Mechanisms of regulation of G11α protein by dexamethasone in osteoblastic UMR 106–01 cells

2002 ◽  
Vol 282 (1) ◽  
pp. E24-E30 ◽  
Author(s):  
Ricky Cheung ◽  
Jane Mitchell
Keyword(s):  
De Novo ◽  
Mrna Level ◽  
Time Lag ◽  
Regulatory Protein ◽  
Protein Synthesis Inhibitor ◽  
Osteosarcoma Cell ◽  
Dependent Manner ◽  
Synthesis Inhibitor ◽  
Glucocorticoid Treatment ◽  
Umr 106

We have previously demonstrated that glucocorticoids increased Gq/11α protein expression and phospholipase C activity in the rat osteosarcoma cell line UMR 106–01. In this study, we demonstrated that G11α is the primary Gq-subtype family member expressed in UMR cells. Dexamethasone treatment increased the expression of G11α protein in both a time- and a dose-dependent manner. Glucocorticoid treatment significantly increased the half-life of G11α protein from 20.3 to 63 h. Steady-state G11α mRNA level was also increased by glucocorticoid treatment by ∼70%. This change was not the result of changes in RNA stability but rather the result of increased transcription, because the glucocorticoid-mediated upregulation of G11α mRNA was blocked by the transcription inhibitor actinomycin D. The dexamethasone induction of G11α mRNA occurred after a time lag of 12–24 h and was blocked by the protein synthesis inhibitor cycloheximide. These results suggest that the dexamethasone-induced rise in G11α protein results primarily from changes in the degradation rate of the protein, whereas changes in G11α mRNA play a smaller role and require de novo synthesis of regulatory protein(s).

scholarly journals Social Fear Memory Requires Two Stages of Protein Synthesis in Mice

2020 ◽  
Vol 21 (15) ◽  
pp. 5537
Author(s):  
Johannes Kornhuber ◽  
Iulia Zoicas
Keyword(s):  
Protein Synthesis ◽  
Temporal Dynamics ◽  
De Novo ◽  
Fear Memory ◽  
Protein Synthesis Inhibitor ◽  
Dependent Manner ◽  
Synthesis Inhibitor ◽  
Systemic Injection ◽  
Social Fear ◽  
Two Stages

It is well known that long-term consolidation of newly acquired information, including information related to social fear, require de novo protein synthesis. However, the temporal dynamics of protein synthesis during the consolidation of social fear memories is unclear. To address this question, mice received a single systemic injection with the protein synthesis inhibitor, anisomycin, at different time-points before or after social fear conditioning (SFC), and memory was assessed 24 h later. We showed that anisomycin impaired the consolidation of social fear memories in a time-point-dependent manner. Mice that received anisomycin 20 min before, immediately after, 6 h, or 8 h after SFC showed reduced expression of social fear, indicating impaired social fear memory, whereas anisomycin caused no effects when administered 4 h after SFC. These results suggest that consolidation of social fear memories requires two stages of protein synthesis: (1) an initial stage starting during or immediately after SFC, and (2) a second stage starting around 6 h after SFC and lasting for at least 5 h.

scholarly journals Signal transduction for chemotaxis and haptotaxis by matrix molecules in tumor cells.

1990 ◽  
Vol 110 (4) ◽  
pp. 1427-1438 ◽  
Author(s):  
S Aznavoorian ◽  
M L Stracke ◽  
H Krutzsch ◽  
E Schiffmann ◽  
L A Liotta
Keyword(s):  
Protein Synthesis ◽  
Matrix Protein ◽  
Type Iv Collagen ◽  
De Novo ◽  
Protein Synthesis Inhibitor ◽  
Dependent Manner ◽  
Recognition Sequence ◽  
Synthesis Inhibitor ◽  
Concentration Dependent Manner ◽  
Type Iv

Transduction of signals initiating motility by extracellular matrix (ECM) molecules differed depending on the type of matrix molecule and whether the ligand was in solution or bound to a substratum. Laminin, fibronectin, and type IV collagen stimulated both chemotaxis and haptotaxis of the A2058 human melanoma cell line. Peak chemotactic responses were reached at 50-200 nM for laminin, 50-100 nM for fibronectin, and 200-370 nM for type IV collagen. Checkerboard analysis of each attractant in solution demonstrated a predominantly directional (chemotactic) response, with a minor chemokinetic component. The cells also migrated in a concentration-dependent manner to insoluble step gradients of substratum-bound attractant (haptotaxis). The haptotactic responses reached maximal levels at coating concentrations of 20 nM for laminin and type IV collagen, and from 30 to 45 nM for fibronectin. Pretreatment of cells with the protein synthesis inhibitor, cycloheximide (5 micrograms/ml), resulted in a 5-30% inhibition of both chemotactic and haptotactic responses to each matrix protein, indicating that de novo protein synthesis was not required for a significant motility response. Pretreatment of cells with 50-500 micrograms/ml of synthetic peptides containing the fibronectin cell-recognition sequence GRGDS resulted in a concentration-dependent inhibition of fibronectin-mediated chemotaxis and haptotaxis (70-80% inhibition compared to control motility); negative control peptide GRGES had only a minimal effect. Neither GRGDS nor GRGES significantly inhibited motility to laminin or type IV collagen. Therefore, these results support a role for the RGD-directed integrin receptor in both types of motility response to fibronectin. After pretreatment with pertussis toxin (PT), chemotactic responses to laminin, fibronectin, and type IV collagen were distinctly different. Chemotaxis to laminin was intermediate in sensitivity; chemotaxis to fibronectin was completely insensitive; and chemotaxis to type IV collagen was profoundly inhibited by PT. In marked contrast to the inhibition of chemotaxis, the hepatotactic responses to all three ligands were unaffected by any of the tested concentrations of PT. High concentrations of cholera toxin (CT; 10 micrograms/ml) or the cAMP analogue, 8-Br-cAMP (0.5 mM), did not significantly affect chemotactic or haptotactic motility to any of the attractant proteins, ruling out the involvement of cAMP in the biochemical pathway initiating motility in these cells. The sensitivity of chemotaxis induced by laminin and type IV collagen, but not fibronectin, to PT indicates the involvement of a PT-sensitive G protein in transduction of the signals initiating motility to soluble laminin and type IV collagen.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Granulocyte/macrophage colony-stimulating factor stimulates the expression of the 5-lipoxygenase-activating protein (FLAP) in human neutrophils.

1994 ◽  
Vol 179 (4) ◽  
pp. 1225-1232 ◽  
Author(s):  
M Pouliot ◽  
P P McDonald ◽  
P Borgeat ◽  
S R McColl
Keyword(s):  
De Novo ◽  
Human Neutrophils ◽  
Protein Synthesis Inhibitor ◽  
Dependent Manner ◽  
Colony Stimulating Factor ◽  
Synthesis Inhibitor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The synthesis of leukotrienes in human blood neutrophils chiefly relies on the activity of two enzymes, phospholipase A2 and 5-lipoxygenase (5-LO). In turn, the activation of the 5-LO requires the participation of a recently characterized membrane-bound protein, the 5-LO-activating protein (FLAP). In this study, we have investigated conditions under which FLAP expression in neutrophils may be modulated. Of several cytokines tested, only granulocyte/macrophage colony-stimulating factor (GM-CSF) (and to a lesser extent tumor necrosis factor alpha) significantly increased expression of FLAP. GM-CSF increased FLAP mRNA steady-state levels in a time- and dose-dependent manner. The stimulatory effect of GM-CSF on FLAP mRNA was inhibited by prior treatment of the cells with the transcription inhibitor, actinomycin D, and pretreatment of the cells with the protein synthesis inhibitor, cycloheximide, failed to prevent the increase in FLAP mRNA induced by GM-CSF. The accumulation of newly synthesized FLAP, as determined by immunoprecipitation after incorporation of 35S-labeled amino acids, was also increased after incubation of neutrophils with GM-CSF. In addition, the total level of FLAP protein was increased in GM-CSF-treated neutrophils, as determined by two-dimensional gel electrophoresis, followed by Western blot. GM-CSF did not alter the stability of the FLAP protein, indicating that the effect of GM-CSF on FLAP accumulation was the consequence of increased de novo synthesis as opposed to decreased degradation of FLAP. Finally, incubation of neutrophils with the synthetic glucocorticoid dexamethasone directly stimulated the upregulation of FLAP mRNA and protein, and enhanced the effect of GM-CSF. Taken together, these data demonstrate that FLAP expression may be upmodulated after appropriate stimulation of neutrophils. The increase in FLAP expression induced by GM-CSF in inflammatory conditions could confer upon neutrophils a prolonged capacity to synthesize leukotrienes.

γ-Glutamylcysteine synthetase: mRNA stabilization and independent subunit transcription by 4-hydroxy-2-nonenal

1998 ◽  
Vol 275 (5) ◽  
pp. L861-L869 ◽  
Author(s):  
Rui-Ming Liu ◽  
Lin Gao ◽  
Jinah Choi ◽  
Henry Jay Forman
Keyword(s):  
De Novo ◽  
Mrna Level ◽  
Protein Synthesis Inhibitor ◽  
Lipid Peroxidation Product ◽  
Synthesis Inhibitor ◽  
Mrna Stabilization ◽  
Subunit Mrna ◽  
Mrna Content ◽  
Glutamylcysteine Synthetase ◽  
The Stability

γ-Glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo glutathione (GSH) synthesis, is composed of one catalytic (heavy) and one regulatory (light) subunit. Although both subunits are increased at the mRNA level by oxidants, it is not clear whether they are regulated through the same mechanism. 4-Hydroxy-2-nonenal (4HNE), a lipid peroxidation product, may act as a mediator for the induction of gene expression by oxidants. In the present study, 4HNE was used to study the mechanism of induction of the two GCS subunits in rat lung epithelial L2 cells. 4HNE increased both the transcription rates and the stability of mRNA for both GCS subunits, resulting in an increased mRNA content for both subunits. Both GCS subunit proteins and enzymatic activities also increased. Emetine, a protein synthesis inhibitor, blocked the increase in GCS light subunit mRNA but not the increase in GCS heavy subunit mRNA. This suggested that although 4HNE increased transcription and stabilization of both GCS subunit mRNAs, the signaling pathways involved in the induction of the two GCS subunits differed.

Determinants of kinin release in isolated rat hindquarters

1998 ◽  
Vol 274 (1) ◽  
pp. R120-R125 ◽  
Author(s):  
Tohru Sakakibara ◽  
Thomas H. Hintze ◽  
Alberto Nasjletti
Keyword(s):  
Protein Synthesis ◽  
Trypsin Inhibitor ◽  
De Novo ◽  
Soybean Trypsin Inhibitor ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Bicarbonate Buffer ◽  
Kallikrein Inhibitor ◽  
Isolated Rat

We studied the determinants of kinin release into the venous effluent of rat hindquarters perfused with Krebs bicarbonate buffer. Kinin release in preparations perfused with control media (14.6 ± 2.5–20.7 ± 6.7 pg/15 min) was surpassed by that in preparations perfused with media containing kininase inhibitors (243 ± 53 to 276 ± 78 pg/15 min). Kinin release increased when purified kininogen (from 242 ± 43 to 3,365 ± 725 pg/15 min) or kallikrein (from 270 ± 49 to 30,649 ± 8,040 pg/15 min) was added to the perfusate. Conversely, kinin release fell when the kallikrein inhibitor aprotinin (from 272 ± 58 to 122 ± 27 pg/15 min) or soybean trypsin inhibitor (from 273 ± 52 to 195 ± 25 pg/15 min) was added. Both basal and kininogen-induced kinin release were attenuated in preparations perfused with media containing cycloheximide, a protein synthesis inhibitor, but kallikrein-induced kinin release was not. These data suggest that kinin release from perfused rat hindquarters reflects the activity of both the kinin-degrading and kinin-generating pathways and that the latter is sustained by a kallikrein manufactured de novo and by preexistent kininogen(s).

scholarly journals Natural and environmental oestrogens induce TGFB1 synthesis in oviduct cells

Reproduction ◽  
2018 ◽  
Vol 155 (3) ◽  
pp. 233-244 ◽  
Author(s):  
Barbara P S Cometti ◽  
Raghvendra K Dubey ◽  
Bruno Imthurn ◽  
Marinella Rosselli
Keyword(s):  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
De Novo ◽  
Embryo Implantation ◽  
Early Embryo ◽  
Biochanin A ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Paracrine Factors ◽  
Aryl Hydrocarbon

Autocrine/paracrine factors generated in response to 17β-oestradiol (E2), within the oviduct, facilitate early embryo development for implantation. Since transforming growth factor beta 1 (TGFB1) plays a key role in embryo implantation, regulation of its synthesis by E2 may be of biological/pathophysiological relevance. Here, we investigated whether oviduct cells synthesize TGFB1 and whether E2 and environmental oestrogens (EOEs; xenoestrogens and phytoestrogens) modulate its synthesis. Under basal conditions, bovine oviduct cells (OCs; oviduct epithelial cells and oviduct fibroblasts; 1:1 ratio) synthesized TGFB1. E2 concentration-dependent induced TGFB1 levels in OCs and these effects were mimicked by some, but not all EOEs (genistein, biochanin A and 4-hydroxy-2′,4′,6′-trichlorobiphenyl, 4-hydroxy-2′,4′,6′-dichlorobiphenyl); moreover, EOEs enhanced (P < 0.05) the stimulatory effects of E2 on TGFB1 synthesis. The OCs expressed oestrogen receptors alpha and beta and aryl hydrocarbon; moreover, co-treatment with ER antagonist ICI182780 blocked the stimulatory effects of E2 and EOEs on TGFB1 synthesis. Treatment with non-permeable E2-BSA failed to induce TGFB1, thereby ruling out the involvement of membrane ERs. Cycloheximide (protein synthesis inhibitor) blocked E2-induced TGFB1 synthesis providing evidence forde novosynthesis. The stimulatory effects of E2 and EOEs, were inhibited (P < 0.05) by MAPK inhibitor (PD98059), whereas intracellular-Ca2+chelator (BAPTA-AM) and adenylyl cyclase inhibitor (SQ22536) abrogated the effects of E2, but not EOEs, suggesting that post-ER effects of E2 and EOEs involve different pathways. Our results provide the first evidence that in OCs, E2 and EOEs stimulate TGFB1 synthesis via an ER-dependent pathway. Exposure of the oviduct to EOEs may result in continuous/sustained induction of TGFB1 levels in a non-cyclic fashion and may induce deleterious effects on reproduction.

Insulin stimulates sodium-dependent phosphate transport by osteoblast-like cells

1991 ◽  
Vol 260 (5) ◽  
pp. E751-E755 ◽  
Author(s):  
K. J. Kunkler ◽  
L. M. Everett ◽  
D. K. Breedlove ◽  
S. A. Kempson
Keyword(s):  
De Novo ◽  
Insulin Receptors ◽  
Phosphate Transport ◽  
Osteosarcoma Cell ◽  
Maximal Velocity ◽  
Stimulatory Action ◽  
Cotransport System ◽  
Umr 106 ◽  
Dose Dependent ◽  
De Novo Protein Synthesis

The rat osteosarcoma cell line UMR-106-01 has an osteoblast-like phenotype. When grown in monolayer culture, these cells transport Pi via a Na(+)-dependent carrier. The Na(+)-Pi cotransport system is stimulated by parathyroid hormone (PTH). Because there are insulin receptors on osteoblast-like cells, we determined possible effects of insulin on Na(+)-Pi cotransport in UMR-106-01 cells. Incubation of cells with 10(-8) M insulin for 3 h produced a 73% increase (P less than 0.025) in Na(+)-Pi cotransport. There was no significant change in Na(+)-L-alanine cotransport or in Na(+)-independent uptake of Pi and alanine. The stimulatory action of insulin on Na(+)-Pi cotransport was present within 2 h and was dose dependent in the range 10(-10) to 10(-7) M. The increase in Na(+)-Pi cotransport was accompanied by an increase in apparent maximal velocity with no change in apparent Michaelis constant for Pi. Use of cycloheximide to block de novo protein synthesis did not interfere with this action of insulin. We conclude that insulin, like PTH, directly stimulates the Na(+)-Pi cotransport system in osteoblast-like cells. The mechanism remains to be determined.

scholarly journals Retinoic acid receptor γ 2 gene expression is up-regulated by retinoic acid in 3T3-L1 preadipocytes

1993 ◽  
Vol 293 (3) ◽  
pp. 807-812 ◽  
Author(s):  
Y Kamei ◽  
T Kawada ◽  
R Kazuki ◽  
E Sugimoto
Keyword(s):  
Adipose Tissue ◽  
Retinoic Acid ◽  
Mrna Expression ◽  
De Novo ◽  
Retinoic Acid Receptor ◽  
Mrna Level ◽  
Fold Increase ◽  
Dependent Manner ◽  
Alpha Beta ◽  
Rat Adipose Tissue

Retinoids, especially all-trans retinoic acid (RA), have been shown to inhibit the differentiation of preadipose cells. In the present study, the expression of retinoic acid receptors (RAR alpha, beta and gamma) and retinoid X receptors (RXR alpha, beta and gamma) was examined by Northern blot analysis in rat adipose tissue and mouse 3T3-L1 adipose cells. The adipose tissue and/or 3T3-L1 cells expressed mRNAs for a number of nuclear retinoid receptors, including RAR alpha, beta and gamma, and RXR alpha, beta and gamma. RAR alpha, RAR gamma, RXR alpha and RXR beta mRNAs were abundant in adipose tissue and 3T3-L1 cells. RXR gamma mRNA was detected in adipose tissue but not in 3T3-L1 cells. Treatment of 3T3-L1 cells with 1 microM RA led to a 4-5-fold increase in the RAR gamma mRNA level, but only a trace amount of RAR beta mRNA was detected. RAR gamma mRNA expression was rapidly (within 2 h) induced by physiological concentrations of RA in a dose-dependent manner. The response of RAR gamma mRNA expression to RA was reversible; rapid disappearance of RAR gamma mRNA occurred on RA removal. In addition, the induction of RAR gamma expression did not require de novo protein synthesis, but was completely abolished by an inhibitor of RNA synthesis. Using RAR gamma 1 and gamma 2 isoform-specific probes, the patterns of RAR gamma 1 and gamma 2 mRNA expression in 3T3-L1 cells in the presence and absence of RA were examined. RAR gamma 1 mRNA was detected in 3T3-L1 cells but was not affected by RA treatment; however, RAR gamma 2 mRNA was strongly induced by RA.

Effect of Cerophyl growth medium on exocytosis in Tetrahymena thermophila

1985 ◽  
Vol 78 (1) ◽  
pp. 23-48
Author(s):  
D.M. Pesciotta ◽  
B.H. Satir
Keyword(s):  
Cell Division ◽  
De Novo ◽  
Tetrahymena Thermophila ◽  
Freeze Fracture ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Particle Array ◽  
Proteose Peptone ◽  
Section Electron ◽  
Cell Changes

Culturing the ciliate Tetrahymena thermophila in Cerophyl has provided an opportunity for studying the assembly and/or synthesis of the intramembrane particle array, the rosette, which marks the site of exocytosis in these cells. Cultures grown in this medium cease cell division after only 12h and enter ‘stationary phase’ earlier (12h of growth) relative to growth in standard medium (proteose peptone). In addition, the cell changes from the normally observed pear-shaped body to a thinner more ellipsoid form. Despite the initial similarities to starving cells, several differences are observed in the Cerophyl-grown cells. One is that cell size remains constant for at least 72h in contrast to starved cells. Secondly, in spite of this block in cell division, results from freeze-fracture replicas of the cell membrane of these cells show that they continue to assemble rosettes, the number of which increases approximately six times, from 0.34 rosette/microgram2 to 2.1 rosettes/microgram2. Addition of the protein synthesis inhibitor, cycloheximide (6h exposure), during growth in Cerophyl shows that 70% of rosettes can be assembled, despite the blockage of translation, by using pre-existing component(s) from a pool. The remaining 30% must involve de novo synthesis of one or more components; this percentage can be increased with longer exposure to the drug. Thirdly, an apparent increase in the number of mucocysts is observed by thin-section electron microscopy. At first (12–24h) only docked mucocysts seem to accumulate in the cell. However, by 36h a considerable increase seems to have taken place, particularly in the number of mucocysts located in the cytoplasm. In the cycloheximide-treated cells this increase in mucocysts begins to be blocked after 6h of exposure to the drug. These observations are in agreement with the results obtained from the freeze-fracture data on the concomitant increase in number of rosettes. This system therefore offers the first possibility of exploring the biosynthesis of these components.

Stimulation of Tetrahydrobiopterin Synthesis by Basic Fibroblast Growth Factor in Vascular Endothelial Cells

Pteridines ◽  
2003 ◽  
Vol 14 (1) ◽  
pp. 9-12 ◽  
Author(s):  
Shunichi Shimizu ◽  
Yoshiyuki Miyasaka ◽  
Shinichiro Yamamoto ◽  
Masakazu Ishii ◽  
Yuji Kiuchi
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
De Novo ◽  
Mrna Level ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Fibroblast Growth ◽  
Concentration Dependent Manner

Abstract The purpose of this study was to examine whether basic fibroblast growth factor (bFGF) stimulates tetrahydrobiopterin (BH4) synthesis in mouse brain microvascular endothelial cells. BH4 content was determined by oxidation under acidic conditions as biopterin and analysed with reversed-phase high Performance liquid chromatography. Measurement of the mRNA level of QTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme of the de novo pathway of BH4 synthesis. The addition of bFGF to endothelial cells increased the BH4 content and GTPCH mRNA levels in an incubation period- and a concentration-dependent manner. 2,4-Diamino-6- hydroxypyrimidine, an inhibitor of GTPCH, strongly reduced the bFGF-induced increase in BH4 content. These findings suggest that bFGF stimulates BH4 synthesis via a de novo pathway with the induction of GTPCH.

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