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)

Gonadotropin releasing hormone (GnRH) contributes to the maintenance of gonadotrope function by increasing extracellular signal-regulated kinase (ERK) activity subsequent to binding to its cognate G-protein-coupled receptor. As the GnRH receptor exclusively interacts with G(q/11) proteins and as receptor expression is regulated in a beta-arrestin-independent fashion, it represents a good model to systematically dissect underlying signaling pathways. In alphaT3-1 gonadotropes endogenously expressing the GnRH receptor, GnRH challenge resulted in a rapid increase in ERK activity which was attenuated by the epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitor AG1478. In COS-7 cells transiently expressing the human GnRH receptor, agonist-induced ERK activation was independent of free Gbetagamma subunits but could be mimicked by short-term phorbol ester treatment. Most notably, G(q/11)-induced ERK activation was sensitive to N17-Ras and to expression of the C-terminal Src kinase but also to other dominant negative mutants of signaling components localized upstream of Ras, like Shc and the EGFR. GnRH as well as phorbol esters led to Ras activation in COS-7 and alphaT3-1 cells, which was dependent on Src and EGFR tyrosine kinases, indicating that both tyrosine kinases act downstream of protein kinase C (PKC) and upstream of Ras. However, Src did not contribute to Shc tyrosine phosphorylation. GnRH or phorbol ester challenge resulted in PKC-dependent EGFR autophosphorylation. Furthermore, a 5-min phorbol ester treatment was sufficient to trigger tyrosine phosphorylation of the platelet-derived growth factor-beta receptor in L cells. Thus, in several cell systems PKC is able to stimulate Ras via activation of receptor tyrosine kinases.
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PMID:Epidermal growth factor receptor tyrosine kinase mediates Ras activation by gonadotropin-releasing hormone. 1076 63

Receptors coupled to heterotrimeric G proteins are linked to activation of mitogen-activated protein kinases (MAPKs) via receptor- and cell-specific mechanisms. We have demonstrated recently that gonadotropin-releasing hormone (GnRH) receptor occupancy results in activation of extracellular signal-regulated kinase (ERK) through a mechanism requiring calcium influx through L-type calcium channels in alphaT3-1 cells and primary rat gonadotropes. Further studies were undertaken to explore the signaling mechanisms by which the GnRH receptor is coupled to activation of another member of the MAPK family, c-Jun N-terminal kinase (JNK). GnRH induces activation of the JNK cascade in a dose-, time-, and receptor-dependent manner in clonal alphaT3-1 cells and primary rat pituitary gonadotrophs. Coexpression of dominant negative Cdc42 and kinase-defective p21-activated kinase 1 and MAPK kinase 7 with JNK and ERK indicated that specific activation of JNK by GnRH appears to involve these signaling molecules. Unlike ERK activation, GnRH-stimulated JNK activity does not require activation of protein kinase C and is not blocked after chelation of extracellular calcium with EGTA. GnRH-induced JNK activity was reduced after treatment with the intracellular calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester), whereas activation of ERK was not affected. Chelation of intracellular calcium also reduced GnRH-induced activation of JNK in rat pituitary cells in primary culture. GnRH-induced induction and activation of the JNK target c-Jun was inhibited after chelation of intracellular calcium, whereas induction of c-Fos, a known target of ERK, was unaffected. Therefore, although activation of ERK by GnRH requires a specific influx of calcium through L-type calcium channels, JNK activation is independent of extracellular calcium but sensitive to chelation of intracellular calcium. Our results provide novel evidence that GnRH activates two MAPK superfamily members via strikingly divergent signaling pathways with differential sensitivity to activation of protein kinase C and mobilization of discrete pools of calcium.
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PMID:Divergent signaling pathways requiring discrete calcium signals mediate concurrent activation of two mitogen-activated protein kinases by gonadotropin-releasing hormone. 1079 94

GnRH has been suggested to regulate hCG secretion in the placenta. In the present study, we report isolation of full-length GnRH receptor (GnRHR) complementary DNA from human placental cells, including a choriocarcinoma cell line (JEG-3), immortalized extravillous trophoblasts (IEVT), and first trimester cytotrophoblast cells in primary culture. Sequence analysis of the placental GnRHR complementary DNA revealed a 100% similarity to its pituitary counterpart. Northern blot analysis using polyadenylated RNA isolated from JEG-3 and IEVT cells revealed a 2.5- and 1.2-kb GnRHR transcripts. Using semiquantitative RT-PCR, regulation ofplacental GnRHR gene expression was examined. In contrast to pituitary gonadotrope alphaT3-1 cells, down-regulation of GnRHR messenger RNA (mRNA) levels was not observed in placental cells after 24 h of 0.1-microM GnRH agonist (GnRHa) treatment. Instead, a 43% (P < 0.01) and 30% (P < 0.05) increase in GnRHR mRNA levels was observed in JEG-3 and IEVT cells, respectively. In addition, 10 microM phorbol ester or forskolin treatments resulted in a significant increase in GnRHR expression in both JEG-3 and IEVT cells. The GnRHa-induced increase in GnRHR expression was shown to be a receptor-mediated process, as cotreatment of GnRH antagonist abolished the effect. It has also been demonstrated that these stimulatory effects on GnRHR gene expression were regulated at least in part at the transcriptional level. Pretreatment of JEG-3 cells with a specific protein kinase C inhibitor (GF109203X), adenylate cyclase inhibitor (SQ22536), or protein kinase A inhibitor [PKI-(14-22) amide, myristylated] reversed GnRHa-induced GnRHR gene expression, suggesting that the placental GnRHR couples to the protein kinase C (PKC) and cAMP/ protein kinase A (PKA) pathways. By Northern blot analysis, we observed a 100% (P < 0.001) increase in hCGbeta mRNA levels after 0.1 microM GnRHa treatment in JEG-3 cells. Again, this effect was prevented in the presence of either protein kinase C inhibitor or adenylate cyclase inhibitor, further supporting the role of the PKC and PKA pathways in GnRHR-coupled signaling in placental cells. In summary, these data strongly support the idea that 1) GnRH plays an autocrine/paracrine role in regulating placental function through a receptor-mediated mechanism; and 2) the placental GnRHR couples to both the PKC and PKA pathways.
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PMID:Regulation of human gonadotropin-releasing hormone receptor gene expression in placental cells. 1087 33

Clinical applications of GnRH agonists (GnRHa) are based primarily on the decrease in gonadotropin release after down-regulation of the GnRH receptor (GnRHR) by continuous GnRHa administration. However, the molecular mechanisms underlying the transcriptional regulation of the human GnRHR gene after prolonged GnRH treatment remain poorly understood. In the present study GnRHa-mediated regulation of human GnRHR gene transcription was studied by transiently transfecting the mouse gonadotrope-derived (alphaT3-1) cells with a 2297-bp human GnRHR promoter-luciferase construct (p2300-LucF). A dose- and time-dependent decrease in human GnRHR promoter activity was observed after GnRHa treatment. An average 71% decrease in promoter activity was observed after 24-h treatment with 0.1 microM GnRHa, which was blocked by cotreatment of the GnRH antagonist, antide. This effect was mimicked by phorbol 12-myristate 13-acetate (TPA) administration. In addition, the GnRHa- and TPA-mediated decrease in the human GnRHR promoter activity was reversed by a specific protein kinase C (PKC) inhibitor, GF109203X, or depletion of PKC by TPA pretreatment. These findings indicate that the activation of the PKC pathway is important in regulating the human GnRHR gene expression. By progressive 5'-deletion studies, we have identified a 248-bp DNA fragment (-1018 to -771, relative to the translation start site) at the 5'-flanking region of the human GnRHR gene that is responsible for the GnRHa-mediated down-regulation of human GnRHR promoter activity. Analysis of this sequence reveals the existence of two putative activating protein-1 (AP-1) sites with 87% homology to the consensus sequence (5'-TGA(G/C)T(C/A)A-3'), located at -1000 to -994 (5'-TTAGACA-3', in complementary orientation) and -943 to 937 (5'-TGAATAA-3'). Using competitive gel mobility shift assays, AP-1 binding was observed within this 248-bp region. Site-directed mutation of the putative AP-1-binding site located at -1000 to -994 abolished the GnRHa-induced inhibition. Further competitive GMSA and supershift experiments confirmed the identity of AP-1 binding in this region. By the use of Western blot analysis, a significant increase in c-Jun (100%; P < 0.05) and c-Fos (50%; P < 0.05) protein levels was observed after GnRHa treatment in alphaT3-1 cells. In addition, our data suggested that a change in AP-1 composition, particularly c-Fos, was important in mediating GnRHa-induced inhibition of human GnRHR gene expression. We conclude that activation of the PKC pathway by GnRH is important in controlling human GnRHR gene expression. In addition, the putative AP-1-binding site located at -1000 to -994 of the human GnRHR5'-flanking region has been functionally identified to be involved in mediating this down-regulatory effect.
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PMID:Transcriptional down-regulation of human gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: role of protein kinase C and activating protein 1. 1101 15

The neuropeptide GnRH is a central regulator of mammalian reproductive function produced by a dispersed population of hypothalamic neurosecretory neurons. The principal action of GnRH is to regulate release of the gonadotropins, LH and FSH, by the gonadotrope cells of the anterior pituitary. Using a cultured cell model of mouse pituitary gonadotrope cells, alphaT3-1 cells, we present evidence that GnRH stimulation of alphaT3-1 cells results in an increase in cap-dependent mRNA translation. GnRH receptor activation results in increased protein synthesis through a regulator of mRNA translation initiation, eukaryotic translation initiation factor 4E-binding protein, known as 4EBP or PHAS (protein, heat, and acid stable). Although the GnRH receptor is a member of the rhodopsin-like family of G protein-linked receptors, we show that activation of translation proceeds through a signaling pathway previously described for receptor tyrosine kinases. Stimulation of translation by GnRH is protein kinase C and Ras dependent and sensitive to rapamycin. Furthermore, GnRH may also regulate the cell cycle in alphaT3-1 cells. The activation of a signaling pathway that regulates both protein synthesis and cell cycle suggests that GnRH may have a significant role in the maintenance of the pituitary gonadotrope population in addition to directing the release of gonadotropins.
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PMID:Activation of translation in pituitary gonadotrope cells by gonadotropin-releasing hormone. 1107 14

Gonadotropin-releasing hormone (GnRH), acting via the GnRH receptor, elicited rapid extracellular acidification responses in mouse gonadotrope-derived alphaT3-1 cells as measured by the Cytosensor microphysiometer, which indirectly monitors cellular metabolic rates. GnRH increased the extracellular acidification rate of the cells in a dose-dependent manner (EC(50) = 1.81 +/- 0.24 nM). The GnRH-stimulated acidification rate could be attenuated by protein kinase C (PKC) down-regulation, extracellular Ca2+ depletion, and the voltage-sensitive Ca2+ channel (VSCC) blocker nifedipine, indicating that the acidification response is activated by both Ca2+ and PKC-mediated pathways. Upon continuous exposure to 100 nM GnRH or periodic stimulation by 10 nM GnRH at 40 min intervals, homologous desensitization was more pronounced in the absence of extracellular Ca2+, suggesting that desensitization of GnRH activity may be mediated via depletion of intracellular Ca2+ stores. We have also compared the potency of eight GnRH analogs on alphaT3-1 cells. No acidification response was detected for GnRH free acid, consistent with the idea that the C-terminal amide is a critical structural determinant for GnRH activity. Replacement of Gly-NH(2) at the C-terminus by N-ethylamide dramatically reduced the EC(50) value, suggesting that substitution of the Gly-NH(2) moiety by N-ethylamide increases the potency of GnRH analogs. Substitution of Gly at position 6 by D-Trp significantly reduced the EC(50) value, whereas D-Lys at the same position slightly increased the EC(50) value, implying that either an aromatic amino acid or a non-basic amino acid at position 6 may be essential for potent GnRH agonists. In summary, our results demonstrate that the Cytosensor microphysiometer can be used to evaluate the actions of GnRH and GnRH analogs in alphaT3-1 cells in a real-time and noninvasive manner. This silicon-based microphysiometric system should provide new information on the structure-function studies of GnRH and is an invaluable tool for the screening of new GnRH agonists and antagonists in the future.
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PMID:Real-time analysis of the activities of GnRH and GnRH analogs in alphaT3-1 cells by the Cytosensor microphysiometer. 1113 59

Normal mammalian sexual maturation and reproductive functions require the integration and precise coordination of hormones at the hypothalamic, pituitary, and gonadal levels. Hypothalamic gonadotropin-releasing hormone (GnRH) is a key regulator in this system; after binding to its receptor (GnRHR), it stimulates de novo synthesis and release of gonadotropins in anterior pituitary gonadotropes. Since the isolation of the GnRHR cDNA, the expression of GnRHR mRNA has been detected not only in the pituitary, but also in extrapituitary tissues, including the ovary and placenta. It has been shown that change in GnRHR mRNA is one of the mechanisms for regulating the expression of the GnRHR. To help understand the molecular mechanism(s) involved in transcriptional regulation of the GnRHR gene, the 5' flanking region of the GnRHR gene has recently been isolated. Initial characterization studies have identified several DNA regions in the GnRHR 5' flanking region which are responsible for both basal expression and GnRH-mediated homologous regulation of this gene in pituitary cells. The mammalian GnRHR lacks a C-terminus and possesses a relatively short third intracellular loop; both features are important in desensitization of many others G-protein coupled receptors (GPCRs), Homologous desensitization of GnRHR has been shown to be regulated by various serine-threonine protein kinases including protein kinase A (PKA) and protein kinase C (PKC), as well as by G-protein coupled receptor kinases (GRKs). Furthermore, GnRHR was demonstrated to couple with multiple G proteins (Gq/11, Gs, and Gi), and to activate cascades that involved the PKC, PKA, and mitogen-activator protein kinases. These results suggest the diversity of GnRHR-G protein coupling and signal transduction systems. The identification of second form of GnRH (GnRH-II) in mammals adds to the complexity of the GnRH-GnRHR system. This review summaries our recent progress in understanding the regulation of GnRHR gene expression and the GnRHR signal transduction pathways.
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PMID:The expression, regulation and signal transduction pathways of the mammalian gonadotropin-releasing hormone receptor. 1114 80

The present study investigated the activation of mitogen-activated protein kinases (MAPKs) by a GnRH agonist (GnRHa) in human granulosa-luteal cells (hGLCs). The phosphorylation state of p44 and p42 MAPK was examined using antibodies that distinguish phospho-p44/42 MAPK (Thr(202)/Tyr(204)) from total p44/42 MAPK (activated plus inactivated). Activation of MAPK by GnRHa was observed within 5 min and was sustained for 60 min after treatment. GnRHa stimulated MAPK activation in a dose-dependent manner, with maximum stimulation (6.7-fold over basal levels) at 10(-7) M. Pretreatment with a protein kinase C (PKC) inhibitor, GF109203X, completely blocked GnRHa-induced MAPK activation. In addition, pretreatment with a PKC activator, phorbol-12-myristate 13-acetate, potentiated GnRH-induced MAPK activation. These results indicate that GnRHa stimulates MAPK activation through a PKC-dependent pathway in hGLCs, possibly coupled to G(q)alpha protein. MAPK activation was also observed in response to 8-bromo-cAMP or cholera toxin, but not pertussis toxin. Forskolin (50 microM) substantially stimulated a rapid cAMP accumulation, whereas GnRHa (10(-7) M) or pertussis toxin (100 mg/ml) did not affect basal intracellular cAMP levels. Cotreatment of GnRHa (10(-7) M) did not attenuate forskolin- or hCG-stimulated cAMP accumulation. These results suggest that the GnRH receptor is probably not coupled to G(s)alpha or G(i)alpha in hGLCs. Finally, GnRHa (10(-7) M) stimulated a significant increase in Elk-1 phosphorylation and c-fos messenger RNA expression, as revealed by an in vitro kinase assay and Northern blot analysis, respectively. These results clearly demonstrate that GnRH activates the MAPK cascade through a PKC-dependent pathway in the human ovary.
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PMID:Stimulation of mitogen-activated protein kinase by gonadotropin-releasing hormone in human granulosa-luteal cells. 1115 38

The aim of these studies was to identify the signaling mechanism(s) that contribute to GnRH-induced expression of MAPK phosphatase (MKP)-2, a dual specificity phosphatase that selectively inactivates MAPKs. GnRH receptor activation induced MKP-2 expression in both clonal (alphaT3-1) and primary gonadotropes. Activation of PKC isozymes was sufficient and required for MKP-2 induction. Inhibition of the extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) but not the p38 MAPK cascade was sufficient to block GnRH-induced MKP-2 expression. Induction of MKP-2 by GnRH was dependent on elevation in intracellular Ca(2+). Inhibition of Ca(2+) influx through L-type voltage-gated calcium channels blocked GnRH-induced MKP-2 expression. Depletion of intracellular Ca(2+) stores with thapsigargin blocked MKP-2 activation by GnRH independent of ERK and JNK activity. These results support the conclusion that MKP-2 induction by GnRH occurs via MAPK-dependent and -independent pathways. One mechanism requires GnRH-induced ERK and JNK activation, while a second MAPK-independent pathway requires a thapsigargin-sensitive calcium signal.
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PMID:Activation of mitogen-activated protein kinase phosphatase 2 by gonadotropin-releasing hormone. 1116 42

In the rat pituitary gland the mechanism responsible for ERalpha regulation has not been fully elucidated. Using transient transfection assays in alphaT3-1 cells, a cell line of gonadotrope origin, we show that GnRH stimulates estrogen response element-containing promoters in an estrogen-independent manner. This effect was strictly ER and GnRH receptor dependent, as no activation of the reporter gene was observed in presence of the anti-estrogen ICI 182,780 or a GnRH antagonist. These data suggest that the GnRH-triggered signaling pathway results in 17beta-estradiol-independent trans-activation of the ERalpha in alphaT3-1 cells. Furthermore, an additive activation was achieved when cells were treated with both GnRH and 17beta-estradiol. In primary pituitary cells, GnRH alone (100 nM) did not cause a significant stimulation of reporter gene activity, presumingly due to the low amount of gonadotropes. Interestingly, the combination of 17beta-estradiol and GnRH resulted in a significant increase in ERalpha trans-activation compared with that in cells treated with 17beta-estradiol alone. This enhancement was prevented by ICI 182,780, showing an ERalpha requirement. Moreover, we show that the effects of GnRH on ERalpha transcriptional activity in gonadotrope cell lines are mediated by the PKC/MAPK pathway. In conclusion, our data demonstrate that GnRH is an important signal in the regulation of ERalpha trans-activation in gonadotrope cells.
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PMID:Steroid-independent activation of ER by GnRH in gonadotrope pituitary cells. 1145 76


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