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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To delineate the scope of the human intraovarian IL-1 system we used a solution hybridization/RNase protection assay to test for expression of the genes encoding IL-1, its type I receptor (IL-1R), and its receptor antagonist (IL-1RA). IL-1 transcripts were not detected in whole ovarian material from days 4 or 12 of an unstimulated menstrual cycle but transcripts (IL-1 beta much greater than IL-11 alpha) were detected in preovulatory follicular aspirates from gonadotropin-stimulated cycles. Concurrently obtained peripheral monocytes did not contain IL-1 beta transcripts but macrophage-depleted follicular aspirates did, thus implicating the granulosa cells as the site of IL-1 expression. IL-1R transcripts were detected in RNA from whole ovaries and follicular aspirates but not in RNA from peripheral monocytes. IL-1RA transcripts were detected in whole ovarian material as well as in macrophage-free follicular aspirates. Cultured human granulosa and theca cells did not contain mRNA for IL-1 beta or IL-1RA but did contain mRNA for IL-1R. Treatment of cell cultures with forskolin (25 microM) induced IL-1 beta transcripts in granulosa but not theca cells. Forskolin also increased the basal levels of IL-1R transcripts in both granulosa and theca cells but did not induce IL-RA transcripts in either cell type. Taken together, these findings reveal the existence of a complete, highly compartmentalized, hormonally dependent intraovarian IL-1 system replete with ligands, receptor, and receptor antagonist.
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PMID:Human intraovarian interleukin-1 (IL-1) system: highly compartmentalized and hormonally dependent regulation of the genes encoding IL-1, its receptor, and its receptor antagonist. 153 16

Glucagon and glucagon-like peptide-1 (GLP-1) are important regulators of glucose homeostasis, and both are involved in regulating pancreatic islet hormone secretion. Since the sensitivity of the endocrine pancreas to regulatory hormones can be influenced by their receptor number, we have examined the regulation of glucagon receptor and GLP-1 receptor messenger RNA (mRNA) expression in cultured rat pancreatic islets by various factors, including glucose, cAMP, and glucocorticoids. By ribonuclease protection assay we have demonstrated the expression of both glucagon and GLP-1 receptor mRNA in cultured rat islets. We observed a dose-dependent increase in glucagon receptor mRNA expression with increasing glucose concentrations: an approximately 3-fold increase in glucagon receptor mRNA in islets cultured in 22 mM glucose as compared to 3.5 mM glucose. GLP-1 receptor mRNA levels, on the other hand, were not affected by culturing the islets in low glucose concentrations; however, a small, but significant, decrease in GLP-1 receptor mRNA levels was detected when islets were cultured in 20 mM glucose. Forskolin and 3-isobuty-1-methylxanthine, which increase intracellular cAMP levels, caused a 75% reduction in glucagon receptor mRNA expression. Somatostatin 14 and 28, both of which can inhibit intracellular cAMP production, stimulated glucagon receptor mRNA expression by 40% and 75%, respectively. GLP-1 receptor mRNA levels remained unchanged under all conditions that altered intracellular cAMP levels. Finally, in islets cultured in the presence of 10 nM dexamethasone an approximately 50% decrease in both glucagon and GLP-1 receptor mRNA expression was observed. These results indicate that the expression of glucagon and GLP-1 receptor mRNA is differentially regulated in rat pancreatic islets and suggest that regulation of receptor mRNA expression may be an important mechanism for controlling the sensitivity of the islets to glucagon and GLP-1.
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PMID:Regulation of glucagon and glucagon-like peptide-1 receptor messenger ribonucleic acid expression in cultured rat pancreatic islets by glucose, cyclic adenosine 3',5'-monophosphate, and glucocorticoids. 753 5

Bone marrow stromal cells synthesize and secrete insulin-like growth factor (IGF)-I and IGF-binding proteins (IGFBP). IGFBPs may modulate the action of IGF-I or IGF-II on haemopoiesis. However, the specific IGFBPs produced by various stromal cell types have not been identified. We examined six different stromal phenotypes for IGFBP protein and IGFBP-1 to -6 mRNA expression. [125I]IGF-I ligand blot analysis of conditioned medium demonstrate different patterns of IGFBP secretion by each cell type. The most prominent IGFBPs were 24 and 29 kD species, consistent with IGFBP4 and IGFBP5, respectively. RNase protection assays demonstrate that, overall, stromal cells express IGFBP-2 to -6 mRNAs, with IGFBP4, IGFBP5 and IGFBP6 mRNAs predominating. Since agents that modulate cAMP levels may influence haemopoiesis via the release of stromal-derived cytokines, we determined the effect of forskolin, a cAMP agonist, on IGFBP4 expression in TC-1 cells. Forskolin (10(-5) M) up-regulated IGFBP4 mRNA and protein secretion in a time-dependent manner. These findings suggest that IGFBP-4, -5 and -6 released by stromal cells may be key modulators of the haemopoietic response to IGFs. Release of IGFBP4 by agents that increase cAMP may be an important mechanism involved in regulating IGF bioavailability in the marrow microenvironment.
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PMID:Characterization of insulin-like growth factor binding proteins (IGFBP) and regulation of IGFBP-4 in bone marrow stromal cells. 754 Aug 52

We examined the effect of PTH on the activity of alpha 1(I) collagen promoter fusion genes in cultured calvariae from transgenic mice. The parent construct, ColCAT 3.6, contains 3520 basepairs of 5' rat alpha 1(I) collagen DNA, 115 basepairs of untranslated alpha 1(I) collagen-coding DNA, and the bacterial chloramphenicol acetyltransferase reporter gene, while the 5'-deletion ColCAT 2.3 contains 2296 kilobases of rat alpha 1(I) collagen promoter sequence. Transgenic mouse lines harboring these collagen promoter fusion genes were developed using the oocyte microinjection technique, and for each construct, three different lines of mice were tested. Calvariae from 6- to 8-day-old transgenic mice were cultured for 48 h with or without bovine PTH-(1-34). ColCAT 3.6 and ColCAT 2.3 were expressed at comparable levels in calvariae and were inhibited by PTH. There were parallel decreases in the incorporation of [3H]proline into collagen and levels of the endogenous alpha 1(I) collagen mRNA and transgene mRNA. Forskolin at 10 microM mimicked the inhibitory effect of PTH on promoter activity in ColCAT 3.6 and ColCAT 2.3 calvariae. A RNase protection assay showed that the transgene was initiated correctly from the transgene promoter. These data show that PTH and cAMP can repress collagen promoter activity in calvariae from transgenic mice, suggesting that the alpha 1(I) collagen promoter may contain cis elements down-stream of -2.3 kilobases that mediate PTH and cAMP repression of collagen gene expression in bone. Cultured bone explants from transgenic mice can be used as a model to study hormonal regulation of alpha 1(I) collagen promoter constructs.
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PMID:Parathyroid hormone represses alpha 1(I) collagen promoter activity in cultured calvariae from neonatal transgenic mice. 848 79

Molecular mechanisms that regulate in situ activation of ryanodine receptors (RY) in different cells are poorly understood. Here we demonstrate that caffeine (10 mM) released Ca2+ from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loaded beta cells from ob/ob mice. Surprisingly, when forskolin, an activator of adenylyl cyclase was present, caffeine induced larger Ca2+ spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release Ca2+ from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca2+ from ER, giving rise to a slow and small increase in [Ca2+]i in beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered Ca2+ release by 4-CEP, giving rise to Ca2+ spikes. In glucose-stimulated beta cells forskolin induced Ca2+ spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting betaTC-3-cells by RNase protection assay, using type-specific RY probes, revealed low-level expression of mRNA for the type 2 isoform of the receptor (RY2). We conclude that in situ activation of RY2 in beta cells requires cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.
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PMID:In situ activation of the type 2 ryanodine receptor in pancreatic beta cells requires cAMP-dependent phosphorylation. 960 Sep 32

The molecular mechanisms involved in regulation of CRH-binding protein (CRH-BP) gene expression were examined using primary rat astrocyte cultures. The cells were treated with various regulators, and CRH-BP messenger RNA (mRNA) levels were determined using ribonuclease protection assays. Forskolin (Fsk, 10 microM) or 12-O-tetradecanoyl-phorbol 13-acetate (TPA, 100 nM) increases CRH-BP mRNA levels up to 30 times control level, and together they act synergistically to increase CRH-BP gene expression up to 100 times control levels. CRH can also positively regulate CRH-BP gene expression to 6.1 times control levels. All of these increases in steady-state CRH-BP mRNA levels can be repressed by dexamethasone, a synthetic glucocorticoid. To determine whether these changes in steady-state CRH-BP mRNA levels are caused by altered transcription or RNA stability, heteronuclear (hn) CRH-BP species were examined using ribonuclease protection assays. CRH-BP hnRNA transcripts can be detected transiently after the addition of Fsk or TPA, and dexamethasone can repress Fsk- or TPA-induced CRH-BP hnRNA levels in this assay. These results demonstrate that CRH, glucocorticoids, and the protein kinase A and protein kinase C signaling pathways are involved in regulation of CRH-BP gene expression in astrocyte cultures, and that this regulation is caused, at least in part, by altered transcription of the gene.
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PMID:Transcriptional regulation of corticotropin-releasing hormone-binding protein gene expression in astrocyte cultures. 1046 81

Granulosa cells from preovulatory follicles show increased expression of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) at the time of ovulation. As ovulation may be an inflammatory process, this may be a mechanism of local enhancement of the activity of anti-inflammatory glucocorticoids. In this study, we examined direct effects of LH, the proinflammatory cytokine, interleukin-1beta (IL-1beta), and pharmacological activators of protein kinase A (PKA) (forskolin and dibutyryl (db) cAMP) and PKC (LH-releasing hormone and phorbol 12-myristate 13-acetate (PMA)) signalling on the expression of 11betaHSD1 mRNA in vitro. Granulosa cells from immature female rat ovaries were cultured (pretreatment) in serum-free medium 199 containing recombinant human (rh) FSH (1 ng/ml) for 48 h to induce responsiveness to LH. Cell monolayers were then washed and cultured (test treatment) for a further 12 h in the presence of rhLH (0-100 ng/ml), IL-1beta (0-50 ng/ml), or both. Total RNA was extracted from granulosa cell monolayers and taken for quantitative ribonuclease protection analysis of 11betaHSD1 mRNA. The low level of 11betaHSD1 mRNA detectable in unstimulated (control) cultures was increased approximately twofold by the 48-h pretreatment with rhFSH. Subsequent exposure to rhLH (1-100 ng/ml) for a further 12 h dose-dependently increased 11betaHSD1 mRNA expression by an additional two- to threefold. Forskolin (10 microM), db-cAMP (2 mM), LH-releasing hormone (LHRH; 1 microM) and PMA (200 nM) were also stimulatory. IL-1beta (0.05-50 ng/ml) stimulated 11betaHSD1 mRNA expression in a dose-related manner, both in the absence and in the presence of rhLH (3 ng/ml). The interaction between IL-1beta (5 ng/ml) and rhLH (3 ng/ml) was additive. Co-treatment with a 50-fold excess of IL-1 receptor antagonist fully reversed the action of IL-1beta. We conclude that 11betaHSD1 mRNA expression in functionally mature granulosa cells is directly stimulated by gonadotrophins and IL-1beta in vitro, potentially involving post-receptor signalling via PKA- and PKC-mediated pathways. Thus both LH and IL-1beta may serve physiological roles in the upregulation of 11betaHSD1 gene expression by granulosa cells in ovulatory follicles.
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PMID:Regulation of 11beta-hydroxysteroid dehydrogenase type 1 gene expression by LH and interleukin-1beta in cultured rat granulosa cells. 1058 15

Bone morphogenetic proteins (BMPs), members of the transforming growth factor beta superfamily, were recently shown to be expressed and to regulate steroidogenesis in rat ovarian tissue. The purpose of this study was to investigate the effect of BMP-4 on androgen production in a human ovarian theca-like tumor (HOTT) cell culture model. We have previously demonstrated the usefulness of these cells as a model for human thecal cells. HOTT cells respond to protein kinase A agonists by increased production of androstenedione and with an induction of steroid-metabolizing enzymes. In this investigation, HOTT cells were treated with forskolin or dibutyryl cyclic AMP (dbcAMP) in the presence or absence of various concentrations of BMP-4. The accumulation of androstenedione, progesterone, and 17alpha-hydroxyprogesterone (17OHP) in the incubation medium was measured by RIA. The expression of 17alpha-hydroxylase (CYP17), 3beta-hydroxysteroid dehydrogenase (3betaHSD), cholesterol side-chain cleavage (CYP11A1), and steroidogenic acute regulatory (StAR) protein was determined by protein immunoblotting analysis using specific rabbit polyclonal antibodies. We also examined the expression of BMP receptor subtypes in our HOTT cells using RT-PCR. In cells treated with medium alone, steroid accumulation and steroid enzyme expression was unchanged. In cells treated with BMP alone there was a modest decrease in androstenedione secretion. In the presence of forskolin, HOTT cell production of androstenedione, 17OHP, and progesterone increased by approximately 4.5-, 35-, and 3-fold, respectively. In contrast, BMP-4 decreased forskolin-stimulated HOTT cell secretion of androstenedione and 17OHP by 50% but increased progesterone production 3-fold above forskolin treatment alone. Forskolin treatment led to an increase in CYP17, CYP11A1, 3betaHSD, and StAR protein expression. BMP-4 markedly inhibited forskolin stimulation of CYP17 expression but had little effect on 3betaHSD, CYP11A1, or StAR protein levels. Similar results were observed with the cAMP analog dbcAMP. In addition, BMP-4 inhibited basal and forskolin stimulation of CYP17 messenger RNA expression as determined by RNase protection assay. Other members of the transforming growth factor beta superfamily, including activin and inhibin, had minimal effect on androstenedione production in the absence of forskolin. In the presence of forskolin, activin inhibited androstenedione production by 80%. Activin also inhibited forskolin induction of CYP17 protein expression as determined by Western analysis. We identified the presence of messenger RNA for three BMP receptors (BMP-IA, BMP-IB, and BMP-II) in the HOTT cells model. In conclusion, BMP-4 inhibits HOTT cell expression of CYP17, leading to an alteration of steroidogenic pathway resulting in reduced androstenedione accumulation and increased progesterone production. These effects of BMP-4 seem similar to those caused by activin, another member of the transforming growth factor-beta superfamily of proteins.
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PMID:Bone morphogenetic protein inhibits ovarian androgen production. 1099 29

Stabilization and correct localization of mRNA are important features of renin synthesis. To elucidate the molecular basis of cAMP-mediated posttranscriptional control via mRNA stabilization, we analyzed the interaction of human preprorenin (hREN) mRNA 3'-untranslated region (3'-UTR) with proteins of renin synthesizing Calu-6 cells and investigated their functional impact on messenger integrity. To identify hREN mRNA binding proteins, electrophoretic mobility shift assays, UV cross-linking and RNA-affinity chromatography with subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were performed. The following six proteins were unambiguously identified as hREN mRNA 3'-UTR binding proteins: hnRNP E1 (synonyms alpha-CP or PCBP), hnRNP K, dynamin, nucleolin, YB-1, and MINT-homologous protein. All proteins contain various RNA binding motifs, and most have been described in the context of mRNA binding and mRNA stabilization. Four proteins for which antibodies were available were verified by immunological techniques (dynamin, nucleolin, hnRNP E1, and YB-1). Forskolin, an activator of cAMP synthesis, considerably stimulates renin synthesis via inhibition of REN mRNA decay. Functionally, this cAMP-based mRNA stabilization is accompanied by a 3- to 6-fold upregulation of REN mRNA binding proteins. RNase degradation assays confirm that 3'-UTR binding proteins are able to protect and stabilize REN mRNA in vitro.
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PMID:Posttranscriptional control of renin synthesis: identification of proteins interacting with renin mRNA 3'-untranslated region. 1260 Aug 97

Forskolin and heregulin synergistically drive human Schwann cell (HSC) proliferation in vitro, but the role of forskolin is not completely understood. To learn how forskolin might affect receptor levels in HSC cultured from adult nerve roots, we first studied expression and localization of HER2 and HER3 in intact roots, using Western blotting and light and electron microscopic immunocytochemistry. We then determined the effect of forskolin and heregulin on receptor expression in HSC cultured from nerve roots using Western blotting and RNase protection assays. HER2 and HER3 were expressed in nonmyelinating Schwann cells in roots and in cultured HSCs before exposure to forskolin. HER2, but not HER3, was also expressed in endoneurial fibroblasts and in cultured nerve root-derived fibroblasts. Treatment with forskolin for 24 h consistently increased HER2 and HER3 protein levels in HSCs but did not alter HER2 and HER3 mRNA levels. In addition, 24-h treatment with heregulin alone decreased HER2 and HER3 protein levels, an effect not previously described. When both heregulin and forskolin were present, HER2 and HER3 protein levels were similar to initial control values. The effect of forskolin on receptor levels was mimicked by dibutyryl-cAMP and receptor levels in both untreated and forskolin treated HSCs were decreased by treatment with the protein kinase A inhibitor H-89. Following pretreatment of HSCs with forskolin, increased receptor levels were correlated with increased rates of thymidine incorporation into HSCs. These results suggest that forskolin/heregulin synergy might derive, at least in part, from post-transcriptional effects leading to increased steady-state receptor levels.
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PMID:Forskolin increases neuregulin receptors in human Schwann cells without increasing receptor mRNA. 1539 Jan 6


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