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

The effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C (PKC), and the PKC inhibitor staurosporine on GnRH secretion and mRNA levels were studied in GT1-7 hypothalamic neuronal cells. Dose-response and time-course studies revealed that TPA (10(-8) M) acutely increased GnRH secretion 3-fold at 3-6 h, which then declined to baseline at 24 h, while it progressively decreased GnRH mRNA levels by 50% and 70% at 6 and 24 h, respectively. To ensure that these effects were due to activation and not down-regulation of PKC, cells were treated for 30 min with TPA (10(-8) M). This brief exposure to TPA also resulted in a decrease (60%) in GnRH mRNA levels at 6 h, with a 1.5- to 2-fold increase in GnRH secretion compared to control values, suggesting that activation of PKC decreases the pretranslational expression of GnRH while increasing GnRH secretion. Additional studies measured PKC activity and documented a shift from a cytosolic to a membrane fraction after incubation with TPA, again supporting PKC activation. Exposure of GT1-7 cells to staurosporine (10(-8) M), a PKC inhibitor, resulted in no change in the level of GnRH mRNA or secretion at 6 h. However, incubation with both TPA and staurosporine prevented the decrease in GnRH mRNA levels and partially blocked the increase in GnRH secretion induced by TPA. We conclude that TPA, by activating the PKC pathway, acutely increases GnRH secretion, but dramatically decreases GnRH gene expression. The exact mechanism of these divergent effects on the synthesis and secretion of GnRH remain to be elucidated.
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PMID:Phorbol ester activation of the protein kinase C pathway inhibits gonadotropin-releasing hormone gene expression. 144 98

We previously demonstrated that down-regulation of protein kinase C (PKC) by prolonged 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment leads to the specific repression of GnRH transcription in GT1-7 hypothalamic neurons. Here we have investigated the regulatory sequences and cognate DNA-binding proteins that mediate this transcriptional response. The promoter-proximal section of the GnRH gene contains an evolutionarily conserved sequence that is bound along its entire length by GT1-7 nuclear proteins in DNase I protection assays. Two distinct regions within this sequence are required for PKC regulation of the GnRH gene, as excision of either region results in loss of TPA repression of transcription. Excision of either of these regions also decreases basal transcription, demonstrating their role in GnRH promoter function. One region encompasses three AT-rich protein-binding sites; the other is an extended region of continuous DNase I protection, 50 nucleotides in length, that contains consensus recognition motifs for the CCAAT/EBP and helix-loop-helix families of transcription factors. Mobility shift analysis of binding to the latter region reveals that TPA treatment of GT1-7 neurons induces the formation of a specific DNA-protein complex with kinetics of appearance consistent with a role in repression of GnRH transcription. Thus, the sequences that mediate PKC regulation of GnRH are proximal to the promoter, evolutionarily conserved, and form TPA-inducible complexes with GT1-7 nuclear proteins.
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PMID:Regulation of gonadotropin-releasing hormone transcription by protein kinase C is mediated by evolutionarily conserved promoter-proximal elements. 747 68

Using a transgenic mouse derived GnRH expressing neuronal cell line, GT1-3, we studied the effects of activation of cAMP, Ca2+ and protein kinase C pathways by forskolin, ionomycin and the phorbol ester phorbol 12-myristate 13-acetate (PMA), respectively, upon gonadotropin-releasing hormone (GnRH) secretion, cellular peptide content, mRNA and RNA primary transcript levels. Forskolin, ionomycin and phorbol ester all caused an increase in GnRH secretion in GT1-3 cells in a time and dose-dependent manner during a short-term (1 h) static incubation. Prolonged treatment with forskolin (10 microM), ionomycin (1 microM) and PMA (10 nM) for 12 or 24 h resulted in significant decreases in GnRH mRNA levels. Time-course studies showed that the increases in GnRH secretion stimulated by forskolin, ionomycin and PMA were gradually attenuated over time in parallel with the decreases in mRNA expression. In contrast, there were only small and variable changes in the GnRH cellular content. Studies using a GnRH antagonist (100 microM) suggested that the released GnRH has a negative feedback effect on its own secretion. However, co-incubation with the GnRH antagonist did not alter the inhibitory effects on GnRH mRNA levels by the secretagogues. Further studies on the transcriptional effects of forskolin, ionomycin and PMA on GnRH gene expression in GT1-3 cells revealed that all three secretagogues suppressed GnRH RNA primary transcript levels, with forskolin having a slower time course of action. Thus, the inhibition of cytoplasmic GnRH mRNA, and presumably its synthesis, after 12-24 h of secretagogue treatment may be due at least in part to a suppression of GnRH gene transcription.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Second messenger regulation of mouse gonadotropin-releasing hormone gene expression in immortalized mouse hypothalamic GT1-3 cells. 752 6

We recently demonstrated that immortalized GT1-7 neurons co-express luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor and gonadotropin releasing hormone (GnRH) genes. Treatment of GT1-7 neurons with LH/hCG resulted in a transcriptional inhibition of GnRH gene. In the present study, we investigated the signaling and transacting factors involved in the action of hCG. Eight-bromo-cyclic AMP can mimic the down-regulating action of hCG on GnRH mRNA levels. H-89, a protein kinase (PK) A inhibitor, but not bisindolylmaleimide, a PKC inhibitor, blocked the down- regulating actions of hCG as well as of 8-bromocyclic AMP. Treatment with the PKA inhibitor alone modestly decreased GnRH mRNA levels suggesting that PKA signaling also controls the basal expression of the GnRH gene. The direct measurement of PK activities revealed that hCG treatment of GT1-7 neurons increased the PKA but not the PKC activity. New protein synthesis is required for the down-regulating action of hCG on GnRH mRNA levels. Since some of the new proteins could be nuclear transcription or transacting factors, we investigated the effects of hCG on cyclic AMP response element binding protein (CREB), c-Fos and c-Jun protein levels. Treatment of GT1-7 neurons with hCG resulted in an increase of 43 kDa phosphorylated CREB, 50 kDa c-Fos and 40 kDa c-Jun proteins compared to the corresponding controls. The kinetics of increases were different and in all cases the increases of the proteins preceded the decrease of GnRH mRNA levels. In summary, PKA signaling and transacting factors such as CREB, Fos and Jun are probably involved in transcriptional inhibition of GnRH gene by hCG in GT1-7 neurons.
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PMID:Signaling and transacting factors in the transcriptional inhibition of gonadotropin releasing hormone gene by human chorionic gonadotropin in immortalized hypothalamic GT1-7 neurons. 766 77

GT1-7 cells, a clonal line derived from specific tumours of gonadotropin-releasing hormone-secreting neurons from mouse hypothalamus, were used as a model system to investigate the cellular mechanisms underlying the histamine H1 receptor-mediated desensitisation. GT1-7 cells contain H1 receptors, acute stimulation of which leads to the desensitisation of histamine-mediated calcium mobilisation and is manifest as a concurrent reduction in both the magnitude of the calcium transient and of the sustained phase. Acute pretreatment of the cells with the phorbol ester, phorbol 12-myristate 13-acetate, can also ablate the histamine-stimulated calcium mobilisation. In addition, acute H1-receptor stimulation and acute phorbol ester treatment result in the attenuation of histamine-mediated inositol phosphate production. Receptor desensitisation resulting from acute stimulation with histamine is not affected by inhibiting protein kinase C (PKC) activity with Ro 31-7549 or staurosporine. In contrast, the desensitisation of H1-receptor responses induced by direct activation of protein kinase C is preventable by PKC inhibitors. Thus, these results imply that a PKC-dependent mechanism and PKC-independent mechanism are involved in the H1-receptor desensitisation cascade in GT1-7 cells and do not support the involvement of PKC in the receptor-mediated desensitisation of H1 receptor-stimulated calcium and inositol phosphate responses.
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PMID:Receptor-mediated desensitisation of histamine H1 receptor-stimulated inositol phosphate production and calcium mobilisation in GT1-7 neuronal cells is independent of protein kinase C. 779 Aug 57

The GT1 GnRH neuronal cell lines exhibit highly differentiated properties of GnRH neurons. We have used GT1-1 cells to study the role of the cyclic AMP/protein kinase A, cyclic GMP/protein kinase G and Ca2+/protein kinase C signaling pathways in the regulation of GnRH secretion. Superfusion of GT1-1 cells with the cyclic AMP analog 8-Br-cyclic AMP (0.5 and 2.5 mM) or the adenylate cyclase activator forskolin (1 and 10 microM) for 100 min increased the amplitude of GnRH secretion 2- to 35-fold. The cyclic GMP analog 8-Br-cyclic GMP (2.5 mM) also stimulated the amplitude of GnRH release from superfused GT1-1 cells, although to a much lesser extent (1.5- to 3-fold). The amplitude of GnRH pulses was also stimulated (5- to 50-fold) by the protein kinase C activator TPA (1 microM). Increasing intracellular Ca2+ with an ionophore (ionomycin, 1 microM) or by the Ca2+ channel activator Bay K 8644 (10 microM) also stimulated GnRH release, while secretion was markedly decreased and spontaneous pulsatility abolished by the L-type Ca2+ channel blocker methoxyverapamil (10 microM). These results demonstrate that in GT1 cells the protein kinase A, protein kinase G and protein kinase C pathways are functionally coupled to regulation of GnRH secretion. Furthermore, pulsatile GnRH secretion is coupled to the entry of extracellular Ca2+ via L-type Ca2+ channels.
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PMID:Signaling pathways involved in GnRH secretion in GT1 cells. 789 36

We previously showed that activation of protein kinase C (PKC) with the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) in GT1-7 hypothalamic cells decreases GnRH mRNA levels in a dose and time dependent fashion. In the present studies, we examined the mechanism of this effect. Analysis of the half-life of GnRH mRNA levels after transcriptional arrest with actinomycin-D (5 micrograms/ml) estimated the half-life of GnRH mRNA to be 22 h. TPA treatment did not alter the GnRH mRNA half-life directly, suggesting that the effects of TPA occur predominantly at the level of gene transcription. Exposure of cells transiently transfected with various deletion constructs of the rat (r)GnRH promoter to TPA resulted in a decrease of 60% in luciferase reporter activity. This repression was maintained in constructs deleted to position -126 and was lost with further deletion to position -73. In conclusion, these experiments suggest that phorbol esters repress GnRH expression at the level of transcription through DNA sequences in the proximal rGnRH promoter.
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PMID:Evidence for transcriptional inhibition of GnRH gene expression by phorbol ester at a proximal promoter region. 820 25

Activation of GnRH receptors in GnRH neuronal (GT1-7) cells causes marked and transient increases in c-fos expression, with peak response at 30 min. GnRH and des-Gly10-[D-Ala6]GnRH N-ethylamide induced concentration-dependent c-fos responses with EC50s of approximately 0.1 and approximately 1 nM, respectively. GnRH action was mimicked by phorbol 12-myristate-13-acetate (PMA), but stimulation of Ca2+ entry by K(+)-induced depolarization and Bay K 8644 was much less effective. In protein kinase C-depleted cells, the c-fos response to GnRH was reduced to that elicited by increased Ca2+ entry, and the effect of PMA was abolished. Thus, GnRH-induced c-fos expression in GT1 cells appears to be mediated predominantly by protein kinase C, and to a lesser extent by Ca2+. These findings demonstrate that c-fos expression can be induced in a peptidergic neuron by activation of receptors for its neurosecretory product. It is possible that the expression of c-fos in GnRH hypothalamic neurons during the proestrous surge of gonadotropins could likewise be stimulated by a positive feedback action of GnRH on its neuronal receptors.
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PMID:Autocrine induction of c-fos expression in GT1 neuronal cells by gonadotropin-releasing hormone. 824 34

As major signal transduction cascades, the protein kinase-A and -C (PKA and PKC) pathways have been implicated in the regulation of GnRH synthesis and secretion in the hypothalamus. We have investigated the roles of these pathways in the regulation of GnRH transcription, mRNA levels, propeptide processing, and secretion in GT1-7 cells, a mouse hypothalamic GnRH neuronal cell line. Forskolin, which activates adenylate cyclase to raise cAMP levels, had no effect on GnRH mRNA levels at 10 microM, but induced c-fos mRNA at 30 min. An activator of PKC, 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nM), also induced c-fos at 30 min, but produced a progressive decline in GnRH mRNA, resulting in a 70% decrease by 16 h. Coadministration of 10 nM TPA and 20 microM of a PKC inhibitor, NPC 15437 [2,6-diamino-N-([1-(1-oxotridecyl)2-piperidinyl]methyl)hexanami de], prevented c-fos induction, but did not antagonize GnRH repression. Instead, the inhibitor itself reduced GnRH mRNA levels by 56% at 16 h (with no effect on c-fos mRNA). Thus, since extended exposure to TPA can down-regulate PKC, suppression of GnRH mRNA by TPA may be due to decreased PKC activity, indicating a role for PKC in the maintenance of the GnRH gene expression (a role that is unlikely to involve c-fos). In transient transfections, the transcriptional activity from 3 kilobases of GnRH 5'-flanking sequence was repressed 2-fold by either 100 nM TPA or 20 microM NPC 15437 at 24 h, demonstrating that suppression of GnRH mRNA is at least, in part, at the level of transcription. In contrast, both TPA (100 nM) and forskolin (10 microM) stimulated secretion. Enhancement of GnRH secretion by TPA was robust and rapid (2.5 min), while the response to forskolin was relatively delayed (2 h). Over a 24-h period, unstimulated cells released primarily unprocessed prohormone, whereas forskolin and TPA stimulated the secretion of processed products. These data indicate that PKC and PKA may influence propeptide processing and/or the route of GnRH secretion. These data demonstrate that the PKA and PKC pathways regulate GnRH at the multiple levels of transcription, pro-GnRH processing, and GnRH secretion.
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PMID:Regulation of gonadotropin-releasing hormone by protein kinase-A and -C in immortalized hypothalamic neurons. 850 41

Growth factors are commonly associated with the regulation of cellular proliferation and differentiation. In established cells, growth factors can also serve as trophic agents. Immortalized LHRH neurons contain basic fibroblast growth factor (bFGF) receptors. Although these receptors are coupled to activation of protein kinase C, and phorbol esters are strong activators of protein kinase C-stimulated LHRH release, bFGF did not influence LHRH secretion from these cells. To clarify this discrepancy, the effects of bFGF and phorbol ester on pro-LHRH biosynthesis, protein processing, and secretion were examined in GT1-7 cells. Phorbol ester stimulated LHRH secretion, whereas bFGF either had no effect or stimulated LHRH release depending upon the antiserum used. Pro-LHRH levels in lysate and medium were depressed by phorbol esters; concentrations in bFGF-treated cells were somewhat lower than those in unstimulated controls. HPLC analyses revealed that both agents enhanced the release of LHRH intermediate products into the medium. C-Terminally extended forms of LHRH, especially LHRH-[Gly11], were prominent in medium from bFGF-stimulated neurons. Levels of LHRH were depressed relative to those in the control or phorbol ester groups. These data indicate that phorbol esters control the biosynthesis, secretion, and, to some extent, processing of pro-LHRH. The effects of bFGF are novel because this factor regulates processing of the prohormone so that LHRH-intermediate products are predominantly secreted instead of LHRH. By enhancing the secretion of these intermediates over that of LHRH, bFGF can control the biological activity of the decapeptide and regulate LHRH neuronal function.
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PMID:Basic fibroblast growth factor regulates the conversion of pro-luteinizing hormone-releasing hormone (Pro-LHRH) to LHRH in immortalized hypothalamic neurons. 864 Dec 15


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