Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This study was performed to assess the effects of activin on intracellular mechanisms involved in GnRH action. When rat pituitary cell cultures were pretreated with activin-A (5-80 ng/ml) for 3 days, subsequent FSH and LH release (percentage of total cellular FSH and LH released during 4 h) in response to GnRH (10(-10)-10(-6) M) was not significantly different from that in cells pretreated with medium alone. In contrast, activin pretreatment increased the potency of both A23187 (Ca2+ ionophore) and phorbol 12-myristate 13-acetate [a protein kinase-C (PKC) activator] as secretagogues for FSH and LH release. FSH or LH release in response to another Ca(2+)-mobilizing secretagogue, maitotoxin (an activator of the GnRH receptor-associated Ca2+ channel), was not increased by activin. Although PKC is capable of influencing the actions of Ca2+, which is believed to be the second messenger for GnRH action, neither GnRH- nor maitotoxin-stimulated gonadotropin release was increased by activin even when the influence of activin on PKC was eliminated by the addition of a PKC inhibitor (staurosporine; 100 nM) during the final 30 min of the 3-day pretreatment period. These results indicate that although activin does not influence GnRH action with regard to gonadotropin release, it increases the sensitivity of the system regulating gonadotropin release to increases in cytosolic Ca2+ concentrations and PKC activation. Furthermore, activin appears to exhibit an inhibitory effect(s) at some point(s) in GnRH action in a PKC-independent manner, which could be responsible for opposing the increased sensitivity of the gonadotrope to Ca2+. The differential effects of activin on gonadotropin release in response to Ca(2+)-mobilizing secretagogues (ionophore and maitotoxin) raise the possibility that the activity of the GnRH receptor-associated Ca2+ channel may be suppressed by activin.
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PMID:Activin modulates the intracellular signaling system activated by gonadotropin-releasing hormone: dual effect on calcium messenger system and protein kinase-C pathway. 827 26

FSH signal transduction in Sertoli cells involves the generation of cAMP and calcium as second messengers; however, the relationship between these two signals is not clear. In order to determine whether these were serial or parallel signals, we studied cytosolic calcium levels in freshly isolated rat Sertoli cells using maneuvers to dissociate generation of endogenous cAMP from cytosolic calcium. Pretreatment with 1 mM MDL 12,330A, an adenylate cyclase inhibitor, reduced by greater than 90% increases in cytosolic calcium induced by FSH (97 +/- 6 vs. 213 +/- 16 nM), whereas, despite adenylate cyclase blockade, 1 mM (Bu)2cAMP continued to elevate cytosolic calcium (from 87 +/- 6 to 182 +/- 23 nM), indicating the involvement of adenylate cyclase in the FSH-induced rise of cytosolic calcium. A cAMP antagonist, 1 mM Rp-cAMP, reduced by 75% the FSH-induced rise of cytosolic calcium (115 +/- 14 vs. 213 +/- 16 nM), suggesting that endogenous cAMP levels generated by FSH are sufficient to activate the cytosolic calcium response to FSH. Pretreatment with pertussis toxin (1 mg/liter) to dissociate the FSH-receptor interaction from its G-protein-mediated linkage to adenylate cyclase also suppressed the FSH-induced rise in cytosolic calcium (97 +/- 11 vs. 213 +/- 16 nM). Sertoli cells preincubated with 1 mM staurosporine, an inhibitor of protein kinases, exhibited a reduced calcium response to FSH (125 +/- 14 vs. 213 +/- 16 nM), suggesting that FSH-induced calcium flux might be mediated by protein kinase, presumably cAMP-dependent protein kinase A. The present findings therefore strengthen the premise that the cytosolic calcium response to FSH in Sertoli cells is predominantly attributable to serial signaling after the generation of endogenous cAMP.
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PMID:The relationship between 3',5'-cyclic adenosine monophosphate and calcium in mediating follicle-stimulating hormone signal transduction in Sertoli cells. 827 46

Human myometrium contains receptors for hCG/human LH (hLH). This suggested the possibility that hCG and hLH might regulate human myometrium, which has not previously been considered a direct target of gonadotropin regulation. To investigate such a possibility, highly pure and viable smooth muscle cells were isolated from nonpregnant human myometrium and cultured as monolayers. The cells contained hCG/LH receptor mRNA transcripts and a 50-kDa immunoreactive protein that can bind 125I-hCG in a ligand-specific manner. The presence of hCG during culture resulted in a significant increase of myometrial smooth muscle cell density. The hCG effect was time- and concentration-dependent and was mimicked by hLH but not by human FSH or human FSH or human thyroid-stimulating hormone. Human CG also greatly increased the size of a subpopulation of myometrial smooth muscle cells without affecting their chromosomal ploidy. Antibodies to hCG/LH receptors and hCG blocked hCG effects. Human prolactin and growth hormone, which do not bind to hCG/LH receptors, also increased the myometrial smooth muscle cell density. A protein kinase A inhibitor (H-89) blocked hCG response whereas calphostin (a protein kinase C inhibitor) and lavendustin A (a tyrosine kinase inhibitor) had no effect on hCG response, suggesting that a cAMP/protein kinase A signaling mechanism is involved in hCG action. Eicosanoids from cyclooxygenase and 5-lipoxygenase pathways of arachidonic acid metabolism are probably not involved, because the inhibitors of these enzymes had no effect on hCG response. While progesterone and estradiol could not mimic or modify hCG action, epidermal growth factor did mimic hCG in increasing myometrial smooth muscle cell density.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Human myometrial smooth muscle cells are novel targets of direct regulation by human chorionic gonadotropin. 828 97

A higher dose of GnRH is required to stimulate release of FSH than of LH, both in vivo and in vitro. Therefore, we tested the hypothesis that secretion of FSH may be mediated via a second messenger pathway different from the one that modulates secretion of LH. Pituitary cells from intact ewes were cultured in suspension in DMEM plus 10% wether serum. After 18 h, cell were washed and challenged for 2 h with agents capable of activating protein kinase A (dibutyryl cAMP), protein kinase C (phorbol 12-myristate 13-acetate; PMA), or increasing intracellular calcium (the calcium ionophore A23187). GnRH (0.01-10 nM) and PMA (0.2-20 nM) stimulated dose-dependent increases in secretion of LH. FSH secretion also was stimulated by GnRH and PMA; however, the percentage of total cellular FSH released was lower (p < 0.05) than the percentage of total cellular LH released. Dibutyryl cAMP (10 mM) induced a modest release (p < 0.05) of both LH and FSH. A23187 (1-10 microM) stimulated secretion of LH in a dose-dependent manner but did not influence secretion of FSH; however, GnRH- and PMA-induced secretion of FSH required the presence of intracellular calcium. On the basis of the results of this study, we suggest that secretion of FSH is less than secretion of LH following direct activation of these second messenger systems. Furthermore, we suggest that in contrast to the situation for LH, increased intracellular calcium is not the primary stimulus for inducing secretion of FSH.
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PMID:Differential secretion of follicle-stimulating hormone and luteinizing hormone from ovine pituitary cells following activation of protein kinase A, protein kinase C, or increased intracellular calcium. 831 50

Follistatin, activin and inhibin proteins are produced by granulosa cells, but the mechanisms controlling their production remain unclear. Here, we examined how the protein kinase A (PKA) and protein kinase C (PKC) pathways act and interact to regulate production of these proteins. Granulosa cells from immature rats were cultured with activators of the PKA pathway (100 ng/ml FSH, 10 microM forskolin) and/or activators of the PKC pathway (100 nM GnRH agonist, 100nM 2-0-tetradecanoyl-phorbol-13-acetate, TPA). Conditioned media were assayed for inhibin and activin by ligand blotting using recombinant human 125I-follistatin and for follistatin by double ligand blotting using cold activin plus 125I-follistatin. FSH and forskolin stimulated inhibin but not activin production. In contrast, GnRH and TPA stimulated activin, and to a lesser degree, inhibin production; significantly, this is the first demonstration of activin dimer production by granulosa cells. Activators of the PKA pathway antagonized the actions of PKC effectors and vice versa. All agents increased follistatin protein production, and the PKA and PKC activators interacted to generate further increases in follistatin production. These results show that the FSH-PKA signalling pathway favors formation of alpha beta inhibin dimers while the GnRH-PKC pathway favors formation of beta-subunit activin dimers. Both pathways act to increase follistatin protein production.
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PMID:Differential control of activin, inhibin and follistatin proteins in cultured rat granulosa cells. 833 40

FSH induces the expression of cholesterol side-chain cleavage cytochrome P450 (P450scc) in rat ovarian granulosa cells. The present study reveals that the tyrphostin AG18, a member of novel protein tyrosine kinase inhibitors, can arrest the FSH-induced synthesis of P450scc with an apparent IC50 of 30 microM. Total inhibition of P450scc expression was achieved at 80 microM AG18. AG18-mediated inhibition of P450scc was also observed when the enzyme was induced by prostaglandin E2, forskolin, or 8-bromo-cAMP. Studies examining functional LH receptors showed that the tyrphostin inhibits the expression of FSH-induced LH receptors. The drug did not affect FSH-induced cAMP accumulation, suggesting that it may interfere with the flow of FSH signal transduction at a site distal intracellular accumulation of cAMP. Control experiments demonstrated that the inhibitory action of AG18 was reversible, did not hamper total protein synthesis in the cells, and did not change the adenine nucleotide (ATP:ADP:AMP) ratio or their levels in the treated cells. A cell-free assay of cAMP-dependent protein kinase showed that the tyrphostin AG18 does not affect this enzyme activity up to concentrations above 200 microM. These results suggest that a putative tyrosine kinase activity is involved in the gonadotropin signal transduction pathway leading to expression of functional genes in ovarian cells.
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PMID:Tyrphostins inhibit follicle-stimulating hormone-mediated functions in cultured rat ovarian granulosa cells. 838 Mar 82

The transcriptional transactivational activities of the phosphoprotein cAMP-response element-binding protein (CREB) are activated by the cAMP-dependent protein kinase A signaling pathway. Dimers of CREB bind to the palindromic DNA element 5'-TGACGTCA-3' (or similar motifs) called cAMP-responsive enhancers (CREs) found in the control regions of many genes, and activate transcription in response to phosphorylation of CREB by protein kinase A. Earlier we reported on the cyclical expression of the CREB gene in the Sertoli cells of the rat testis that occurred concomitant with the FSH-induced rise in cellular cAMP levels and suggested that transcription of the CREB gene may be autoregulated by cAMP-dependent transcriptional proteins. We now report the structure of the 5'-flanking sequence of the human CREB gene containing promoter activity. The promoter has a high content of guanosines and cytosines and lacks canonical TATA and CCAAT boxes typically found in the promoters of genes in eukaryotes. Notably, the promoter contains three CREs and transcriptional activities of a promoter-luciferase reporter plasmid transfected to placental JEG-3 cells are increased 3- to 5-fold over basal activities in response to either cAMP or 12-O-tetradecanoyl phorbol-14-acetate, and give 6- to 7-fold responses when both agents are added. The CREs bind recombinant CREB and endogenous CREB or CREB-like proteins contained in placental JEG-3 cells and also confer cAMP-inducible transcriptional activation to a heterologous minimal promoter. Our studies suggest that the expression of the CREB gene is positively autoregulated in trans.
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PMID:The promoter of the gene encoding 3',5'-cyclic adenosine monophosphate (cAMP) response element binding protein contains cAMP response elements: evidence for positive autoregulation of gene transcription. 838 Oct 74

To determine the cellular signaling pathways involved in granulosa cell luteinization, known activators of protein kinase-A (LH and FSH) and protein kinase-C [GnRH and phorbol 12-myristate 13-acetate (PMA)] as well as inhibitors of tyrosine kinases (AG18 and genistein) were tested in an in vitro system using specific markers of luteinization (cell hypertrophy, side-chain cleavage cytochrome P450, and progesterone) and ovulation [prostaglandin endoperoxide synthase-2 (PGS-2)]. When preovulatory follicles were incubated in the presence of an ovulatory (500 ng/ml) dose of LH or high GnRH (1 microM), the granulosa cells harvested from these follicles assumed and maintained a stable luteal cell phenotype in vitro. Granulosa cells harvested from follicles incubated in subovulatory doses of LH (5 and 50 ng/ml), lower doses of GnRH (5, 50, and 500 nM), or PMA alone were unable to form a stable luteal cell phenotype. When PMA was combined with subovulatory doses of LH, granulosa cells luteinized, and PGS-2 protein was induced. AG18 (or genistein) blocked agonist induction of luteinization and of PGS-2 mRNA and protein when present during the first 2 h (0-2 h) of follicle incubation, but failed to block these events if added for the last 2 h (5-7 h of incubation). Combined, these results provide evidence to support a primary role for cAMP and protein kinase-A, a supportive but essential role for protein kinase-C, and an obligatory role for tyrosine kinases acting at an early stage in the cascade of events required for luteinization and ovulation.
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PMID:Hormone induction of luteinization and prostaglandin endoperoxide synthase-2 involves multiple cellular signaling pathways. 839 74

cAMP regulation of gonadotropin secretion and subunit mRNA levels was studied in pituitary cells perifused with pulses of GnRH. Pituitary cells from 7-week-old male rats castrated at 5 weeks of age were stimulated hourly for 9-24 h with 1-min pulses of GnRH, the adenylate cyclase activator forskolin, the cell-permeable cAMP analog 8-bromo-cAMP (8Br-cAMP), or control medium. Cells were also treated with the nonsteroidal antiinflammatory drug flufenamic acid, which reduces pituitary cAMP levels. During perifusion, the effluent was collected in 10-min fractions for FSH and LH assay. At the completion of perifusion, total RNA was extracted, and gonadotropin subunit mRNA levels were quantitated by Northern analysis. Continuous administration of flufenamic acid gradually reduced the amplitude of GnRH-stimulated FSH and LH pulses to nadir values of 40 +/- 4.7% and 62 +/- 12% of the control value, respectively. Flufenamic acid decreased (P < 0.05) FSH beta and alpha-subunit mRNA levels and blocked the effect of GnRH to lengthen LH beta mRNA. Pulses of forskolin or 8Br-cAMP released LH and FSH, and continuous forskolin or 8Br-cAMP potentiated the gonadotropin stimulatory effect of GnRH. Forskolin or 8Br-cAMP increased (P < 0.05) FSH beta mRNA and alpha-subunit mRNA levels when administered in pulses, but not when administered continuously, and lengthened LH beta mRNA. The Nal-Glu GnRH antagonist blocked the effects of GnRH pulses, but not the effects of 8Br-cAMP or forskolin. In conclusion, lowering intracellular cAMP levels with flufenamic acid attenuated GnRH-stimulated gonadotropin secretion, decreased alpha-subunit and FSH beta mRNA levels, and blocked the effect of GnRH to lengthen LH beta mRNA, whereas 8Br-cAMP or forskolin produced the opposite effect. These data extend previous results which suggested that cAMP modulates gonadotropin secretion and indicate that the cAMP/A-kinase pathway regulates each of the gonadotropin subunit mRNAs.
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PMID:Evidence for a role for the cyclic adenosine 3',5'-monophosphate/protein kinase-A pathway in regulation of the gonadotropin subunit messenger ribonucleic acids. 840 51

Follistatin is a 35-kilodalton monomer isolated from follicular fluid by virtue of its ability to suppress FSH secretion from cultured pituitary cells. Experiments were designed to test the hypothesis that the accumulation of follistatin RNA in the ovary is regulated by epidermal growth factor (EGF) and activation of the protein kinase-C (PKC) pathway. Follistatin mRNA was quantitated by slot blot hybridization of total RNA from primary cultures of porcine granulosa cells treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA), an activator of PKC. PMA (0.1, 1.0, 10, and 100 nM) induced a dose-dependent increase in follistatin mRNA accumulation after 2 h, with a maximal increase of 40-fold over that in untreated control cultures at a dose of 10 nM. PMA (10 nM) induced a time-dependent increase in follistatin mRNA levels, with a maximal response at 2 h. Follistatin gene expression was induced by a 2-h incubation with EGF (3 nM), but not by LH (100 ng/ml), GnRH (10 nM) or prostaglandin F2 alpha (80 micrograms/ml). EGF (0.01, 0.1, 1, and 10 nM) induced a dose-dependent induction of follistatin gene expression in granulosa cells after 2-h incubation, with maximal stimulation of 33-fold at a dose of 1 nM. The time course of induction of follistatin mRNA by EGF was very similar to that induced by PMA, with maximal stimulation occurring at 2 h and declining thereafter. Pretreatment of granulosa cells for 24 h with PMA abrogated the EGF-induced stimulation of follistatin mRNA accumulation. However, cotreatment of granulosa cells with EGF and PMA for 2 h resulted in additive stimulation of follistatin mRNA. These results demonstrate that 1) follistatin gene expression in cultured porcine granulosa cells is acutely stimulated by PMA and EGF in a time- and dose-dependent manner; 2) follistatin gene expression may be regulated by the PKC pathway; and 3) the stimulatory effect of EGF on follistatin gene expression may require PKC.
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PMID:Regulation of follistatin messenger ribonucleic acid in porcine granulosa cells by epidermal growth factor and the protein kinase-C pathway. 846 62


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