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)

The release of gonadotropins is effected by GnRH and regulated by steroids. The classical mechanism of steroid hormone action, which implies the binding of hormone receptor complexes to regulatory elements of nuclear genes, is derived largely from the well-studied and familiar steroids such as progesterone, testosterone, and estradiol. Their effects on gonadotropin release generally have been examined following hours or days of exposure and therefore cannot account for the rapid effects of steroids on gonadotropin release. Moreover, tissues such as gonad, pituitary, and hypothalamus can produce a variety of hormonally active steroids in addition to these well-studied, traditional ones. The recently discovered allylic steroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP), is readily interconverted from/to progesterone and is found at appreciable levels in serum, gonads, pituitary, hypothalamus, and other tissues. 3 alpha HP has provided the "missing link" in the progesterone biosynthetic/ metabolic pathways, allowing cyclical 4-pregnene and 5 alpha-pregnane pathways to be described for steroidogenic tissues. Among the functions ascribed to 3 alpha HP is the ability to selectively and rapidly (within seconds or minutes) suppress GnRH-provoked FSH release. In vitro studies using pituitary gonadotropes in culture and in perifusion paradigms suggest that suppression of FSH release by 3 alpha HP occurs as a result of nongenomic mechanisms of action. These mechanisms are discussed and include interaction at the level of receptors in the gonadotrope membrane and the cell-signaling pathway involving protein kinase C, phospholipase C, or IP3-induced Ca2+ mobilization and Ca2+ channels. This may be the first evidence of a gonadal steroid regulating gonadotropin release by nongenomic mechanisms of action. In order to understand the critical role of steroids in the rapid regulation of secretory (and bence, circulating) levels of gonadotropins, other gonadal steroids will need to be examined for their nongenomic action on gonadotropes.
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PMID:Nongenomic actions of steroids on gonadotropin release. 923 48

In teleosts, ovarian steroidogenesis is under the control of two gonadotropic hormones, GTH I and GTH II, that are structurally and functionally homologous to FSH and LH. The intracellular mechanisms by which GTH I and GTH II stimulate steroidogenesis in the teleost ovary are not well understood. The purpose of this study was to investigate the involvement of the cAMP/protein kinase A (PKA) and protein kinase C (PKC)/Ca2+ signaling pathways in the steroidogenic actions of GTH I and GTH II in the ovary of the brook trout (Salvelinus fontinalis). The cAMP/PKA pathway mediated the actions of GTH I before germinal vesicle breakdown (preGVBD) and GTH II after GVBD and before ovulation (preOV). Experimental increases in intracellular cAMP concentration mimicked the steroidogenic effects of GTH I and GTH II, and an antagonistic analog of cAMP partially blocked them. In addition, GTH I and GTH II stimulated the production of cAMP in preGVBD and preOV follicles, respectively. Activation of the PKC/Ca2+ pathway by a phorbol ester or a Ca2+ ionophore blocked the GTH I- and GTH II-induced steroid production, whereas inhibition of PKC by specific inhibitors potentiated the effects of GTH I. These results suggest that the cAMP/PKA signaling pathway mediates the stimulatory effects of GTH I and GTH II on steroidogenesis, and they also suggest the additional involvement of the PKC/Ca2+ signaling pathway in modulating the actions of gonadotropins in brook trout ovarian follicles.
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PMID:Stimulation of brook trout ovarian steroidogenesis by gonadotropins I and II is mediated by the cyclic adenosine 3',5'-monophosphate/protein kinase A pathway. 928 3

Ligand- and second messenger-regulated expression of the gene for steroidogenic acute regulatory protein (StAR) was evaluated in luteinized porcine granulosa cells. For comparison, cytochrome P450 side-chain cleavage (P450scc) was examined. Northern hybridization with homologous cDNA probes demonstrated three StAR mRNA species, of 2.7, 1.6, and 0.8 kilobases (kb), with the smallest variably present, and a single P450scc band at 1.9 kb. FSH elevated both STAR and P450scc messages in a dose-dependent manner over 6 h and continually stimulated both over 24 h (p < 0.001). STAR message induction depended on transcription, as did that of P450scc. Over 6 h, actinomycin D eliminated constitutive StAR message and reduced that of P450scc by two thirds, indicating briefer persistence of StAR. Pretreatment with cycloheximide prevented FSH induction of StAR and P450scc mRNA, implicating intermediate protein synthesis in expression of both genes. Dibutyryl cAMP caused time-dependent increases in StAR and P450 mRNAs over 24 h (p < 0.001), indicating the importance of the protein kinase A (PKA) pathway in their gene expression. Activation of the protein kinase C (PKC) pathway by a phorbol ester eliminated FSH induction of STAR mRNA increases (p < 0.01) while only reducing P450scc induction (p < 0.05). Thus, StAR gene expression, as reflected in mRNA abundance, is regulated by FSH via the PKA pathway and is dependent on transcription and translation. Conversely, the PKC pathway inhibits induction of these important steroid synthetic genes in luteinized granulosa cells.
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PMID:Follicle-stimulating hormone and intracellular second messengers regulate steroidogenic acute regulatory protein messenger ribonucleic acid in luteinized porcine granulosa cells. 928 5

Granulosa cells have been used to study the regulation of LH/hCG receptor protein and mRNA expression. Phorbol 12-myristate 13-acetate (PMA) dose-dependently attenuates the increases in LH/hCG receptor mRNA and protein induced by FSH and forskolin (FSK). The presence of PMA caused a decrease in cAMP production stimulated by FSH and FSK. These results suggest that PMA-mediated decreases in cAMP are a major factor in PMA-mediated decreases in LH/hCG receptor mRNA. On the other hand, in the presence of 8-Br-cAMP, PMA significantly increased LH/hCG receptor mRNA and protein, with maximal stimulation between PMA concentrations of 3 to 30 nM (1.5 fold) with 8-Br-cAMP. These findings suggest that activation of protein kinase C by PMA attenuates the increase in cAMP accumulation induced by FSH but enhances the effect of cAMP on LH/hCG receptor expression, and that the inhibitory and stimulatory effects of PMA on LH/hCG receptor content are correlated with regulation of LH/hCG receptor mRNA levels. Since the half-life study revealed no change in the stability of the LH/hCG receptor mRNA following PMA treatment, a change in the rate of LH/hCG receptor gene transcription must be responsible for the change in the LH/hCG receptor mRNA levels.
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PMID:Effect of phorbol ester on the regulation of LH/hCG receptors. 933 33

The development of ovarian follicles and subsequent corpus luteum formation is accompanied by very active angiogenesis. Ovarian granulosa cells produce vascular endothelial growth factor (VEGF), which is a potent endothelial cell mitogen and an angiogenic agent. The complementary DNAs of two other factors structurally related to VEGF, namely VEGF-B and VEGF-C, were recently cloned, but little is known of their regulation in the ovary. We first studied the expression of the messenger RNAs (mRNAs) of the three VEGF isotypes in freshly isolated human granulosa-luteal (GL) cells obtained at oocyte retrieval for in vitro fertilization. The hormonal regulation of these mRNAs was subsequently studied in primary cultures of human GL cells. Analysis of cultured GL cell RNA by reverse transcription-PCR revealed that these cells express the alternatively spliced transcripts representing 121-, 145-, and 165-amino acid VEGF isoforms. Northern blot hybridization analyses indicated that transcripts of 4.5 and 3.7 kilobases for VEGF, and 1.4 and 2.4 kilobases for VEGF-B and VEGF-C, respectively, are expressed in human GL cells. The basal VEGF mRNA levels declined steadily, whereas VEGF-B mRNA levels were rather invariant over a 10-day culture period of GL cells. In contrast, VEGF-C mRNA levels increased toward the end of culture. For studying the hormonal regulation of VEGF isotype mRNAs, GL cells were treated with hCG, recombinant human FSH, PGE2, as well as 8-bromo-cAMP and 12-O-tetradecanoylphorbol 13-acetate, which activate protein kinase A- and protein kinase C-dependent signaling pathways, respectively. All test agents stimulated the expression of VEGF mRNA levels in a concentration-dependent manner. Time-course studies indicated that all treatments induced VEGF mRNA levels as early as incubation for 2 h, and the effect was sustained up to 48 h. VEGF-B mRNA levels were not regulated by any of the test agents. However, we found that hCG and 8-bromo-cAMP decreased VEGF-C mRNA levels with a maximal response observed at 24 and 48 h after cellular treatment. We conclude that the mRNAs of VEGF, VEGF-B, and VEGF-C are expressed in human GL cells and that their mRNA steady state levels are regulated in cultured human GL cells in an isotype-specific manner. The differential regulation of VEGF, VEGF-B, and VEGF-C in human GL cells suggests that distinct VEGF isotypes may play different roles during the vascularization of the human ovarian follicle and corpus luteum.
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PMID:Differential hormonal regulation of vascular endothelial growth factors VEGF, VEGF-B, and VEGF-C messenger ribonucleic acid levels in cultured human granulosa-luteal cells. 934 2

gamma-Glutamyl transpeptidase (gamma-GTP) activity in Sertoli cells can be stimulated by FSH. This cAMP-dependent metabolic event can be enhanced when Sertoli cells are co-cultured with germ cells, suggesting that different signal transduction pathways may be involved in the regulation of gamma-GTP activity. In this study we examined the participation of Ca(2+)- and protein kinase C (pkC)-dependent signal transduction pathways in the regulation of basal and FSH-stimulated gamma-GTP activity. Under basal conditions, the increase in extracellular Ca2+ concentration or the addition of the Ca2+ ionophore 4Br-A23187 produced a decrease in gamma-GTP activity. Conversely, blockage of voltage-dependent Ca2+ channels with verapamil or nifedipine or inhibition of Ca(2+)-calmodulin dependent processes with trifluoperazine resulted in an increase in gamma-GTP activity. To study the role of a pkC-dependent pathway the effects of low doses of staurosporine were evaluated. Under these experimental conditions an increase in gamma-GTP activity was observed. It was then investigated whether these signal transduction pathways could interact with the FSH-stimulated cAMP-dependent pathway to regulate gamma-GTP activity. Increase in extracellular Ca2+ concentration, the addition of 4Br-A23187 or the blockage of voltage-dependent Ca2+ channels did not modify FSH-stimulated gamma-GTP activity. However, staurosporine produced an additional increase in FSH-stimulated gamma-GTP activity and this effect was also observed when cells were stimulated with dbcAMP. In summary, our data are consistent with an inhibitory role of Ca(2+)-calmodulin- and pkC-dependent pathways in the regulation of basal gamma-GTP activity. Similar to what has been shown for other Sertoli cell parameters, a pkC-dependent pathway can interact with the FSH-stimulated cAMP-dependent pathway. The precise steps involved in this interaction are still unknown.
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PMID:Regulation of gamma-glutamyl transpeptidase activity by Ca(2+)- and protein kinase C-dependent pathways in Sertoli cells. 940 20

Our studies using immature rat granulosa cells cultured in serum-free medium on collagen-coated dishes indicated that FSH receptor mRNA levels do not change for at least 4 days of culture in the absence of hormone treatment. Addition of FSH (30 ng ml[-1]) led to a reduction of FSH receptor mRNA for a short time (6 h), followed by an increase in FSH receptor mRNA levels that reached maximum of around 200% of the initial level within 2-3 days after the addition of FSH. Following the addition of 10 nM PMA, FSH receptor mRNA levels were decreased to 50% of the pretreatment levels. During prolonged exposure to PMA, gradual recovery of the FSH receptor mRNA level was observed, and it was significantly higher than the control level at 48 h. The inactive phorbol ester 4 alpha-phorbol-12,13-didecanoate did not depress FSH receptor mRNA levels. Downregulation of the FSH receptor mRNA was detectable at a PMA concentration of 1 nM. The two predominant FSH receptor mRNA transcripts, ca. 5.5 and 2.4 kb, respectively, appeared to be equally affected by SH and PMA treatments. To examine the role of PKC mediation of the effect of FSH on FSH receptor mRNA levels, granulosa cells were treated with the PKC inhibitor, H-7, and FSH. Although, FSH receptor mRNA levels decreased to 50% of control in the cells treated with FSH alone, the addition of H-7 (0.1 nM) caused no decline in FSH receptor mRNA levels relative to the control in the cells treated with FSH. On the other hand, inhibition of FSH receptor mRNA by FSH was partially suppressed by the PKC-selective inhibitor bisindolylmaleimide. The mRNA turnover experiments showed that the half-life of FSH receptor transcripts was unaffected by PMA exposure.
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PMID:Follicle-stimulating hormone regulation on its receptor messenger ribonucleic acid levels in cultured rat granulosa cells. 940 13

The regulation of LH and FSH subunit gene expression is under the control of GnRH. Physiological changes in the frequency of pulsatile GnRH release from the hypothalamus result in differential stimulation of alpha-, LHbeta-, and FSHbeta-gene expression. Previous studies indicate that the GnRH receptor couples to G proteins of the G(q/11) family, with phosphoinositide turnover and its resultant increase in intracellular calcium concentration and protein kinase C (PKC) activation, to stimulate secretion of LH and FSH. However, the molecular mechanisms by which GnRH mediates its transcriptional effects remain largely unknown. We used GH3 cells, constitutively expressing the rat GnRH receptor (GGH(3)-1' cells) and transiently transfected with a luciferase reporter gene controlled by either the alpha, LHbeta, or FSHbeta gene regulatory region (alphaLUC, LHbetaLUC, and FSHbetaLUC, respectively), to examine the roles of several signal transduction pathways in the GnRH-mediated stimulation of gonadotropin subunit gene expression. Activation of PKC by phorbol, 12-myristate, 13-acetate resulted in an increase in the luciferase activity of all three gonadotropin subunit gene reporter constructs. Phorbol, 12-myristate, 13-acetate had a greater stimulatory effect, relative to the maximal stimulation with GnRH, for the beta-subunit genes than for the alpha-subunit gene. Depletion of PKC, or inhibition of PKC by GF109203X, demonstrated that PKC-dependent pathways play a larger role in the GnRH-mediated transcriptional control of the LHbeta- and FSHbeta-genes than the alpha-subunit gene. In contrast, an L-type calcium channel agonist, Bay K 8644, was able to stimulate alphaLUC but not LHbetaLUC or FSHbetaLUC. Nimodipine, an L-type calcium channel antagonist, had a larger inhibitory effect on the GnRH response of alphaLUC, relative to LHbetaLUC or FSHbetaLUC. We conclude from these results that the differential regulation of gonadotropin subunit gene expression by GnRH is caused, in part, by differential use of signal transduction pathways, activated upon GnRH binding.
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PMID:Differential use of signal transduction pathways in the gonadotropin-releasing hormone-mediated regulation of gonadotropin subunit gene expression. 952 69

FSH is an alpha/beta heterodimeric glycoprotein, the formation of which is regulated primarily by expression of its beta-subunit. Recent studies on transcriptional regulation of the ovine FSH beta-subunit gene (oFSHbeta) have defined two functional activating protein-1 (AP-1) enhancers in the proximal promoter (located at -120 and -83 bp) that are probably physiologically important for FSHbeta expression. As GnRH is a major regulator of FSHbeta expression and is also known to stimulate the synthesis of Jun and Fos family members (AP-1), we investigated the possibility that oFSHbeta transcription may be regulated by GnRH through AP-1. Here we report the use of an in vitro cell system involving transient transfection of GnRH receptors (GnRHR) into HeLa cells to define regulatory elements involved in GnRH-mediated induction of oFSHbeta. This system was used to show that expression of luciferase constructs containing either the -4741/+759 region of the oFSHbeta gene (-4741oFSHbeta-Luc) or the -846/+44 region of the human alpha gene (alpha-Luc; a positive control) was stimulated 3.1 +/- 0.3- and 7.7 +/- 1.9-fold, respectively, by 100 nM GnRH. Another luciferase expression plasmid containing the Rous sarcoma virus promoter (a negative control) showed no response to GnRH. Similar results with these constructs were obtained in COS-7 cells. Studies with progressive 5'-deletion constructs and site-specific mutations demonstrated that this stimulation was dependent on each AP-1 site in the proximal promoter of oFSHbeta. Gel shift assays demonstrated the ability of GnRHR in HeLa cells to increase AP-1 binding activity. Responses in the HeLa cell system were dependent on GnRH (ED50 = 0.5 nM) and GnRHR, which was identified by photoaffinity labeling. In addition, GnRHR-expressing HeLa cells exhibited a normal GnRH-dependent mobilization of intracellular calcium. Finally, as protein kinase C (PKC) is a known target of GnRH action in gonadotropes, the role of PKC in transcriptional regulation of oFSHbeta and alpha-subunit genes by GnRH in HeLa cells was investigated. Although 12-O-tetradecanoyl 13-acetate induction of alpha-Luc and -215oFSHbeta-Luc could be completely blocked in a dose-dependent manner by the specific PKC inhibitor bisindolylmaleimide I, only 57-65% of the GnRH-mediated stimulation of these promoters was blocked, demonstrating the involvement of PKC as well as other signaling systems in GnRH induction. These data define a molecular action of GnRH on oFSHbeta gene transcription that involves two proximal AP-1 enhancer elements and PKC activation. Furthermore, these studies establish the usefulness of HeLa and COS-7 cells to investigate specific aspects of GnRH action on gonadotropin subunit gene expression, as similar signaling pathways and transcription factors that are activated by GnRH in gonadotropes (such as PKC, mitogen-activated protein kinase, Ca2+, and AP-1) exist in these cells.
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PMID:Transcriptional activation of the ovine follicle-stimulating hormone beta-subunit gene by gonadotropin-releasing hormone: involvement of two activating protein-1-binding sites and protein kinase C. 979 52

Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel neuropeptide with considerable homology to vasoactive intestinal peptide and GH-releasing hormone, exists in two biologically active forms, PACAP-38 and -27. The presence of PACAP in the ovary has been demonstrated, where it stimulates steroidogenesis and cAMP accumulation in cultured granulosa cells. In the present study, gonadotropin regulation of PACAP gene expression was examined in PMSG/human (h)CG-treated immature rat ovaries and cultured preovulatory follicles. Northern blot analysis of ovaries obtained from PMSG/hCG-treated immature animals revealed the transient induction of PACAP transcripts by hCG, reaching a maximum at 6 h. The major cell types expressing PACAP messenger RNA were granulosa cells of preovulatory follicles and some theca/interstitial cells. In preovulatory follicles cultured in serum-free medium, PACAP transcripts were transiently induced by LH and FSH, reaching a maximum 6-9 h after stimulation in granulosa cells but not in theca cells. Treatment with cycloheximide or alpha-amanitin abolished LH-induced PACAP transcripts, indicating that new protein synthesis and transcription are necessary. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, inhibited LH-induced PACAP messenger RNA, and forskolin mimicked the LH action, implying the role of adenylate cyclase activation. In contrast, treatment with chelerythrine, an inhibitor of protein kinase C, and 2-O-tetradecanol-phorbol-13-acetate had no effect. We further tested the role of PACAP in follicle apoptosis using apoptotic DNA fragmentation analysis. Treatment with PACAP-38 suppressed follicle apoptosis in a dose-dependent manner. Moreover, the LH suppression of follicle apoptosis was partially blocked by cotreatment with PACAP-38 antagonist, indicating mediation by endogenous PACAP-38. These results suggest that PACAP, transiently induced by the gonadotropin surge, could be a local regulator of a number of events and may act as a follicle survival factor during the periovulatory period.
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PMID:Gonadotropin stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP) messenger ribonucleic acid in the rat ovary and the role of PACAP as a follicle survival factor. 992 11


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