZ, firefly luciferase [luc], and Salmonella typhimurium histidinol dehydrogenase [his]) driven by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat. Adenine, adenosine, and caffeine were also effective inducers, but other purine or pyrimidine derivatives were ineffective. Experiments with mutant derivatives of the HIV-1 long terminal repeat revealed no specific eukaryotic promoter elements necessary for 2-AP induction but indicated the need for some minimum combination of such elements. Induction of HIV-1-directed gene expression appeared not to require action of the transcription factor NF-kappa B. The mechanism of induction was investigated by using the luc and his genes linked to the HIV-1 long terminal repeat. 2-AP induced marked, steady rises in mRNA accumulation from both transfected and chromosomally integrated HIV-1 constructs but no increases from an endogenous gene encoding gamma-actin or glucose 6-phosphate dehydrogenase. Thus, induction is selective and not an artifact induced by transfecting DNA into cells. In run-on transcription experiments, the rates of transcription initiation of both transfected and integrated copies of the his gene increased about sixfold in cells treated with 2-AP. Thus, while increased initiation accounted for a portion of 2-AP induction, it could not cause the far greater increase in steady-state mRNA levels. 2-AP induction did not change mRNA decay rates and differed from the phorbol ester (phorbol myristate acetate)-induced activation of the protein kinase C-NF-kappa B pathway in its time course and in its requirement for new protein synthesis. Gel retardation assays showed that unlike phorbol myristate acetate induction, 2-AP induction is enhancer independent. Whereas many previous studies have implicated the activation of various protein kinases in gene induction, we here describe a mechanism of gene activation that appears to involve protein kinase inhibition as a component of the induction response.
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PMID:Inducible transcriptional activation of the human immunodeficiency virus long terminal repeat by protein kinase inhibitors. 835 80

Transcriptional activation of the human CYP1A1 gene by halogenated and polycyclic aromatic hydrocarbons is mediated by the aryl hydrocarbon receptor (AhR) complex, a ligand-dependent transcription factor. A competent AhR comprises at least two components following nuclear translocation and DNA binding, the AhR and the AhR nuclear translocator (Arnt) protein, whose combined action on human CYP1A1 gene transcription is shown to be dependent upon functional protein kinase C (PKC). In the present study, we examined the effects of phorbol 12-myristate 13-acetate, a potent PKC activator, on the ligand-induced transcriptional activation of the CYP1A1 gene and cellular function of the AhR in human HepG2 101L cells. The 101L cells carry a stable transgene consisting of 1800 bases of 5'-flanking DNA and the promoter of the human CYP1A1 gene linked to the firefly luciferase structural gene (Postlind, H., Vu, T. P., Tukey, R. H. & Quattrochi, L. C. (1993) Toxicol. Appl. Pharmacol. 118, 255-262). Pretreatment of cells with 12-myristate 13-acetate enhanced ligand-induced CYP1A1 gene expression 2-3-fold. Inhibition of PKC activity blocked directly the transcriptional activation and the transactivation of the CYP1A1 gene, indicating a role for PKC in the AhR-mediated transcriptional activation process. However, the DNA binding activities of the in vitro activated and the induced nuclear AhR as measured by electrophoretic mobility shift analysis were not affected when CYP1A1 transcription was inhibited, indicating the actions of PKC to be a nuclear event that works in concert with or precedes AhR binding to the gene. These results illustrate that PKC is absolutely essential for the cellular and molecular events that control induction of CYP1A1 gene transcription.
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PMID:Protein kinase C modulates regulation of the CYP1A1 gene by the aryl hydrocarbon receptor. 882 76

We cloned a cDNA for rat TX receptor, and observed its expression in the kidney, including vascular smooth muscle. The aim of the present study was to clone the 5'-flanking region (5'-FL) of rat TX receptor gene, and to examine its transcriptional gene expression regulation. The 5'-FL was cloned by a PCR method, and the nucleic acid structure of 5'-FL (approximately 1 Kb) was disclosed. The transcription initiation site was shown to be 63 bases upstream of the 5' end of the cDNA by the primer extension. In the 5'-FL, putative AP-1 binding sites, glucocorticoid-responsive elements, NF-kappa B binding sites, GATA box, and shear stress-responsive elements were identified. The 5'-FL was then fused upstream of firefly luciferase cDNA in an expression vector, and we examined its transcriptional activity in transiently transfected cultured vascular smooth muscle cells (VSMC). Luciferase expression was dependent on the length of 5'-FL, and it was significantly stimulated by phorbol 12-myristate 13-acetate (PMA), dexamethasone (Dex), tumor necrosis factor-alpha, and interleukin (IL). By a semi-quantitative RT-PCR method, TX receptor mRNA was shown to be induced by Dex, IL-6, and PMA in cultured VSMC. In conclusion, we have revealed the structure of transcription regulatory region of TX receptor. Expression of TX receptor gene is possibly up-regulated by activation of protein kinase C, glucocorticoid excess, and IL-6, in vascular smooth muscle.
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PMID:Structure and transcriptional function of the 5'-flanking region of rat thromboxane receptor gene. 951 39

Regulation of the mitogen-activated protein kinase (MAPK) family by prolactin-releasing peptide (PrRP) in both GH3 rat pituitary tumor cells and primary cultures of rat anterior pituitary cells was investigated. PrRP rapidly and transiently activated extracellular signal-regulated protein kinase (ERK) in both types of cells. Both pertussis toxin, which inactivates G(i)/G(o) proteins, and exogenous expression of a peptide derived from the carboxyl terminus of the beta-adrenergic receptor kinase I, which specifically blocks signaling mediated by the betagamma subunits of G proteins, completely blocked the PrRP-induced ERK activation, suggesting the involvement of G(i)/G(o) proteins in the PrRP-induced ERK activation. Down-regulation of cellular protein kinase C did not significantly inhibit the PrRP-induced ERK activation, suggesting that a protein kinase C-independent pathway is mainly involved. PrRP-induced ERK activation was not dependent on either extracellular Ca(2+) or intracellular Ca(2+). However, the ERK cascade was not the only route by which PrRP communicated with the nucleus. JNK was also shown to be significantly activated in response to PrRP. JNK activation in response to PrRP was slower than ERK activation. Moreover, to determine whether a MAPK family cascade regulates rat prolactin (rPRL) promoter activity, we transfected the intact rPRL promoter ligated to the firefly luciferase reporter gene into GH3 cells. PrRP activated the rPRL promoter activity in a time-dependent manner. Co-transfection with a catalytically inactive form of a MAPK construct or a dominant negative JNK, partially but significantly inhibited the induction of the rPRL promoter by PrRP. Furthermore, co-transfection with a dominant negative Ets completely abolished the response of the rPRL promoter to PrRP. These results suggest that PrRP differentially activates ERK and JNK, and both cascades are necessary to elicit rPRL promoter activity in an Ets-dependent mechanism.
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PMID:Prolactin-releasing peptide activation of the prolactin promoter is differentially mediated by extracellular signal-regulated protein kinase and c-Jun N-terminal protein kinase. 1065 64

In order to improve transient gene transfer into PLB-985 cells, we treated cells with 12-tetradecanoylphorbol 13-acetate (TPA) 4 h before transfection, and increased by 540-fold the reporter activity of the firefly luciferase gene transfected by TFL-01, a cationic liposome. Dioctanolyglycerol added before TPA addition inhibited the TPA-dependent increase in activity, suggesting it to be a TPA competitor for PKC binding. H7, staurosporine, GO 6976, and H8, but not GF 109203X and forskolin, inhibited the TPA-dependent increase in reporter activity when added 8 h after TFL-01/gene addition. Forskolin and GF 109203X also inhibited the increase when added before TPA. Therefore, for the potentiation of transfection by the TPA/TFL-01 method, conventional PKC activity with significant but low protein kinase A (PKA) activity are first required, and then a novel PKC activity with significant PKA activity. TPA enhanced the uptake of FITC-labeled phosphorothioated oligonucleotides and their prolonged maintenance by cells, suggesting increased TFL-01-assisted plasmid uptake and its stabilization in TPA-treated PLB-985 cells. This method was used successfully for the sensitive analysis of the promoter function of the gp91(phox) gene, implying the method to be generally useful for promoter analyses of various genes expressed in differentiated human monocytic cells.
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PMID:Phorbol ester-potentiated liposomal transfection to monocytic PLB-985 cells. 1109 42

The Y1 receptor for neuropeptide Y (NPY-Y1) is constitutively expressed in PC12 cells. In this study, we examined the role of nerve growth factor (NGF), pituitary adenylyl cyclase activating polypeptide (PACAP) and dexamethasone on the expression of the gene encoding the rat NPY-Y1 receptor in PC12 cells. A fusion gene (pY1-Luc) was constructed where the reporter enzyme firefly luciferase was placed under the control of 700 bp of the promoter region of the rat NPY-Y1 receptor gene. This promoter region contains recognition consensus sequences for various transcription factors, including one activation protein-1 (AP-1) site, two cyclic AMP responsive element sites, one estrogen receptor element site and four glucocorticoid receptor element sites. NGF increased luciferase activity in a concentration dependent manner. This increase was inhibited by K-252a, a trk A receptor inhibitor, and calphostin C, a PKC inhibitor. PACAP-38 increased luciferase activity in a concentration dependent manner. This activation was inhibited by H-89. Dexamethasone increased transcription of NPY-Y1 gene in PC12 cells. These results indicate that differentiation of PC12 cells into endocrine-like phenotype by dexamethasone and into a neuronal-like phenotype by either NGF or PACAP-38 increases the transcriptional activity of the NPY-Y1 receptor gene in PC12 cells.
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PMID:Regulation of the Y1 neuropeptide Y receptor gene expression in PC12 cells. 1140 93


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