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

---

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

Granulosa cells play an essential role in follicular development and formation of corpora lutea. Many functions of granulosa-lutein cells are controlled by activation of G protein-coupled receptors and the formation of cyclic AMP (cAMP) by adenylyl cyclase. There are at least nine mammalian adenylyl cyclase isoenzymes, which show different sensitivities towards other signalling systems. The aim of this study was to identify the types of adenylyl cyclase present in human granulosa cells and to investigate its functional regulation by G proteins, calcium and the protein kinase C and A pathways. Granulosa cells were obtained from women undergoing IVF. The cells were maintained in primary culture and they consistently expressed mRNA coding for adenylyl cyclase I, III, VI, VII and IX. The signals for adenylyl cyclase V and VIII were more variable among patients and there was no signal for adenylyl cyclase II. The expression of multiple adenylyl cyclase proteins was confirmed by immunochemistry with subtype-specific antibodies. The formation of cAMP in cultured cells was stimulated many times by hCG (EC(50) value 4.2 iu ml(-1)) and by prostaglandin E(2) (PGE(2); EC(50) = 0.75 micromol l(-1)) in a concentration-dependent manner, thus confirming the presence of receptors coupled positively to G(s). The diterpene forskolin, which stimulates all isoforms of adenylyl cyclase except for adenylyl cyclase IX, increased cAMP formation to higher levels than hCG or PGE(2). The strong stimulation by forskolin indicates that adenylyl cyclase IX is unlikely to be the major source of cyclase activity in these cells. Basal and forskolin- or PGE(2)-stimulated adenylyl cyclase activity was amplified 1.5-2.0 times by phorbol-12,13-dibutyrate, indicating that protein kinase C-sensitive enzymes (for example, adenylyl cyclase types IV, V, VI or VII) may be active in the cells. In contrast, hCG-stimulated activity was inhibited (76 +/- 6%) by phorbol ester. Stimulation of G(i) with the alpha-adrenoceptor agonist clonidine inhibited hCG-induced cyclase activity. This finding indicates that adenylyl cyclase II and IV subtypes, which are stimulated by betagamma subunits released from G(i), are not predominant. Increases in intracellular free calcium concentrations by the ionophore A23187, the calcium-ATPase inhibitor thapsigargin or by fluprostenol, a selective prostanoid FP receptor agonist, which is known to open calcium channels in granulosa cells, or removal of calcium by EGTA, had no significant effects on basal or forskolin-stimulated formation of cAMP. These results indicate that subtypes adenylyl cyclase I, III and VIII, which are activated by calcium, and adenylyl cyclase V and VI, which are inhibited by calcium, are not dominant isoforms in granulosa-lutein cells. The protein kinase A inhibitor H89 had no effects on formation of cAMP; this finding rules out the involvement of adenylyl cyclase V and VI subtypes, which are subjected to negative feedback by protein kinase A. These results indicate that adenylyl cyclase VII is the dominant functional isoenzyme in human granulosa-lutein cells.
...
PMID:Characterization of adenylyl cyclases in cultured human granulosa cells. 1122 46

During the human menstrual cycle, serum inhibin concentrations fluctuate in a cyclic fashion. To examine the regulation of inhibin/activin beta(B) subunit gene expression in ovarian granulosa-luteal cells, the levels of beta(B) subunit mRNA were determined in primary cultures of human granulosa-luteal cells treated with gonadotrophins and protein kinase modulators. Granulosa cells were obtained from women undergoing an IVF programme. The cells were enzymatically dispersed, separated from red blood cells, and maintained in culture for 5--10 days before addition of different agents. Northern blot analysis with specific oligonucleotide probes was performed to study inhibin/activin beta(B) subunit mRNA levels. Both LH and FSH reduced the accumulation of beta(B) subunit mRNA in a dose-dependent manner. The protein kinase A activator, (Bu)(2)cAMP, and the protein kinase inhibitor staurosporine also inhibited beta(B) subunit mRNA expression dose-dependently. Activin A increased dose-dependently beta(B) subunit mRNA expression. Our study suggests that activin-induced and gonadotrophin-inhibited beta(B) subunit expression in granulosa cells might be key factors in the transition from inhibin B to inhibin A dominance during the menstrual cycle.
...
PMID:Gonadotrophins inhibit and activin induces expression of inhibin/activin beta(B) subunit mRNA in cultured human granulosa-luteal cells. 1127 93

Insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) have been shown to be involved in ovarian follicular growth/development and steroidogenesis. Recently, a number of low-affinity IGFBP-related proteins (IGFBP-rP) have been characterized. In this study, we investigated the expression of the gene for IGFBP-rP2 (also known as connective tissue growth factor, CTGF) in human granulosa cells in vitro and in vivo. Northern blot analysis demonstrated that IGFBP-rP2 mRNA is expressed in cultured human granulosa-luteal cells obtained from women undergoing an IVF programme. Accumulation of IGFBP-rP2 mRNA was dose-dependently down-regulated by FSH and LH after 24 h treatment (both P < 0.05) in cultured granulosa-luteal cells. The inhibitory effects of gonadotrophins were mimicked by treatment with the protein kinase A activator, (Bu)(2)cAMP. Protein kinase C inhibitor staurosporine reduced, whereas protein kinase C activator TPA (12-O-tetradecanoyl phorbol 13-acetate) increased, IGFBP-rP2 mRNA accumulation. These results suggest that the inhibitory effects of gonadotrophins on IGFBP-rP2 gene expression may involve signal transduction via both protein kinase A and C pathways. Immunohistochemical analysis revealed positive staining for IGFBP-rP2 in the granulosa and theca cells of normal human ovarian follicles. Corpus luteum and ovarian surface epithelial cells were also positively stained. Modulation of IGFBP-rP2 expression by gonadotrophic hormones may have a role in ovarian follicular development and in the ovulatory process.
...
PMID:Gonadotrophins inhibit the expression of insulin-like growth factor binding protein-related protein-2 mRNA in cultured human granulosa-luteal cells. 1181 16

Activin and its binding protein follistatin may act as local regulators of cell growth and steroidogenesis in the human ovary. The recently identified follistatin-related gene (FLRG) is expressed abundantly in the human ovary, has high affinity for activin, and is able to inhibit activin-induced transcriptional responses. However, little is known about the regulation of FLRG expression in specific cell types in the ovary, while it is known that gonadotrophins induce follistatin gene expression in human granulosa-luteal cells. In this study, we investigated the expression of FLRG mRNA in granulosa-luteal cells of preovulatory follicles obtained from women undergoing IVF. FLRG mRNA was detected by RT-PCR in fresh and cultured granulosa-luteal cells, as well as in normal ovarian stroma, theca and granulosa cells. Northern blot analysis revealed a 2.5 kb transcript of the FLRG in cultured granulosa-luteal cells. The protein kinase C activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA, 160 nmol/l), and prostaglandin E(2) (PGE(2), 1 micromol/l) increased FLRG mRNA accumulation up to 3-8 fold over the control level after 24 h of treatment, and these stimulatory effects were dose-dependent. Co-treatment with the protein kinase C inhibitor, Ro-31-8220 (3 micromol/l), blocked the stimulatory effect of TPA. Although short term treatment with the protein kinase A activator, (Bu)(2)cAMP (1 mmol/l), slightly reduced FLRG mRNA expression in most experiments, long term treatment with FSH (100 IU/l), LH (100 IU/l), or (Bu)(2)cAMP had no significant effect on the FLRG mRNA levels. As expected, gonadotrophins, protein kinase A and C activators and PGE(2) increased granulosa-luteal cell progesterone secretion into the culture media. Taken together, previous and our present data suggest that protein kinase C and A signal transduction pathways differently regulate the expression of FLRG and follistatin genes in human ovarian granulosa-luteal cells.
...
PMID:Regulation of follistatin-related gene (FLRG) expression by protein kinase C and prostaglandin E(2) in cultured granulosa-luteal cells. 1239 11

p27 is a cyclin-dependent kinase (CDK) inhibitor whose specific late G(1) destruction allows progression of the cell across the G(1)/S boundary. The protein is ubiquitinated by S-phase kinase-interacting protein-2 (Skp2) following its specific phosphorylation, and is subsequently degraded by the 26s proteasome. There is a direct relationship between low level of p27 and rapid proliferation occurring in several benign states and in many malignancies. In the glandular cells of the normal endometrium, the level of p27 is exceedingly low during the proliferative phase, whereas it is markedly increased during the secretory phase. The expression of p27 in endometrial carcinoma is very low but has been found to increase following treatment with progesterone. However, estrogen exposure is considered as a major risk factor in developing endometrial cancer. The implications of the high dose of estrogen and progesterone induced during IVF treatment are still unknown. We have examined the expression of p27 and Skp2 as well as of Ki67 proliferation marker by using endometrial extracts and cells from normal endometrium, from ovarian hyperstimulated patients, and from endometrial carcinoma patients. The expression of p27, Skp2 and Ki67 was found to be similar in both normal secretory endometrium and endometrium from ovarian hyperstimulated patients. In striking contrast, p27 is significantly lower while Skp2 and Ki67 are significantly higher in the endometrial carcinoma and in endometrium from the proliferative phase compared with their normal secretory counterpart tissue.
...
PMID:Decreased level of the cell cycle regulator p27 and increased level of its ubiquitin ligase Skp2 in endometrial carcinoma but not in normal secretory or in hyperstimulated endometrium. 1522 Apr 66

Locally synthesized atrial natriuretic peptide (ANP) and its receptors have been found in reproductive tissues of various mammals, and play an important role in the acrosome reaction of human sperm. The objective of the present study was to examine the expression of ANP and its receptors in pig spermatozoa and oviduct, and the effect of ANP on pig spermatozoa function. The expression of ANP and its receptors was analyzed by RT-PCR. Only natriuretic peptide receptors-A (NPRA) mRNA was detected in fresh sperm. While the levels of natriuretic peptide receptors-C (NPRC) mRNA were low with no obvious change among different oviductal phases, the levels of ANP mRNA were high in oviduct(OT)1 , OT3 and OT5, but were very low in OT2. On the other hand, the levels of NPRA mRNA were low in OT1 and OT2, increased in OT3 and reached a maximum in OT4 and OT5. Western blot analysis revealed that the level of ANP was high in OT1, decreased in OT2 and OT3, and arrived at the nadir in OT4 and OT5. The effect of ANP on spermatozoa function was studied by the acrosome reaction and IVF. Incubation with ANP for 1 h significantly induced acrosome reaction of preincubated spermatozoa, and maximal response of acrosome reaction (34.1 +/- 2.3%) was achieved at 1 nM ANP treatment. Both C-ANP-(4-23), a selective ligand of NPRC, and caffeine had no effect on the acrosome reaction. The stimulatory effect of ANP on acrosome reaction could be mimicked by the permeable cGMP analog, 8-Br-cGMP. ANP and caffeine had a similar effect on improving the oocytes penetration rate, polyspermy rate and the average number of sperm per penetrated oocyte. Also, ANP treatment had a similar effect on cleavage rate, blastocyst formation rate and the number of cells per blastocyst as that of caffeine treatment. The effects of ANP on the acrosome reaction and the parameters of oocyte penetration could be blocked by cGMP-dependent protein kinase (PKG) inhibitors KT5823 and/or Rp-8-pCPT-cGMPS. These results suggest that the expression of ANP in the oviduct may be involved in the regulation of the acrosome reaction and the fertilising ability of pig spermatozoa, and the PKG pathway possibly participates in the process.
...
PMID:The expression of atrial natriuretic peptide in the oviduct and its functions in pig spermatozoa. 1673 81

High concentrations of cyclic AMP in germinal vesicle oocytes generally inhibit GVBD. Thus, maintaining the GV stage in growing oocytes is essential for the developmental competence of the eggs. In this study, we traced the effects of dibutyryl cyclic AMP on meiotic maturation and early embryonic development in pigs. We also investigated several blastocyst qualities, including structural integrity, mitochondrial membrane potential, and apoptosis, which are affected by dbcAMP. To determine whether increased concentrations of cAMP inhibit GVBD, we explored the meiotic patterns and during maturation of pig oocytes. When treated with dbcAMP for 22h, 91.1% of the oocytes were arrested in the GV stage compared to only 38.8% of the oocytes in the control group (P<0.05). After completion of IVM, a higher proportion of the dbcAMP-treated oocytes were in metaphase II than the untreated ones (91.3% vs. 72.8%, P<0.05). Western blot analysis showed a reduction (at 22h) and/or increase (at 44h) in MPF and MAP kinase activities in porcine oocytes treated with dbcAMP for the first 22h of IVM compared to the untreated control. We also confirmed that protein kinase A activity increased in dbcAMP-treated oocytes, indicating an elevated intracellular concentration of cAMP. After IVF, the frequency of polyspermy in the dbcAMP-treated group decreased compared to that in the control group (22.4% vs. 47.4%, P<0.05). Furthermore, blastocyst formation, the blastocyst cell number, mitochondrial membrane potential, and apoptosis were enhanced and/or reduced by dbcAMP in both IVF and SCNT embryos. We concluded that synchronizing meiotic resumption by dbcAMP treatment improved the developmental capacity and embryonic qualities of IVF and SCNT embryos by increasing the mitochondrial membrane potential and decreasing the incidence of apoptosis in preimplantation-stage porcine embryos.
...
PMID:Exogenous dibutyryl cAMP affects meiotic maturation via protein kinase A activation; it stimulates further embryonic development including blastocyst quality in pigs. 1797 89

GDF-9 stimulates granulosa cell proliferation and plays important roles during folliclogenesis. However, its molecular mechanisms are still far from clear, particularly its roles in human granulosa cells around the periovulatory stage. Therefore, we investigated the effects of GDF-9 on cell cycle distribution, regulatory molecules, and signaling pathways involved in human luteinized granulosa (hLG) cells in vitro. Primary cultures of hLG cells obtained from women undergoing IVF and treated with and without recombinant GDF-9 were evaluated with and without a specific inhibitor to activin receptor-like kinase 5 (ALK5; SB-431542), ERK42/44 (PD-098059), or Smad3 (SIS3). Cell proliferation, cell cycle distribution, mRNA expression, and protein expression of relevant cell cycle molecules were determined by [(3)H]thymidine incorporation, flow cytometry, quantitative PCR, and immunoblotting, respectively. GDF-9 stimulated [(3)H]thymidine incorporation, enhanced cell transition from G(0)/G(1) to S and G(2)/M phases (whereas both SB-431542 and PD-098059 attenuated these changes), increased mRNA and protein expression of cyclin D(1) and E, and decreased those of the cyclin-dependent kinase (CDK) inhibitors p15(INK4B) and p16(INK4A). GDF-9 also activated Rb protein (a critical G(1) to S-phase regulator), ERK42/44, and Smad3. PD-098059 blocked Rb protein phorsphorylation and the increase in cyclin D(1) and E but not the decrease in p15(INK4B) and p16(INK4A) induced by GDF-9. In contrast, SIS3 reversed the decrease in p15(INK4B) and p16(INK4A) but not the increase in cyclin D(1) and E induced by GDF-9. GDF-9 stimulates hLG cell proliferation by stimulating cyclin D(1) and E and suppressing p15(INK4B) and p16(INK4A) via both Smad-dependent and Smad-independent pathways.
...
PMID:Effects of growth differentiation factor 9 on cell cycle regulators and ERK42/44 in human granulosa cell proliferation. 1936 76

We tested whether microgravity affects mouse development during a period when gravity cues chick and frog embryo development. A rotating vessel developed approximately 0.1% simulated microgravity (MGS) for embryos. Microgravity simulation resulted in blocked cell accumulation in E2.5 embryos. E1.5 and E3.5 embryos showed lesser effects. For E1.5/2.5 embryos, cell accumulation block was followed by lethality at 48 hours after MGS. For E3.5 embryos, MGS blocked development without lethality but with apoptosis. E1.5-3.5 embryos from the rotational control developed lesser effects than MGS embryos. Embryonic stress-activated protein kinase (SAPK) was phosphorylated during MGS and mediated apoptosis. Increased pSAPK suggested that lethality is due to cellular stress induced by MGS, unlike the dysfunctional development after gravitational disorientation in frog and chick embryos. Thus, MGS causes lethality, a novel phenotype not often observed in microgravity or MGS. Embryonic lethality at E2.5 and apoptosis at E3.5 are associated with SAPK function, suggesting that MGS causes a general stress response that immediately affects many aspects of development. In addition, MGS and many aspects of In vitro fertilization/assisted reproductive technologies (IVF/ART) produce nonphysiological, nonevolutionary stresses that are mediated by SAPK, suggesting the primacy of this protein kinase in a wide range of mechanisms mediating negative reproductive outcomes in IVF/ART and potentially in spaceflight.
...
PMID:A major effect of simulated microgravity on several stages of preimplantation mouse development is lethality associated with elevated phosphorylated SAPK/JNK. 1954 24

The effect of various capacitation inducers, i.e. heparin, superoxide anion, bicarbonate, adenosine, and caffeine, and their role in intracellular mechanisms involved in capacitation, were studied in cryopreserved bovine sperm. Capacitation was determined by epifluorescence chlortetracycline, protein tyrosine phosphorylation, and the ability of capacitated sperm to undergo an acrosome reaction and fertilize in vitro matured oocytes. Participation of membrane adenylate cyclase and protein kinases (protein kinase A, protein kinase C, and protein tyrosine kinase) was evaluated indirectly (with specific inhibitors). Involvement of reactive oxygen species (ROS) was determined with scavengers of superoxide anion, hydrogen peroxide, or nitric oxide. Percentages of capacitated (27-29%) and acrosome-reacted sperm (23-26%) did not differ (P > 0.05) among various capacitation inducers. Significantly higher rates of IVF were obtained with heparin (43%) or bicarbonate plus caffeine (45%), when compared with control samples (17%). Adding the membrane adenylate cyclase inhibitor diminished capacitation rates with heparin (8%) or adenosine (10%). There was differential protein kinase participation in response to inducers; protein kinase inhibitors diminished cleavage rates in heparin-capacitated sperm relative to controls. There were differences between and within the studied inducers in protein tyrosine phosphorylation patterns. We inferred that capacitation in cryopreserved bovine sperm was promoted through diverse pathways. Mechanisms triggered by heparin, or caffeine plus bicarbonate-induced capacitation, involved activation of intracellular pathways to optimize fertilizing capability of cryopreserved bovine sperm.
...
PMID:Capacitation inducers act through diverse intracellular mechanisms in cryopreserved bovine sperm. 2058 81


1 2 Next >>

---