Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this report we demonstrate glucocorticoid receptors in seminiferous tubules of the rat testis, and that these receptors are localized in Sertoli cells and peritubular cells. The receptors had high affinity for [3H]dexamethasone (Kd = 0.5 - 1 x 10(-9) M), and similar Kd values were calculated from equilibrium analysis and from rate studies (k1 = 1.5 x 10(6) M-1 min-1 and k-1 = 1.4 x 10(-3) min-1, O C). Binding specificity was typical for glucocorticoid receptors (affinity: dexamethasone greater than corticosterone greater than cortisol approximately R5020 approximately progesterone greater than aldosterone = R1881 greater than 17 beta-estradiol approximately cortisone approximately testosterone greater than 5 alpha-dihydrotestosterone). The concentration of glucocorticoid receptors in rat seminiferous tubules revealed an age-dependent decrease, coinciding with the increase in the number of germ cells. Glucocorticoid receptor levels were higher in Sertoli cells from immature rats than in cells from adult rats. Cultured peritubular cells from immature rats contained levels of glucocorticoid receptors similar to cultured Sertoli cells from rats of the same age. With a nick-translated human glucocorticoid receptor complementary DNA probe, a messenger RNA (mRNA) species of approximately 7 kilobase was clearly detected in both Sertoli cells and peritubular cells. In peritubular cells, a smaller mRNA species (5 kilobase) was also clearly detectable. In mRNA from whole testis tissue, a similar developmental pattern as for dexamethasone binding was found. Dexamethasone caused a concentration-dependent stimulation of mRNA levels for androgen binding protein and for the cAMP-dependent protein kinase regulatory subunit type II beta in cultured immature rat Sertoli cells. On the other hand, mRNA levels for glucocorticoid receptor decreased, whereas mRNA levels for beta-actin remained constant. This report documents for the first time the presence of glucocorticoid receptors and glucocorticoid effects in rat Sertoli cells, and is also the first demonstration of glucocorticoid receptors in peritubular cells of the rat testis.
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PMID:Glucocorticoid receptors and glucocorticoid effects in rat Sertoli cells. 290 75

Cytosolic casein kinase type II activity has been identified in MCF-7 and MDA-MB-231 human breast cancer cells heterotransplanted into athymic nude mice. Sephacryl S-300 chromatography of MCF-7 and MDA-MB-231 tumor cytosols revealed a major peak of casein kinase activity with an estimated molecular weight of 150,000. This peak was further characterized and optimal conditions for breast tumor casein kinase activity were established. Polylysine (10 micrograms) acted as a potent stimulator with casein as the phosphate acceptor protein. This enzyme used both ATP and GTP as phosphate donors and the Km for GTP was 10 microM. The rate of phosphorylation with increasing concentrations of [gamma-32p]GTP revealed typical Michaelis-Menten kinetics and Vmax was approached at a concentration of 30 microM GTP. MgCl2 stimulated enzyme activity at concentrations between 10-20 mM. Quercetin, a bioflavonoid, inhibited casein kinase type II activity in a dose dependent manner. MCF-7 (hormone-dependent) human breast cancer cells (2-3 X 10(6)) were inoculated into the mammary fat pads of nude mice, supplemented with a 0.5 mg estradiol pellet. To determine the influence of various regulatory agents on casein kinase activity in vivo, tumor-bearing mice were treated for five days with estradiol, progesterone, dexamethasone or tamoxifen. Casein kinase type II was partially purified by gel filtration on a Sephacryl S-300 column and assayed in the presence of polylysine and casein. Dexamethasone treatment significantly decreased casein kinase II activity in MCF-7 tumors, which are receptor-positive for estrogen, androgen and glucocorticoid receptors.
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PMID:Characterization and hormonal regulation of casein kinase II activity in heterotransplanted human breast tumors in nude mice. 348 45

Milk production during lactation is a consequence of the suckling stimulus and the presence of glucocorticoids, prolactin, and insulin. After weaning the glucocorticoid hormone level drops, secretory mammary epithelial cells die by programmed cell death and the gland is prepared for a new pregnancy. We studied the role of steroid hormones and prolactin on the mammary gland structure, milk protein synthesis, and on programmed cell death. Slow-release plastic pellets containing individual hormones were implanted into a single mammary gland at lactation. At the same time the pups were removed and the consequences of the release of hormones were investigated histologically and biochemically. We found a local inhibition of involution in the vicinity of deoxycorticosterone- and progesterone-release pellets while prolactin-release pellets were ineffective. Dexamethasone, a very stable and potent glucocorticoid hormone analogue, inhibited involution and programmed cell death in all the mammary glands. It led to an accumulation of milk in the glands and was accompanied by an induction of protein kinase A, AP-1 DNA binding activity and elevated c-fos, junB, and junD mRNA levels. Several potential target genes of AP-1 such as stromelysin-1, c-jun, and SGP-2 that are induced during normal involution were strongly inhibited in dexamethasone-treated animals. Our results suggest that the cross-talk between steroid hormone receptors and AP-1 previously described in cells in culture leads to an impairment of AP-1 activity and to an inhibition of involution in the mammary gland implying that programmed cell death in the postlactational mammary gland depends on functional AP-1.
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PMID:Glucocorticoid and progesterone inhibit involution and programmed cell death in the mouse mammary gland. 749 Feb 85

This study analyzes cyclooxygenase II (COX-2) gene expression, protein synthesis, and PGE2 release in normal human articular chondrocytes. Stimulation of chondrocytes in primary culture resulted in a dose-dependent induction of COX-2 mRNA in response to IL-1 with an ED50 between 0.1 and 1 ng/ml. COX-2 mRNA was detectable after 2 h, reached high levels at 6 h, and showed a remarkably long duration of expression for at least 72 h. Analysis of other extracellular stimuli showed that COX-2 mRNA was inducible by other cytokines including TNF-alpha, IL-6, and LIF and by bacterial LPS. Dexamethasone completely inhibited IL-1-induced COX-2 mRNA expression. Analysis of signaling pathways showed that PMA and calcium ionophore A23187, but not dibutyryl cAMP, induced COX-2 mRNA. The combination of IL-1 and A23187 resulted in synergistic increases. IL-1 effects were not reduced by the protein kinase C inhibitor staurosporine or by the protein kinase A inhibitor H89 but blocked by the protein tyrosine kinase inhibitor herbimycin A. COX-2 protein was detected at 71 kDa by Western blotting in IL-1-stimulated, and to almost similar levels in A23187-treated, cells. Flow cytometric analysis showed that after IL-1 stimulation 78% of the chondrocytes expressed COX-2 protein. The patterns of COX-2 protein expression and the levels of PGE2 release correlated with the effects of the different stimuli and inhibitors on mRNA expression.
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PMID:Regulation of cyclooxygenase-2 expression in normal human articular chondrocytes. 760 56

The somatostatin (SS) gene is transcriptionally regulated via the cyclic AMP (cAMP) response element (CRE), located in the proximal promoter (-41 to -48 bp). We have previously reported that glucocorticoids induce dose-dependent cell-specific alterations in the steady-state SS mRNA level. Here we have investigated direct transcriptional control of the SS gene by glucocorticoids. We have examined transcriptional interaction between glucocorticoids and the cAMP signalling pathway and mapped the 5' upstream regulatory region of the SS gene involved in glucocorticoid transactivation. Transcriptional regulation was determined by analysis of chloramphenicol acetyltransferase (CAT) activity in PC12 rat pheochromocytoma cells and A126-1B2 (protein kinase A-deficient mutant PC12) cells, by acute transfection of 5' flanking SS DNA (- 750, -250 and -71 bp) ligated to the reporter (CAT) gene. Dexamethasone (DEX) induced a dose-dependent 2.2-fold stimulation of SS gene transcription in PC12 cells, but not in A126-1B2 cells. Other steroid and thyroid hormones tested, and retinoic acid, were ineffective, while cAMP and forskolin stimulated gene transcription 4-5-fold in PC12 cells but not in A126-1B2 cells. DEX exerted an additive effect on cAMP-induced gene transcription. Deletion of the promoter from -750 to -71 bp (but not from -750 to -250 bp) abolished all stimulatory effects of DEX without affecting cAMP responsiveness. Mutation of the CRE abrogated both DEX- and cAMP-dependent gene enhancement. Gel electrophoretic mobility shift assays confirmed that the -250 to -71 bp region of the SS promoter (but not the -71 to +55 bp domain) binds specifically to a glucocorticoid response element-sensitive nuclear protein(s) from PC12 cells, suggesting a putative glucocorticoid receptor interaction with SS promoter DNA. We conclude that glucocorticoids regulate SS gene transcription positively. Glucocorticoid-induced transactivation shows dependence on protein kinase. A activity, and may be mediated via protein-protein interaction between the glucocorticoid receptor and the CRE binding protein. DNA sequences upstream from the CRE between -250 and -71 bp in the SS promoter appear to be the target of glucocorticoid action.
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PMID:Glucocorticoids activate somatostatin gene transcription through co-operative interaction with the cyclic AMP signalling pathway. 791 2

Recombinant forms of the human thromboxane A2 (TXA2) receptor composed of the carboxyl-terminal amino acid residues 220-343 were phosphorylated in vitro by both cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC), and these phosphorylations were competed for by synthetic peptides corresponding to the proposed carboxyl-terminal cytoplasmic tail and/or the third extracellular loop of the receptor, respectively. Exogenous addition of PKA or PKC to membrane preparations of human embryonic kidney 293 cells, transfected with the TXA2 receptor, typically reduced TXA2 receptor binding by 10 and 30%, respectively. In vivo inhibition of PKC or PKA in the transfected human embryonic kidney 293 cells increased TXA2 receptor binding to 121.4% (+/- 5.3%) and 110.4% (+/- 4.6%), respectively, relative to control cells. In vivo activation of PKC in the platelet-like human erythroleukemia (HEL) cells by the phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in an initial reduction followed by a time-dependent increase in TXA2 receptor ligand binding. Stimulation of HEL cells with the TXA2 receptor agonist [15-(alpha,2 beta(5Z)-3 alpha(E,3S)-4 alpha)]-7-[3- (3-hydroxy-4-(p-iodophenoxy)-1-butenyl)-7-oxabicyclo-[-2.2,1-]hept -2-yl]- 5-heptenoic acid or basic fibroblast growth factor, alone or together, resulted in a marked decrease in TXA2 receptor binding. Northern blot studies in HEL cells demonstrated that PMA stimulation induced the expression of the TXA2 receptor gene with mRNA levels peaking following PMA stimulation for 4-8 h. This induction is consistent with the presence of a phorbol ester response element in promoter I of the TXA2 receptor gene. Dexamethasone did not induce the expression of the receptor gene, despite the presence of a glucocorticoid response element in promoter II of the TXA2 receptor gene. In summary, our results indicate that the cellular responses to TXA2 are mediated both by phosphorylation of the TXA2 receptor by different protein kinases and by regulated expression of the TXA2 receptor gene.
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PMID:Phosphorylation and regulated expression of the human thromboxane A2 receptor. 796 88

Human phaeochromocytomas abundantly express insulin-like growth factor-II (IGF-II), but its regulation and biological role in these neoplasms is not known at present. To clarify the regulation of IGF-II gene expression in phaeochromocytomas, we studied the effects of glucocorticoids, nerve growth factor (NGF), and protein kinase A and C regulators in primary cultures of human phaeochromocytoma cells. Cytoplasmic RNA was extracted and analysed by Northern and dot blotting with a 32P-labelled cDNA probe for IGF-II. Dexamethasone treatment (500 ng/ml) for 3 and 7 days resulted in a 260% and 515% increase in the accumulation of IGF-II mRNA respectively. The stimulatory effect of dexamethasone was time- and dose-dependent. The increases in the 6.0 and 2.2 kb species of IGF-II mRNAs were the most apparent. Cortisol (1 microgram/ml) increased the amount of IGF-II mRNA by threefold compared with the control. NGF (200 ng/ml), dibutyryl cyclic AMP (1 mM) and 12-O-tetradecanoyl phorbol-13-acetate (a protein kinase C activator; 100 ng/ml) had no significant effect on IGF-II mRNA levels. These data suggest that IGF-II gene expression in human phaeochromocytomas may be regulated by microenvironmental glucocorticoids.
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PMID:Glucocorticoids increase insulin-like growth factor-II mRNA accumulation in cultured human phaeochromocytoma cells. 796 81

The vasoactive peptide endothelin-1 (ET-1) dose-dependently increased release of 51Cr from human cerebromicrovascular endothelial cells (HBEC), without affecting cell viability as assessed by lactate dehydrogenase release. ET-1 also induced transient accumulation of inositol triphosphate (IP3) and release of [3H] arachidonic acid (AA) from HBEC. The ET-1-induced 51Cr release, formation of IP3, and AA release from HBEC were competitively inhibited by selective ETA subtype receptor antagonist BQ-123. ET-1-stimulated 51Cr- and AA release from HBEC were potentiated by proteinkinase C (PKC) activator phorbol-myristate ester, and abolished by H7, an inhibitor of PKC. Dexamethasone, indomethacin, acetylsalicylic acid, imidazole, as well as the inhibitor of protein kinase A, H8, had no effect on 51Cr release. The results suggest that ETA-receptor mediated activation of PKC and increase in the HBEC 'permeability' for low molecular weight molecules in response to excessive release of endothelins from either HBEC or surrounding tissues during pathologic conditions may contribute to the formation of cerebral edema.
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PMID:Arachidonic acid release and permeability changes induced by endothelins in human cerebromicrovascular endothelium. 797 60

Early glucocorticoid feedback in sheep anterior pituitary (AP) cells was compared and contrasted with that in mouse pituitary tumor AtT-20 cells. Dexamethasone (DEX) inhibited corticotropin-releasing hormone (CRH)-stimulated adrenocorticotropin (ACTH) release in a concentration- and time-dependent manner with similar potency amongst cell types. This inhibition was mediated through type II glucocorticoid receptors and required the synthesis of new protein. However, stimulation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) resulted in greater ACTH release and greater inhibition by DEX in sheep AP cells. In contrast to sheep AP cells, AtT-20 cells were insensitive to glucocorticoids when secretion was stimulated by KCl depolarization or the voltage-dependent calcium channel agonist, maitotoxin (MTX). In both cell types, CRH-, KCl-, and MTX-stimulated ACTH release was inhibited by the calcium channel blocker, nifedipine (NIF). Whereas NIF also inhibited PMA-induced ACTH secretion in AtT-20 cells, it did not in sheep AP cells. These data demonstrate that early glucocorticoid feedback is operative in sheep corticotrophs and that AtT-20 cells appear to serve as an appropriate mechanistic model for aspects of negative feedback when the CRH-protein kinase A pathway is activated but may not be appropriate when ACTH secretion is activated via other intracellular signaling pathways.
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PMID:Glucocorticoid negative feedback in sheep corticotrophs: a comparison with AtT-20 corticotroph tumor cells. 806 55

Contradictory results have been reported on the question of the role of Ca2+ in glucocorticoid-induced apoptosis in thymocytes. To resolve this problem, we investigated the effect of dexamethasone, a synthetic glucocorticoid, on intracellular Ca2+ concentration ([Ca2+]i), by microscopic fluorometry that enables us to monitor real-time [Ca2+]i of cells loaded with fura-2, a fluorescent Ca2+ indicator, on a single cell basis. The results indicated that dexamethasone does not induce an increase in [Ca2+]i above control level both in murine and rat thymocytes at least for 1 h after the start of the culture. We also investigated whether the depletion of extracellular Ca2+ with EGTA or buffering intracellular Ca2+ with quin-2/AM inhibited glucocorticoid-induced apoptosis as reported on rat thymocytes. Dexamethasone-induced apoptosis in both murine and rat thymocytes, however, was not inhibited by EGTA. High concentrations (25 microM and over) of quin-2/AM inhibited DNA fragmentation, but failed to inhibit cytolysis. Calmodulin inhibitors, trifluoperazine and calmidazolium, also inhibited DNA fragmentation as reported, although they markedly enhanced cytolysis. Therefore, glucocorticoid-induced death is not inhibited by quin-2/AM or calmodulin inhibitors. Furthermore, we have previously found that a proper combination of the calcium ionophore, ionomycin, and the protein kinase activator, PMA, inhibits corticosterone-induced apoptosis. These results suggest that an early increase in [Ca2+]i is neither induced by glucocorticoids nor responsible for glucocorticoid-induced apoptosis in thymocytes.
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PMID:Early mobilization of Ca2+ is not required for glucocorticoid-induced apoptosis in thymocytes. 822 18


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