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)

FSH promotes the differentiation of ovarian follicular granulosa cells via a cAMP-dependent mechanism. Based upon the presence of a prominent phospholipid/diolein/Ca2+-independent myelin basic protein kinase activity in soluble extracts of proliferating immature rat granulosa cells, we determine whether this activity was attributable to the mitogen-activated protein kinases (MAPKs), one of the ubiquitous families of myelin basic protein kinases, and whether FSH acutely regulated the MAPKs in rat granulosa cells. Granulosa cells were obtained from large preantral follicles in ovaries of immature rats treated with 17beta-estradiol to promote granulosa cell proliferation. Exposure of granulosa cells, cultured overnight in serum-free medium containing 10 nM 17beta- estradiol, to 50 ng/ml FSH for 10 min promoted a 2- to 5- fold increase in MAPK activity. The effects of FSH were mimicked by forskolin and inhibited by the inhibitor of cAMP- dependent protein kinase H89, but were not inhibited by the tyrosine kinase inhibitor Ag-18. FSH also promoted increased phosphorylation of the 90-kDa ribosomal S6 protein kinase and phosphorylation of exogenous S6 protein. These results suggest that the cAMP-directed pathway by which FSH initiates granulosa cell differentiation includes activation of MAPKs.
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PMID:A stimulatory role of cyclic adenosine 3',5'-monophosphate in follicle-stimulating hormone-activated mitogen-activated protein kinase signaling pathway in rat ovarian granulosa cells. 860 10

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

Previous studies have shown that inhibitors of protein tyrosine kinases, tyrphostins, can markedly attenuate the steady-state levels of mRNAs of hormone-induced genes expressed in ovarian cells. To further elucidate the mechanism of tyrphostin action, rat granulosa cells were electroporated with chimeric expression vectors containing the promoters of two key steroidogenic genes, cholesterol side chain cleavage cytochrome P450 (CYP11A; P450scc) and aromatase cytochrome P450 (CYP19; P450arom), ligated to the CAT reporter gene. The electroporation method of transfection documents that the respective promoter-reporter constructs, -379sccCAT and -534aromCAT, can confer greater than 10-fold FSH/cAMP responsiveness to the reporter genes expressed in naive granulosa cells. Furthermore, the electroporation approach allows transfection of DNA into small numbers of cells and facilitates the assay of expression in cells isolated from follicles at advanced stages of differentiation. In naive granulosa cells, the functional activities of -379sccCAT, -534aromCAT, and -169 alpha CGCAT were abolished by the A-kinase specific inhibitor, H89, supporting the notion that activation of protein kinase A is obligatory for transcriptional activation of the promoter regions within these genes. Similar inhibitory effects were also observed for tyrphostin AG18, thus implicating a tyrosine kinase in the regulation of the steroidogenic genes. As a result of eCG/hCG treatments, a gradual loss of transfection efficiency accompanied by decreasing forskolin induction of CAT expression was observed in the differentiating granulosa-lutein cells. Although the reason(s) for the apparent loss in the ability of hormones to regulate chimeric gene expression remains to be determined, cell and promoter refractoriness to hormone treatment appears to reflect a fundamental change in the mechanism of promoter activation in the differentiated cells compared to the naive granulosa cells.
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PMID:Effects of hormones and protein kinase inhibitors on expression of steroidogenic enzyme promoters in electroporated primary rat granulosa cells. 883 18

We have studied the changes in membrane potential induced by LH in cumulus and granulosa cells isolated from sheep antral follicles. The investigation was carried out by using a non-invasive technique based on the use of a membrane potential sensitive probe, bis-oxonol. The membrane potential of mural granulosa cells was totally unaffected by LH, while that of cumulus or corona cells showed a marked depolarisation, starting 2-3 min after the addition of the hormone and plateauing after 5-10 min. None of the cells tested reacted to FSH. In the second part of the experiment the role of protein kinase A (PKA) and protein kinase C (PKC) in mediating the effect of LH was studied. The selective activation of PKA or PKC induced in cumulus-corona cells a rapid hyperpolarisation due to increased Cl and K conductance respectively. By contrast, the simultaneous activation of the two kinases induced a rapid membrane depolarisation due to the progressive decrease in K conductance. The activation of each kinase or their combined stimulation did not induce any change in the membrane potential of mural granulosa cells. These data demonstrated that LH has a depolarising effect regionally circumscribed to cumulus-corona cells and that this depolarisation depends on a reduction of K conductance caused by the activation of PKA and PKC.
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PMID:Activation of protein kinase A and protein kinase C mediates the depolarising effect of LH in ovine cumulus-corona cells. 888 63

Gonadotropin and TSH receptors represent a subgroup of seven transmembrane-spanning, G protein-coupled receptors with a large extracellular ligand-binding region. After ligand binding to their receptors, the majority of actions of gonadotropins and TSH are believed to be mediated by the cAMP-protein kinase A pathway. Although formation of inositol phosphates (IP) has been reported after stimulation of rodent gonadotropin receptors, activation of phospholipase C after ligand binding of human LH or FSH receptors has not been investigated. Human gonadotropin receptors were transiently expressed in 293 cells, and the agonist-induced stimulation of IP formation was measured. The LH receptor responded to a saturating dose of human CG (hCG) with a 5.2-fold increase of IPs whereas the FSH receptor responded to a saturating dose of FSH with only a 50% increase. On the basis of these differences and in view of the homologous nature of the two gonadotropin receptors, chimeric receptors were constructed using domain transfer to identify the regions in the human LH receptor important for phosphatidylinositol hydrolysis. Chimeric receptors containing the entire extracellular region of the FSH receptor and the seven transmembrane region plus the cytoplasmic tail of the LH receptor responded to FSH treatment with a 4.7-fold increase in IP accumulation. In contrast, the chimeric receptor with the extracellular region of the LH receptor and the TM region plus the cytoplasmic tail of the FSH receptor responded minimally (50%) to hCG treatment. When the C-terminal third (from TM V to the cytoplasmic tail) of the FSH receptor was replaced with the LH receptor sequence, the chimeric receptor still responded to FSH treatment with a large (6.2-fold) increase in IP release, similar to that of the wild type LH receptor (to hCG), suggesting that C-terminal third of the human LH receptor confers IP signaling ability. This functional domain was further divided into two areas, namely TM V to TM VI and TM VII to the cytoplasmic tail. The chimeric receptors F(I-IV)L(V-VI)F(VII-C)R and F(I-VI)L-VII-C)R, in which these two regions of the FSH receptor were replaced by the corresponding sequences of the LH receptor, responded to FSH treatment with partial increases in phosphatidylinositol hydrolysis (2.0- and 3.7-fold, respectively). Furthermore, when TM VII and the cytoplasmic tail of the LH receptor were replaced with the corresponding sequence of the FSH receptor, this chimeric receptor showed a diminished (2.0-fold) response to hCG in IP release. For all the chimeric receptor constructs analyzed, overall expression, equilibrium binding constants, and adenyl cyclase activation were not altered. Thus, unlike studies using chimeric muscarinic and dopaminergic receptors in which the second and third intracellular loops were found to be important for IP signaling, the entire C-terminal third of the human LH receptor is important for IP release. Future analysis using the chimeric receptor approach should provide new information on the structure-function relationship of gonadotropin, TSH, and other seven transmembrane-spanning receptors.
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PMID:The C-terminal third of the human luteinizing hormone (LH) receptor is important for inositol phosphate release: analysis using chimeric human LH/follicle-stimulating hormone receptors. 888 47

Follistatin (FS) is the specific binding protein of activin; it has a broad tissue distribution and is also found in serum. The ovary has the highest level of FS expression, but ovariectomy does not cause a permanent reduction in the serum FS level. Therefore, the source of FS in serum is still elusive. As a regulatable, nongonadal source of serum FS could influence ovarian and pituitary-derived hormone secretion and thus reproductive function, we searched for a source of extragonadal FS expression that might contribute to the FS protein level in serum. We found that endothelial cells from blood vessels express FS messenger RNA (mRNA) and protein; therefore, we studied the regulation of steady state levels of FS mRNA in porcine endothelial cells from aorta (AEC) and brain microvessels (BMVEC) in tissue culture. For detection of FS mRNA, a specific 32P-radiolabeled antisense probe and a S1-nuclease protection assay were used. FS steady state levels of AEC decreased with time in culture, i.e. postconfluent AEC had lower FS mRNA levels than confluent cultures, which, in turn, had lower FS mRNA levels than subconfluent cell cultures. FS mRNA levels in AEC were induced by increasing concentrations of FCS and stimulated by 30 micrograms/ml endothelial cell growth supplement. FS mRNA levels in AEC and BMVEC increased approximately 20-fold within 4 h during incubation of the cells with 100 nM phorbol 12-myristate, 13-acetate, whereas 0.5 nmol/ml forskolin tested in AEC for between 4-48 h had no significant effect. Furthermore, 0.1 microM ocadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, caused a significant increase in FS mRNA levels. FS mRNA levels in AEC were not significantly affected by various concentrations of porcine FSH, epidermal growth factor, or retinoic acid for between 4-48 h. Treatment of the cells with 0.01-10 micrograms/ml bacterial lipopolysaccharides (LPS) caused a dose-dependent increase (up to 10-fold) in FS mRNA steady state level in AEC, whereas 1-1000 nM RU 28362, a synthetic glucocorticoid, inhibited FS mRNA steady state levels in a dose-dependent manner. The induction of FS mRNA with 1 microgram/ml LPS was completely blocked by 100 nM RU 28362, and the stimulatory effects of LPS were only visible after 4 h of treatment, not after 24 or 48 h. The same effects were observed with BMVEC. We, furthermore, analyzed FS protein secretion of AEC by Western blotting and demonstrated that FS proteins were secreted into the culture medium upon stimulation with LPS. None of these treatments had an obvious effect on the ratio of the two different forms of FS mRNA (FS 344:FS 317). Besides the expression of FS mRNA in AEC and BMVEC, FS mRNA is also expressed in uncultured plexus choroideus epithel and meninges, and FS protein is found in human cerebrospinal fluid. From this study it is concluded that 1) endothelial cells from different tissues produce FS mRNA; 2) the FS mRNA levels of AEC and BMVEC are subjected to regulation by FCS, endothelial cell growth supplement, bacterial LPS, and the glucocorticoid RU 28362; 3) phosphatases and the protein kinase C-dependent, but not the protein kinase A-dependent, pathway are involved in regulating the steady state levels of FS mRNA in AEC and BMVEC; and 4) endothelial cells produce and secrete FS protein and are thus a likely source of FS in serum.
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PMID:Production of follistatin in porcine endothelial cells: differential regulation by bacterial compounds and the synthetic glucocorticoid RU 28362. 889 65

To address a possible role of type 1 and 2A serine/threonine protein phosphatases (PP1 and PP2A) in regulating granulosa cell hormonal responses, we investigated the effects of okadaic acid (OA) on FSH- and cAMP-induced steroidogenesis in these cells. When added alone (0.01-1 nmol/l), the cell-permeant phosphatase inhibitor did not affect progesterone and 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase (3 beta-HSD) enzyme activity, whereas when added with FSH it dose-dependently augmented (minimal effective dose, 0.1 nmol/l) gonadotropin-stimulated steroidogenesis in cultured granulosa cells. A similar stimulatory effect of the toxin was observed in cells cultured for 48 h with the cell-permeant analogue dibutyryl cAMP (1 mmol/l), or when granulosa cells were stimulated with the cAMP-inducing agents cholera toxin (1 microgram/ml), forskolin (15 mumol/l) or 1-methyl-3-isobutyl-xanthine (0.1 mmol/l). The observed effect of OA on FSH-supported granulosa cell steroidogenesis was not a consequence of increased cAMP generation, and time course experiments also revealed that a minimal time period of 12 h was necessary for OA (0.1 and 1 nmol/l) to significantly enhance FSH-induced progesterone and 3 beta-HSD enzyme activity. Since OA also inhibits the dephosphorylation of protein kinase C (PKC) substrates, we also compared the effect of OA and the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) on FSH-induced granulosa cell steroidogenic activity. While activation of the PKC pathway with the tumor promoter TPA (10 nmol/l) inhibited progesterone and cAMP accumulation in FSH-stimulated granulosa cells, treatment with OA augmented steroidogenesis and did not affect gonadotropin-induced cAMP generation. Collectively these results suggest that PP1 and PP2A may be important in regulating the phosphorylation state of proteins implicated in the cAMP-protein kinase A-stimulated steroidogenic activity of these cells.
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PMID:Effect of the protein phosphatase inhibitor okadaic acid on FSH-induced granulosa cell steroidogenesis. 901 48

The aims of this study were to apply enzyme-linked immunosorbent assays (ELISA) for human follistatins (FS) to measure total immunoreactive (ir-) rat FS and free rat FS, and investigate the regulation of production of total ir-FS and free FS by rat granulosa cells (GC) in vitro. Production of ir-inhibin was monitored as an index of GC function. The ELISAs for total ir-FS, based on an immunoradiometric assay developed recently for human FS, and free FS, based on capture of FS by a monoclonal antibody and detection by activin A binding, had sensitivities of 0.4 and 0.8 ng recombinant human (rh-) FS 288/ml, respectively, and did not cross-react with inhibin A, rLH, or FSH. rh-Activin did not cross react in the total ir-FS ELISA, but interfered with the measurement of free FS. Dilutions of GC-conditioned medium were parallel to the standard curve of rh-FS 288 for each assay. The values obtained in the free FS assay were 10- to 20-fold higher than those in the total ir-FS ELISA, suggesting that rat FS may be recognized by the antibodies differently than the human standard. Both total ir-FS and free FS production by undifferentiated GC from diethylstilbestrol (DES)-treated, immature rats increased with cell number and time in culture and were stimulated dose dependently by FSH, rh-activin A (except free FS, which was not measured because of interference), forskolin, and phorbol 12-myristrate. The effects of FSH and activin on FS production by undifferentiated GC were additive. There were significant effects of degree of differentiation of GC on basal FS production and responsiveness to FSH, LH, and rh-activin A. Both total ir-FS and free basal FS production increased up to 4-fold with the degree of differentiation of GC, produced by treating rats in vivo with DES (undifferentiated), DES plus FSH (partially differentiated), or DES plus FSH plus hCG (fully differentiated). The addition of FSH in vitro increased FS production by undifferentiated and partially differentiated GC, but not by fully differentiated GC. The only detectable effect of LH on FS production was on partially differentiated GC. Activin A stimulated total ir-FS production by undifferentiated and partially differentiated GC, but inhibited total ir-FS production by fully differentiated GC. Ir-inhibin production in these experiments was similar to that of FS with the following exceptions; phorbol 12-myristrate inhibited ir-inhibin production by undifferentiated GC, basal ir-inhibin decreased in fully differentiated GC, FSH stimulated ir-inhibin only in undifferentiated GC, and rh-activin A stimulated ir-inhibin at all stages. It is concluded that 1) FS protein production by cultured undifferentiated rat GC is up-regulated by FSH and activin, possibly via both protein kinase A and C pathways; 2) increasing GC differentiation is associated with a significant increase in basal FS production by rat GC and a change in the hormonal regulation of FS production; and 3) FS and ir-inhibin production by cultured rat GC can be differentially regulated. The results are consistent with the hypothesis that activin tone decreases within follicles as they develop due to increased production of the activin-binding protein FS.
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PMID:Regulation of follistatin production by rat granulosa cells in vitro. 916 47

Thyroid disorders have been frequently associated with menstrual disturbances and impaired fertility. To characterize the nature of thyroid hormone action in the ovary, the direct effects of T3-gonadotropin interactions were investigated in vitro using a culture system of human luteinized granulosa cells in serum-free medium. Although FSH alone was devoid of any significant effect on cell proliferation, it inhibited T3-stimulated cell growth. The electrophoretic profiles of the radiolabeled proteins induced by the different hormonal treatments revealed similarity in overall protein patterns but differences in intensity of labeling. Human CG, alone or combined with T3, had no major influence on the total intensity of labeling compared with control, whereas T3 or FSH alone reduced total labeling intensity but a 30,000 Da protein band was increased. FSH combined with T3 augmented the total intensity of labeling, including the 30,000-Da protein band. Western blot analysis revealed the presence of the tissue inhibitor of metalloproteinases-1 (TIMP-1), mol wt 30,000, known to play a key role in ovarian function. TIMP-1 was dose dependently stimulated by T3 and FSH, and an additive effect was obtained when both hormones were combined. This is the first report of TIMP-1 modulation by FSH in ovarian cells and of an effect by thyroid hormone on TIMP-1 levels. The study shows TIMP-1 induction in human ovarian cells not only by FSH, i.e. via a probable protein kinase A mechanism, but also demonstrates an additional mode of TIMP-1 hormonal induction: via thyroid hormone stimulation, acting by modulation of gene transcription. The present study provides novel data on TIMP-1 hormonal modulation and of direct T3 in vitro ovarian effects that may account for the in vivo indications of a thyroid-ovarian connection.
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PMID:Triiodothyronine and follicle-stimulating hormone, alone and additively together, stimulate production of the tissue inhibitor of metalloproteinases-1 in cultured human luteinized granulosa cells. 917 98

It is reported that oxytocin (OT) receptors in bovine granulosa cells decrease in concentration during follicular development. However, the factor or factors that regulate OT receptors are not known. In the present study, we evaluated hormonal control of OT receptors in bovine granulosa cells obtained from small antral follicles (3-5 mm in diameter). Granulosa cells were cultured for 48 h and exposed to FSH, LH, progesterone, and/or estradiol-17beta (estradiol) in the final 15 h of culture. The relative binding of OT decreased to 63% of the control value following treatment with FSH (100 ng/ml). The inhibitory effect of FSH was mimicked by an adenylate cyclase activator, forskolin. In contrast, estradiol (10(-7) M) increased the number of OT receptors by 77% compared with that in untreated controls, without changing binding affinity. The effects of estradiol were dose dependent and were diminished by an estradiol antagonist, tamoxifen (10(-6) to 10(-5) M). Although tamoxifen (10(-5) M) alone did not change OT binding, the stimulatory effects of 10(-9) M and 10(-8) M estradiol were inhibited by treatment with tamoxifen (10(-5) M). Furthermore, when the granulosa cells were exposed to FSH (10 ng/ml) and estradiol (10(-10) to 10(-7) M) in various combinations, estradiol inhibited the reduction of OT receptors by FSH. On the other hand, LH and progesterone did not affect OT binding in the cultured granulosa cells. Additionally, OT secretion from cultured granulosa cells was not changed by any treatment used in the present study. These findings suggest that both FSH and estradiol are significant regulators of OT receptors in granulosa cells during follicular development. FSH might down-regulate OT receptors in this phase, and the inhibitory effects of FSH are mediated by the adenylate cyclase-cAMP-protein kinase A system. Furthermore, estradiol seems to play a role in neutralizing the effects of FSH.
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PMID:Regulation of oxytocin receptors in bovine granulosa cells. 928 92


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