EC:2.7.11.13 - FACTA Search

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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 bovine FSH-suppressing protein (FSP) or follistatin on activin- and GnRH-stimulated FSH synthesis and secretion have been studied using cultured pituitary cells from adult male Sprague-Dawley rats. Exposure to FSP (0.001-10 nM) for 3 days dose-dependently suppressed basal FSH secretion (IC50 = 146 +/- 21 pM., mean +/- SE), cellular content (IC50 = 269 +/- 8 pM) and total FSH (IC50 = 181 +/- 25 pM), with no effect on LH. Activin (0.3 nM) increased FSH secretion 2.1-fold, cellular content 1.3-fold, and total FSH 1.9-fold during a 3-day incubation, but these increases were dose-dependently inhibited by concomitant treatment with 35-kDa bovine FSP (0.1-3 nM), with complete inhibition occurring at concentrations between 1 and 3 nM. The 31- and 39-kDa forms of bovine FSP also antagonized the actions of activin. GnRH (1 nM) increased FSH secretion 1.8-fold and total FSH 1.6-fold during a 3-day incubation, effects that were dose-dependently inhibited by concomitant treatment with 35-kDa bovine FSP. The highest tested concentration of FSP (3 nM) suppressed GnRH-stimulated FSH secretion and total FSH to 59 and 57%, respectively, of the levels found in untreated cultures. All three forms of bovine FSP produced a significant inhibition of FSH secretion and total FSH stimulated by GnRH. FSP also suppressed FSH secretion and total FSH in response to activators of protein kinase C including 100 nM phorbol 12-myristate 13-acetate (43 and 59%, respectively) and 100 nM mezerein (40 and 60%, respectively). Finally, treatment of cultured pituitary cells with 35-kDa FSP at 1 and 3 nM for 3 days resulted in 21 and 24% decreases in GnRH binding sites, respectively. It is concluded that (i) FSP inhibits not only the secretion but also the synthesis of FSH induced by activin and GnRH in long-term culture, and (ii) FSP may cause its inhibitory effects on GnRH by suppression of the protein kinase C system, and possibly by reduction of GnRH binding sites.
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PMID:Chronic inhibitory effect of follicle-stimulating hormone (FSH)-suppressing protein (FSP) or follistatin on activin- and gonadotropin-releasing hormone-stimulated FSH synthesis and secretion in cultured rat anterior pituitary cells. 211 27

Follicle-stimulating hormone (FSH)-suppressing protein (FSP) or follistatin, a novel gonadal glycoprotein hormone, has been shown to have chronic inhibitory effects on the secretion of both FSH and luteinizing hormone (LH) in response to gonadotropin-releasing hormone (GnRH) in vitro. The present study was designed to investigate the acute effects of bovine FSP on GnRH-stimulated gonadotropin secretion and to examine the potential subcellular sites of this action of FSP using cultured pituitary cells. Anterior pituitaries from adult male Sprague-Dawley rats were enzymatically dispersed and cultured for 48 h, after which the cells were treated with bovine FSP for 6 h, followed by a 4 h stimulation with secretagogues in the continued presence of FSP. Results showed that the 35 kDa form of bovine FSP (0.1-3 nM) dose-dependently suppressed GnRH-stimulated FSH and LH secretion, with inhibition of 38 and 25%, respectively, at 3 nM. In addition, FSP suppressed gonadotropin secretion in response to activators of protein kinase C (phorbol 12-myristate 13-acetate (PMA) and mezerein) and a calcium ionophore (A23187). However, FSP had no effect on gonadotropin secretion evoked by melittin, an activator of phospholipase A2. Furthermore, 35 kDa bovine FSP did not compete with GnRH for GnRH binding sites in a direct competition study and treatment of cultured pituitary cells with FSP (0.1-3 nM) for 10 h did not alter the number of GnRH binding sites on the cell membranes. Finally, similar inhibitory effects on gonadotropin secretion in response to GnRH, PMA and mezerein were obtained with the 31 and 39 kDa forms of bovine FSP, each at a concentration of 1 nM. We conclude from the present study that FSP acutely inhibits GnRH-stimulated gonadotropin secretion in cultured pituitary cells, and that FSP exerts its action beyond the GnRH receptor, possibly by affecting the protein kinase C and/or the calcium-calmodulin systems.
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PMID:Acute inhibitory effect of follicle-stimulating hormone-suppressing protein (FSP) on gonadotropin-releasing hormone-stimulated gonadotropin secretion in cultured rat anterior pituitary cells. 212 65

The actions of FSH on ovarian follicular development and steroidogenesis are mediated through its binding to FSH receptors and required in the differentiation and maturation of ovarian follicles. Therefore it is clear that the ability of the gonadotropins to modulate ovarian function depends not only on the circulating levels of the gonadotropins, but also on the expression of appropriate receptor proteins by potential target cells in the ovary. We have observed a rapid and tentative down-regulation of FSH receptor mRNA after FSH treatment first time in granulosa cell culture. Using this culture cells, we investigated the mechanism of hormonal regulation of FSH receptor mRNA. The fact that both FSH and PMA show tentative down-regulation of FSH receptor mRNA suggests that these agents could be acting, at least in part, through a common mechanism, namely the activation of protein kinase C. Indeed, a role of the C kinase in cellular responses to hCG and TSH is consistent with findings that exposure to hCG or TSH stimulate the accumulation of inositol phosphates in nongonadal cells transfected with LH or TSH receptor. Activin, a stimulator or FSH secretion from the cultured pituitary cells, has been isolated from the mammalian follicular fluids and found to be a dimer of beta-subunits of inhibin. Activin has been shown to induce FSH receptor expression and FSH receptor mRNA on cultured rat granulosa cells. Since activin alone did not increase intracellular cAMP accumulation, to further clarify the role of activin and cAMP in the induction of FSH receptors, we examined the interactive effect of activin with cAMP on the induction of FSH receptors on cultured rat granulosa cells by 125I-FSH binding analysis. We have shown activin can up-regulate the expression of FSH receptor mRNA and protein by increasing both stability of mRNA and transcription rate. Using the cDNA clones of activin receptor type I and II, we have shown that both receptors are expressed in this cultured cells. This fact indicates that activin acts through autocrine/paracrine mechanism. On the other hands, follistatin can decrease the number of FSH binding sites elevated by addition of activin. Therefore, we examined the effects of endocrine manipulations (cAMP and PMA), FSH and activin, that are known to modulate granulosa cell function, on the follistatin productions. According to our data, these factors stimulate the production of follistatin mRNA in this cell culture, particularly the effect of activin on the follistatin mRNA indicates that the existence of negative regulation of activin effect by activin itself.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:[The regulation of gonadotropin receptor by the nonsteroidal intraovarian regulatory molecules during ovarian folliculogenesis]. 759 84

The regulation of the follistatin mRNA by hormones and endocrine manipulations was examined in granulosa cell cultures. The follistatin mRNA accumulation was stimulated in a dose-dependent manner by follicle-stimulating hormone (FSH) with a maximal response twice as great as in control cultures at a dose of 100 ng/ml FSH. The time course of the FSH effect on follistatin mRNA had a biphasic effect in which FSH increased follistatin mRNA within 2 h, and subsequently reduced it to below the control level. 8-Br-8 brom-adenosine 3,5-cyclic monophosphate (cAMP) (2 mM) and phorbol 12-myristate 13-acetate (PMA) (10 nm) induced a time-dependent increase in follistatin mRNA levels, with the maximal response at 6 h and 2 h, respectively. Co-treatment of the granulosa cells with cAMP and PMA demonstrated that 0.2 mM of 8-Br-cAMP suppressed the follistatin mRNA of the control and the samples with a small amount of PMA in the granulosa cells. Follistatin expression is therefore regulated by protein kinase A and protein kinase C pathways in rat granulosa cells. A more dramatic stimulation of follistatin mRNA was observed when this culture was treated with activin, and follistatin also blocked the effect of activin on the follistatin mRNA.
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PMID:Regulation of follistatin messenger ribonucleic acid in cultured rat granulosa cells. 766 79

Follistatin is a 35-kilodalton monomer isolated from follicular fluid that acts on pituitary gonadotropes to suppress the production of FSH. Transfection of rat granulosa cells with specific oncogenes, such as simian virus 40 (SV40) DNA and Ha-ras oncogene, leads to their immortalization concomitant with preservation of their capacity for inducible steroidogenesis. Experiments were designed to investigate the regulation of follistatin messenger RNA (mRNA) accumulation upon stimulation with forskolin, 2-O-tetradecanol-phorbol-13-acetate (TPA), FSH, and hCG in four different granulosa cell lines. Granulosa cells were transfected with SV40 DNA alone (POGS5); with SV40 DNA and Ha-ras oncogene (POGRS1); with SV40 DNA, Ha-ras oncogene, and LH/CG receptor (GLHR15); or with FSH receptor (GFSHR17) expression plasmid. Cells were cultured to reach confluence and then stimulated for 6 or 24 h with ovine FSH (oFSH; 0.004-4 nM), hCG (9 nM), forskolin (50 microM), and TPA (50 nM), alone or in combination. In the POGS5 cell line, forskolin caused a 15-fold stimulation of follistatin mRNA after 24-h incubation. The POGRS1 cell line showed a time-dependent stimulation of follistatin gene expression induced by both forskolin (5.7-fold) and TPA (9.4-fold). In the GFSHR17 cells, forskolin, oFSH, and TPA induced an increase in follistatin mRNA. When oFSH (1.6 nM) was added to cells treated with forskolin (50 microM) or TPA (50 nM), no additional stimulation was observed. The GLHR15 cell line treated with hCG showed a 2.7-fold increase in follistatin mRNA accumulation within 6 h. Our data demonstrate that 1) follistatin mRNA is detectable and induced by forskolin and TPA in transformed granulosa cell lines that do not express the FSH or LH receptors; 2) in the GFSHR17 cell line, FSH, forskolin, and TPA caused a time- and dose-dependent regulation of the gene; and 3) follistatin gene expression is up-regulated by hCG in the GLHR15 cell line. We conclude that these transformed steroidogenic cell lines can serve as a useful model to study the regulation of follistatin gene expression, a peptide known to regulate pituitary and ovarian hormone secretion and differentiation of granulosa cells by its activin-binding action. Moreover, this gene can be regulated in immortalized granulosa cells by both the protein kinase A and protein kinase C pathways, although these cells express the large T antigen and the Ha-ras oncogenic proteins.
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PMID:Regulation of follistatin messenger ribonucleic acid in steroidogenic rat granulosa cell lines. 778 14

In this study the localization and regulation of steady-state follistatin messenger ribonucleic acid (mRNA) levels in testicular cell cultures were examined with a solution-hybridization assay using a specific 32P-labelled cytosolic RNA antisense probe for follistatin and a 35S-labelled cytosolic RNA antisense probe for cyclophilin as internal standard. Testes from immature rats were dispersed with collagenase and fractionated in Sertoli and Leydig cell-enriched cultures. Follistatin mRNA was mainly localized to the Sertoli cell-enriched fraction and the expression of follistatin mRNA could be stimulated in vitro with fetal calf serum, epidermal growth factor or phorbol-12-myristate-13-acetate (an activator of protein kinase C), whereas follicle-stimulating hormone and forskolin (an activator of protein kinase A) had no effect. Neither prostaglandin E2, the synthetic glucocorticoid RU 28362 or all-trans-retinoic acid, which all regulate follistatin mRNA levels in non-testicular cell types, nor extracellular adenosine triphosphate (a purinergic receptor agonist) or testosterone had any obvious influence on follistatin mRNA levels in Sertoli cell-enriched cultures. From this study it is concluded that Sertoli cells are likely to be the source of follistatin expression in the rat testis, that follistatin mRNA levels in Sertoli cell-enriched cultures are subjected to regulation by epidermal growth factor and the protein kinase C-dependent pathway but are not regulated by extracellular adenosine triphosphate, follicle-stimulating hormone, all-trans-retinoic acid, prostaglandin E2, forskolin, testosterone or the glucocorticoid RU 28362 and that the regulation of follistatin mRNA is sex- and tissue-specific.
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PMID:Expression of follistatin messenger ribonucleic acid in Sertoli cell-enriched cultures: regulation by epidermal growth factor and protein kinase C-dependent pathway but not by follicle-stimulating hormone and protein kinase A-dependent pathway. 810 86

The production of inhibin/activin by cultured rat anterior pituitary cells was evaluated using specific antisera to inhibin/activin alpha, beta A, and beta B subunit proteins (anti-alpha, anti-beta A, and anti-beta B). Cellular or secreted proteins recognized by the antisera were immunoprecipitated from metabolically labeled cells then analyzed by denaturing polyacrylamide gel electrophoresis. Immunoreactive inhibin/activin beta B proteins were visualized in both cell lysates and the media. Experiments with anti-beta B confirmed that activin-B (beta B beta B) is a local secretory product of cultured rat anterior pituitary cells. The secreted beta B-immunoreactive protein band had an apparent size of 24-25 kilodaltons (kDa) or 14-15 kDa, consistent with the size of unreduced beta B dimer or reduced monomer, respectively. Cell lysates contained two proteins that were specifically immunoprecipitated by anti-beta B. One of these had a mobility of greater than 95 kDa (unreduced) or 55-60 kDa (reduced), probably representing dimers or monomers of the beta B precursor, respectively. The second 14- to 15-kDa (reduced and unreduced) immunoreactive beta B protein band was verified to be the mature beta B monomer. Mature heterodimeric inhibin-B (alpha beta B) was not detected by either anti-alpha or anti-beta B. Multiple protein species, however, were observed to be specifically immunoprecipitated by incubation of cell lysates with anti-alpha. Mature beta A monomer was not detected in any of the samples. The regulation of cellular beta B production was monitored by evaluating its rate of synthesis in pulse-labeled cells. Treatment with either forskolin or 12-O-tetradecanoylphorbol acetate enhanced the rate of [35S]cysteine incorporation into the cellular 14- to 15-kDa beta B monomer, indicating that the activation of either protein kinase A or protein kinase C regulates its production. The rate of cellular beta B accumulation was also regulated by activin-A, inhibin-A, and follistatin; activin-A caused a 30% inhibition in contrast to the 70% stimulation by treatment with either inhibin-A or follistatin. Equimolar concentrations of activin-A and follistatin prevented the net effect produced by either factor alone. None of the immunoreactive alpha-forms was detectable under similar pulse-labeling conditions, and there was no apparent change in their level after labeling to equilibrium (up to 48 h). The observed changes in beta B accumulation may, therefore, reflect the regulated production of pituitary activin-B. Taken together, these results suggest that locally produced activin-B or gonadal activins exert an inhibitory tone on the production of pituitary activin-B and that this negative-feedback control is in turn modulated by inhibins and follistatins. The relative importance of pituitary and gonadal activins, inhibins, and follistatins in the proposed regulatory loop remains to be established.
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PMID:Characterization and the regulation of inhibin/activin subunit proteins of cultured rat anterior pituitary cells. 824 76

The activin-binding protein, follistatin (FS), was immunoprecipitated from metabolically labeled rat anterior pituitary cells or their media using a specific antiserum to purified porcine FS (anti-FS). Several immunoreactive proteins, including one that had a mobility in the range of 42-44 kilodaltons (kDa), were detected in the cell lysates. When immunoprecipitates of the culture medium were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, a broad 35- to 46-kDa or 39- to 53-kDa band was visualized under unreducing or reducing conditions, respectively. Upon deglycosylation by treatment with N-glycosidase-F, the secreted product migrated as a sharp protein band with an apparent size of 35 kDa. The identity or the relatedness of the immunoprecipitated proteins to FS was verified by the ability of the C-terminally truncated form of recombinant human FS (rhFS288) to compete for binding to anti-FS. When the cultured rat anterior pituitary cells were treated with either forskolin or 12-O-tetradecanoylphorbol acetate, the accumulation of FS in the culture medium was stimulated by approximately 2.5-fold. These observations suggest that the activation of either the protein kinase A or the protein kinase C signaling pathway has a stimulatory effect on anterior pituitary FS production. A more dramatic stimulation of FS secretion (up to 7-fold) was observed when the rat anterior pituitary cells were treated with activin-A. The concentration dependence for this effect was within the same range that has been reported for most of the actions of activin-A. Inhibin-A suppressed basal FS secretion and blocked its stimulation by activin-A. To determine if locally produced FS exerts an influence on the response of gonadotropes to activins, the effects of anti-FS on FSH secretion were monitored. The ability of this FS antiserum to immunoneutralize the activity of FS was initially confirmed; anti-FS attenuated the inhibitory action of exogenous follistatin on FSH secretion. Treatment of cells with the antiserum increased the apparent sensitivity of gonadotropes to submaximal concentrations of activin-A. Moreover, the presence of the antiserum lowered the concentration of activin-A that was required to produce the maximum amount of FSH secretion, without changing the magnitude of the response. These results suggested that locally produced FS interferes with the secretory response of gonadotropes to activins. Changes in locally secreted FS may, therefore, represent a mechanism by which the response of rat anterior pituitary cells to incoming stimuli are tightly regulated.
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PMID:Activin-A regulates follistatin secretion from cultured rat anterior pituitary cells. 824 77

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

Primary cultures of gonadal cells from chicken embryos were used to explore the paracrine role of inhibin and activin during gonadal development. Fetal testicular and ovarian cells secreted high amounts of immunoactive inhibin. FSH caused a dose-related increase of cAMP and immunoactive inhibin concentrations in testicular cell cultures. Postreceptor signalling through the protein kinase A (PKA) pathway was confirmed by additional experiments with 8-bromo-cAMP, 3-isobutyl-1-methyl-xanthine (MIX), prostaglandins, forskolin, and choleratoxin. The relative ability of these agonists to stimulate cAMP production did not always correlate with their ability to stimulate inhibin secretion. Experiments with phorbol 12-myristate 13-acetate suggested that the regulation of immunoactive inhibin secretion also involves the protein kinase C (PKC) pathway. In addition, it was shown that recombinant human (rh)-inhibin increases the conversion of pregnenolone to androgens whereas rh-activin has the opposite effect. Recombinant human follistatin, an activin-binding protein, antagonized the actions of rh-activin and to a lesser extent those of rh-inhibin. In conclusion, these results show that during the development of the chicken embryo, gonadal inhibin secretion may be regulated by hormones and by local factors such as prostaglandins. Cross talk between the PKA and PKC pathways may be involved in this regulation. Recombinant human inhibin and rh-activin may have antagonistic roles in the paracrine control of gonadal steroidogenesis.
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PMID:Inhibin and activin have antagonistic paracrine effects on gonadal steroidogenesis during the development of the chicken embryo. 872 49

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