Mediation of the hormone- and serum-dependent regulation of thyroglobulin gene expression by thyroid-transcription factors in rat thyroid FRTL-5 cells

1996 ◽  
Vol 150 (2) ◽  
pp. 287-298 ◽  
Author(s):  
F Kambe ◽  
H Seo
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Gene Transcription ◽  
Mrna Level ◽  
Binding Activity ◽  
Mrna Levels ◽  
Mobility Shift ◽  
Cell Extracts ◽  
Rat Thyroid ◽  
Serum Dependent

Abstract The molecular mechanism for hormone- and serum-dependent regulation of thyroglobulin (TG) gene expression was studied. A construct of rat TG promoter (−178 to −3) linked to a luciferase gene was transfected into TSH-, insulin- and serum-deprived FRTL-5 cells. Addition of TSH, insulin or serum augmented the luciferase activity. The endogenous TG mRNA level was also increased, indicating that the promoter used confers responsiveness of TG gene to these additives. The possible involvement of thyroid-transcription factors, TTF-1, TTF-2 and Pax-8, in the induction of TG gene transcription was studied using an electrophoretic mobility shift assay. Since the protein/DNA ratio in FRTL-5 cell extracts was significantly increased by these additives, binding activities of these factors per unit of DNA were examined. It was demonstrated that TSH, insulin or serum increased not only TTF-2 binding activity but also the binding activities of TTF-1 and Pax-8. However, the magnitude of the increase in TTF-1 and Pax-8 mRNA levels per unit of DNA was less than that of the binding activity. Taken together, our results suggest that TSH, insulin and serum increase the binding activities of TTF-1 and Pax-8 to the TG promoter presumably through the posttranslational modification of the factors, thereby enhancing TG gene transcription. Journal of Endocrinology (1996) 150, 287–298

scholarly journals Glucocorticoid repression of human with-no-lysine (K) kinase-4 gene expression is mediated by the negative response elements in the promoter

2007 ◽  
Vol 40 (1) ◽  
pp. 3-12 ◽  
Author(s):  
Chunyi Li ◽  
Yan Li ◽  
Yinghui Li ◽  
Hong Liu ◽  
Zhijun Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Level ◽  
Gene Promoter ◽  
Initiation Site ◽  
Binding Activity ◽  
Hek293 Cells ◽  
Mobility Shift ◽  
Transcriptional Initiation ◽  
Response Elements ◽  
Real Time Quantitative Pcr

With-no-lysine (K) kinase-4 (WNK4) is a serine/threonine kinase that plays an essential role in the regulation of fluid and electrolyte homeostasis. The effects of glucocorticoids, key physiological regulators, on the WNK4 gene expression are still unknown. Here, we used dexamethasone (Dex) to treat the human embryo kidney 293 (HEK293) cells and found a decrease of human WNK4 (hWNK4) mRNA level by northern blot and real-time quantitative PCR. After an hWNK4 transcriptional initiation site was located by 5′ rapid amplification of cDNA end assay, a series of 5′-deleted hWNK4 promoter–luciferase constructs were generated by PCR. Transfection of these constructs in COS-7 and HEK293 cells revealed that Dex inhibited the hWNK4 transcriptional activity in glucocorticoid receptor (GR)-dependent pattern. Two negative glucocorticoid response elements (nGREs) were identified at −285 and −337 of the hWNK4 gene promoter and the GR binding activity to them was increased by Dex as shown by electrophoretic mobility shift assay and chromatin immunoprecipitation. In summary, these data demonstrated that hWNK4 was a new glucocorticoid-regulated gene whose expression was inhibited through the interaction of GR with nGREs in the promoter region.

scholarly journals GATA augments GNRH-mediated increases in Adcyap1 gene expression in pituitary gonadotrope cells

2013 ◽  
Vol 51 (3) ◽  
pp. 313-324 ◽  
Author(s):  
Robin L Thomas ◽  
Natalie M Crawford ◽  
Constance M Grafer ◽  
Weiming Zheng ◽  
Lisa M Halvorson
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Proximal Promoter ◽  
Mrna Levels ◽  
Paracrine Factor ◽  
Mobility Shift ◽  
Subunit Expression ◽  
Mobility Shift Assay

Pituitary adenylate cyclase-activating polypeptide 1 (PACAP or ADCYAP1) regulates gonadotropin biosynthesis and secretion, both alone and in conjunction with GNRH. Initially identified as a hypothalamic-releasing factor, ADCYAP1 subsequently has been identified in pituitary gonadotropes, suggesting it may act as an autocrine–paracrine factor in this tissue. GNRH has been shown to increase pituitaryAdcyap1gene expression through the interaction of CREB and jun/fos with CRE/AP1cis-elements in the proximal promoter. In these studies, we were interested in identifying additional transcription factors and cognatecis-elements which regulateAdcyap1gene promoter activity and chose to focus on the GATA family of transcription factors known to be critical for both pituitary cell differentiation and gonadotropin subunit expression. By transient transfection and electrophoretic mobility shift assay analysis, we demonstrate that GATA2 and GATA4 stimulateAdcyap1promoter activity via a GATAcis-element located at position −191 in the ratAdcyap1gene promoter. Furthermore, we show that addition of GATA2 or GATA4 significantly augments GNRH-mediated stimulation ofAdcyap1gene promoter activity in the gonadotrope LβT2 cell line. Conversely, blunting GATA expression with specific siRNA inhibits the ability of GNRH to stimulate ADCYAP1 mRNA levels in these cells. These data demonstrate a complex interaction between GNRH and GATA on ADCYAP1 expression, providing important new insights into the regulation of gonadotrope function.

Transcriptional and posttranscriptional control of c-myc gene expression in WEHI 231 cells

1986 ◽  
Vol 6 (11) ◽  
pp. 4112-4116
Author(s):  
R A Levine ◽  
J E McCormack ◽  
A Buckler ◽  
G E Sonenshein
Keyword(s):  
Gene Expression ◽  
Gene Transcription ◽  
Mrna Level ◽  
B Cell Lymphoma ◽  
Initial Increase ◽  
Mrna Levels ◽  
Posttranscriptional Control ◽  
Inhibition Of Growth ◽  
Wehi 231 ◽  
Myc Gene

Incubation of WEHI 231 cells, derived from a murine B-cell lymphoma, with antisera directed against its surface immunoglobulin results in the inhibition of growth within 24 h. Previously, we demonstrated that this treatment selectively affects cytoplasmic levels of c-myc mRNA (J. E. McCormack, V. H. Pepe, R. B. Kent, M. Dean, A. Marshak-Rothstein, and G. E. Sonenshein, Proc. Natl. Acad. Sci. USA 81:5546-5550, 1984). An initial increase in the cytoplasmic mRNA level is followed by a precipitous drop. We now show that the early increase results from a dramatic increase in the rate of c-myc gene transcription, as well as from partial stabilization of the mRNA in the cytoplasm. The later decrease results from a shutdown in transcription of the c-myc gene and a return to the normal lability of the cytoplasmic c-myc mRNA. Treatment with phorbol ester, like treatment with anti-immunoglobulin sera, inhibited WEHI 231 cell growth and caused similar changes in cytoplasmic c-myc mRNA levels, which can also be related to alterations in c-myc gene transcription. These results indicate that the control of c-myc gene expression in B cells is effected through regulation at multiple levels.

scholarly journals Cell-Cycle–Dependent Regulation of Erythropoietin Receptor Gene

Blood ◽  
1997 ◽  
Vol 89 (4) ◽  
pp. 1182-1188 ◽  
Author(s):  
Norio Komatsu ◽  
Keita Kirito ◽  
Yoshifumi Kashii ◽  
Yusuke Furukawa ◽  
Jiro Kikuchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Growth ◽  
Receptor Gene ◽  
Mrna Level ◽  
State Level ◽  
Erythropoietin Receptor ◽  
Cycle Phase ◽  
Mrna Levels ◽  
Mobility Shift

Abstract To understand the regulatory mechanism of erythropoietin (EPO) receptor (EPOR) gene expression, the effect of EPO on the steady-state level of EPOR mRNA was examined using the human EPO-dependent cell line UT-7 as a model system. We found that the treatment of UT-7 cells with EPO resulted in a transient decrease of the EPOR mRNA level. This transient downregulation was also induced by stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF ), another stimulator of UT-7 cell growth. These results raised the possibility that EPOR gene expression is in part related to cell growth. Moreover, it was found that EPO-induced downregulation of EPOR mRNA level was preceded by a transient downregulation of GATA-1 mRNA. To examine the relationship between the expression of EPOR, GATA-1, and GATA-2 mRNA levels and the cell cycle, logarithmically growing UT-7 cells were centrifugically fractionated according to the cell-cycle phase. Both EPOR and GATA-1 mRNA levels, but not the GATA-2 mRNA level, concomitantly decreased at the G0/G1 phase and increased at the S and G2/M phases. An electrophoretic mobility shift assay (EMSA) showed that in EPO-stimulated UT-7 cells, the dynamic changes in EPOR gene expression paralleled the GATA-1 DNA-binding activity to the oligonucleotide probe containing a GATA-binding site located at the promoter region of the EPOR gene. These findings suggest that the regulation of EPOR mRNA level is mainly associated with GATA-1 gene expression in UT-7 cells undergoing proliferation, and that these serial events are under the control of, or related to, the cell cycle.

scholarly journals Transcriptional and posttranscriptional control of c-myc gene expression in WEHI 231 cells.

1986 ◽  
Vol 6 (11) ◽  
pp. 4112-4116 ◽  
Author(s):  
R A Levine ◽  
J E McCormack ◽  
A Buckler ◽  
G E Sonenshein
Keyword(s):  
Gene Expression ◽  
Gene Transcription ◽  
Mrna Level ◽  
B Cell Lymphoma ◽  
Initial Increase ◽  
Mrna Levels ◽  
Posttranscriptional Control ◽  
Inhibition Of Growth ◽  
Wehi 231 ◽  
Myc Gene

Incubation of WEHI 231 cells, derived from a murine B-cell lymphoma, with antisera directed against its surface immunoglobulin results in the inhibition of growth within 24 h. Previously, we demonstrated that this treatment selectively affects cytoplasmic levels of c-myc mRNA (J. E. McCormack, V. H. Pepe, R. B. Kent, M. Dean, A. Marshak-Rothstein, and G. E. Sonenshein, Proc. Natl. Acad. Sci. USA 81:5546-5550, 1984). An initial increase in the cytoplasmic mRNA level is followed by a precipitous drop. We now show that the early increase results from a dramatic increase in the rate of c-myc gene transcription, as well as from partial stabilization of the mRNA in the cytoplasm. The later decrease results from a shutdown in transcription of the c-myc gene and a return to the normal lability of the cytoplasmic c-myc mRNA. Treatment with phorbol ester, like treatment with anti-immunoglobulin sera, inhibited WEHI 231 cell growth and caused similar changes in cytoplasmic c-myc mRNA levels, which can also be related to alterations in c-myc gene transcription. These results indicate that the control of c-myc gene expression in B cells is effected through regulation at multiple levels.

scholarly journals NF-κB activity in muscle from obese and type 2 diabetic subjects under basal and exercise-stimulated conditions

2010 ◽  
Vol 299 (5) ◽  
pp. E794-E801 ◽  
Author(s):  
Puntip Tantiwong ◽  
Karthigayan Shanmugasundaram ◽  
Adriana Monroy ◽  
Sangeeta Ghosh ◽  
Mengyao Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Animal Studies ◽  
Acute Exercise ◽  
Human Subjects ◽  
Mrna Level ◽  
Binding Activity ◽  
Moderate Intensity ◽  
Mrna Levels ◽  
Insulin Resistant ◽  
Dna Binding Activity

NF-κB is a transcription factor that controls the gene expression of several proinflammatory proteins. Cell culture and animal studies have implicated increased NF-κB activity in the pathogenesis of insulin resistance and muscle atrophy. However, it is unclear whether insulin-resistant human subjects have abnormal NF-κB activity in muscle. The effect that exercise has on NF-κB activity/signaling also is not clear. We measured NF-κB DNA-binding activity and the mRNA level of putative NF-κB-regulated myokines interleukin (IL)-6 and monocyte chemotactic protein-1 (MCP-1) in muscle samples from T2DM, obese, and lean subjects immediately before, during (40 min), and after (210 min) a bout of moderate-intensity cycle exercise. At baseline, NF-κB activity was elevated 2.1- and 2.7-fold in obese nondiabetic and T2DM subjects, respectively. NF-κB activity was increased significantly at 210 min following exercise in lean (1.9-fold) and obese (2.6-fold) subjects, but NF-κB activity did not change in T2DM. Exercise increased MCP-1 mRNA levels significantly in the three groups, whereas IL-6 gene expression increased significantly only in lean and obese subjects. MCP-1 and IL-6 gene expression peaked at the 40-min exercise time point. We conclude that insulin-resistant subjects have increased basal NF-κB activity in muscle. Acute exercise stimulates NF-κB in muscle from nondiabetic subjects. In T2DM subjects, exercise had no effect on NF-κB activity, which could be explained by the already elevated NF-κB activity at baseline. Exercise-induced MCP-1 and IL-6 gene expression precedes increases in NF-κB activity, suggesting that other factors promote gene expression of these cytokines during exercise.

Nitric oxide increases IL-8 gene transcription and mRNA stability to enhance IL-8 gene expression in lung epithelial cells

2004 ◽  
Vol 287 (4) ◽  
pp. L764-L773 ◽  
Author(s):  
Loretta Sparkman ◽  
Vijayakumar Boggaram
Keyword(s):  
Gene Expression ◽  
Nitric Oxide ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
Gene Transcription ◽  
Mrna Stability ◽  
Inflammatory Responses ◽  
Lung Epithelial Cells ◽  
Mrna Levels ◽  
Lung Epithelial

Interleukin (IL)-8, a C-X-C chemokine, is a potent chemoattractant and an activator for neutrophils, T cells, and other immune cells. The airway and respiratory epithelia play important roles in the initiation and modulation of inflammatory responses via production of cytokines and surfactant. The association between elevated levels of nitric oxide (NO) and IL-8 in acute lung injury associated with sepsis, acute respiratory distress syndrome, respiratory syncytial virus infection in infants, and other inflammatory diseases suggested that NO may play important roles in the control of IL-8 gene expression in the lung. We investigated the role of NO in the control of IL-8 gene expression in H441 lung epithelial cells. We found that a variety of NO donors significantly induced IL-8 mRNA levels, and the increase in IL-8 mRNA was associated with an increase in IL-8 protein. NO induction of IL-8 mRNA was due to increases in IL-8 gene transcription and mRNA stability. NO induction of IL-8 mRNA levels was not inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and KT-5823, inhibitors of soluble guanylate cyclase and protein kinase G, respectively, and 8-bromo-cGMP did not increase IL-8 mRNA levels. This indicated that NO induces IL-8 mRNA levels independently of changes in the intracellular cGMP levels. NO induction of IL-8 mRNA was significantly reduced by inhibitors of extracellular regulated kinase and protein kinase C. IL-8 induction by NO was also reduced by hydroxyl radical scavengers such as dimethyl sulfoxide and dimethylthiourea, indicating the involvement of hydroxyl radicals in the induction process. NO induction of IL-8 gene expression could be a significant contributing factor in the initiation and induction of inflammatory response in the respiratory epithelium.

scholarly journals Feedback Inhibition of Human Scavenger Receptor Class B Type I Gene Expression by Glucocorticoid in Adrenal and Ovarian Cells

Endocrinology ◽  
2010 ◽  
Vol 151 (7) ◽  
pp. 3214-3224 ◽  
Author(s):  
Sofia Mavridou ◽  
Maria Venihaki ◽  
Olga Rassouli ◽  
Christos Tsatsanis ◽  
Dimitris Kardassis
Keyword(s):  
Gene Expression ◽  
Gene Transcription ◽  
Feedback Inhibition ◽  
Steroid Hormone ◽  
Scavenger Receptor ◽  
Type I ◽  
Mrna Levels ◽  
Ovarian Cells ◽  
Scavenger Receptor Class ◽  
Class B

Scavenger receptor class B type I (SR-BI) facilitates the reverse transport of excess cholesterol from peripheral tissues to the liver via high-density lipoproteins. In steroidogenic tissues, SR-BI supplies cholesterol for steroid hormone production. We show here that the transcription of the human SR-BI gene is subject to feedback inhibition by glucocorticoid in adrenal and ovarian cells. SR-BI mRNA levels were increased in adrenals from corticosterone-insufficient Crh−/− mice, whereas corticosterone replacement by oral administration inhibited SR-BI gene expression in these mice. SR-BI mRNA levels were increased in adrenals from wild-type mice treated with metyrapone, a drug that blocks corticosterone synthesis. Experiments in adrenocortical H295R and ovarian SKOV-3 cells using cycloheximide and siRNA-mediated gene silencing revealed that glucocorticoid-mediated inhibition of SR-BI gene transcription requires de novo protein synthesis and the glucocorticoid receptor (GR). No direct binding of GR to the SR-BI promoter could be demonstrated in vitro and in vivo, suggesting an indirect mechanism of repression of SR-BI gene transcription by GR in adrenal cells. Deletion analysis established that the region of the human SR-BI promoter between nucleotides −201 and −62 is sufficient to mediate repression by glucocorticoid. This region contains putative binding sites for transcriptional repressors that could play a role in SR-BI gene regulation in response to glucocorticoid. In summary, this is the first report showing that glucocorticoid suppress SR-BI expression suggesting that steroidogenic tissues maintain steroid hormone homeostasis by prohibiting SR-BI-mediated high-density lipoprotein cholesterol uptake when the endogenous levels of glucocorticoid are elevated.

scholarly journals Characterization of the Crithidia fasciculata mRNA Cycling Sequence Binding Proteins

2001 ◽  
Vol 21 (14) ◽  
pp. 4453-4459 ◽  
Author(s):  
Riaz Mahmood ◽  
Bidyottam Mittra ◽  
Jane C. Hines ◽  
Dan S. Ray
Keyword(s):  
Cell Cycle ◽  
Protein Complexes ◽  
Coiled Coil ◽  
High Specificity ◽  
Binding Activity ◽  
Mrna Levels ◽  
Crithidia Fasciculata ◽  
Cysteine Motif ◽  
Cell Extracts ◽  
Sequence Elements

ABSTRACT The Crithidia fasciculata cycling sequence binding protein (CSBP) binds with high specificity to sequence elements in several mRNAs that accumulate periodically during the cell cycle. Mutations in these sequence elements abolish both cycling of the mRNA and binding of CSBP. Two genes, CSBPA andCSBPB, encoding putative subunits of CSBP have been cloned and were found to be present in tandem on the same DNA molecule and to be closely related. CSBPA andCSBPB are predicted to encode proteins with sizes of 35.6 and 42.0 kDa, respectively. Both CSBPA and CSBPB proteins have a predicted coiled-coil domain near the N terminus and a novel histidine and cysteine motif near the C terminus. The latter motif is conserved in other trypanosomatid species. Gel sieving chromatography and glycerol gradient sedimentation results indicate that CSBP has a molecular mass in excess of 200 kDa and an extended structure. Recombinant CSBPA and CSBPB also bind specifically to the cycling sequence and together can be reconstituted to give an RNA gel shift similar to that of purified CSBP. Proteins in cell extracts bind to an RNA probe containing six copies of the cycling sequence. The RNA-protein complexes contain both CSBPA and CSBPB, and the binding activity cycles in near synchrony with target mRNA levels.CSBPA and CSBPB mRNA and protein levels show little variation throughout the cell cycle, suggesting that additional factors are involved in the cyclic binding to the cycling sequence elements.

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