scholarly journals Sex Differences in Estrogen-Dependent Transcription of Gonadotropin-Releasing Hormone (GnRH) Gene Revealed in GnRH Transgenic Mice

Endocrinology ◽  
2003 ◽  
Vol 144 (8) ◽  
pp. 3351-3358 ◽  
Author(s):  
Niren R. Thanky ◽  
Ruth Slater ◽  
Allan E. Herbison
Keyword(s):  
Transgenic Mice ◽  
Mrna Expression ◽  
Gene Transcription ◽  
Transgene Expression ◽  
Gonadal Steroids ◽  
Critical Element ◽  
Sexually Dimorphic ◽  
Mrna Levels ◽  
Estrogen Replacement

Abstract The mechanisms through which gonadal steroids exert feedback actions on the activity of the GnRH neurons are not understood. Using a series of GnRH-LacZ transgenic mice we have examined the manner in which gonadal steroids suppress GnRH mRNA expression in male and female mice. The long-term gonadectomy of 5.5-GNZ-3.5 transgenic mice resulted in significant increases in cellular GnRH mRNA expression (P < 0.05) and plasma LH concentrations (P < 0.01) in both sexes. However, cellular levels of LacZ mRNA and β-galactosidase, which provide an index of GnRH gene transcription, were only elevated in males after gonadectomy. This sexually differentiated response was also observed in mice gonadectomized for 2 wk. Estrogen replacement in gonadectomized males returned transgene expression to intact levels. Experiments in transgenic mice with 3′ and 5′ deleted GnRH-LacZ constructs revealed that the suppressive influence of estrogen on LacZ transcription in the male required a critical element located between −5.2 and −1.7 kb of the GnRH promoter. These studies show that the suppression of GnRH mRNA expression by estrogen in the male involves a decrease in GnRH gene transcription that is dependent on a distal GnRH promoter element. The same mechanism does not exist in females, indicating that gonadal steroids suppress GnRH mRNA levels in a sexually dimorphic manner.

The Corfu δβ thalassemia deletion disrupts γ-globin gene silencing and reveals post-transcriptional regulation of HbF expression

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 2154-2160 ◽  
Author(s):  
Lyubomira Chakalova ◽  
Cameron S. Osborne ◽  
Yan-Feng Dai ◽  
Beatriz Goyenechea ◽  
Anna Metaxotou-Mavromati ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Gene Transcription ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Critical Threshold ◽  
Critical Element ◽  
Mrna Levels ◽  
Globin Genes ◽  
Mrna Accumulation ◽  
Post Transcriptional Regulation

Abstract The 7.2 kilobase (kb) Corfu δβ thalassemia mutation is the smallest known deletion encompassing a region upstream of the human δ gene that has been suggested to account for the vastly different phenotypes in hereditary persistence of fetal hemoglobin (HPFH) versus β thalassemia. Fetal hemoglobin (HbF) expression in Corfu heterozygotes and homozygotes is paradoxically dissimilar, suggesting conflicting theories as to the function of the region on globin gene regulation. Here, we measure γ- and β-globin gene transcription, steady-state mRNA, and hemoglobin expression levels in primary erythroid cells cultured from several patients with Corfu δβ thalassemia. We show through RNA fluorescence in situ hybridization that the Corfu deletion results in high-level transcription of the fetal γ genes in cis with a concomitant reduction in transcription of the downstream β gene. Surprisingly, we find that elevated γ gene transcription does not always result in a corresponding accumulation of γ mRNA or fetal hemoglobin, indicating a post-transcriptional regulation of γ gene expression. The data suggest that efficient γ mRNA accumulation and HbF expression are blocked until β mRNA levels fall below a critical threshold. These results explain the Corfu paradox and show that the deleted region harbors a critical element that functions in the developmentally regulated transcription of the β-globin genes.

Role of glucocorticoids in activation of hepatic PEPCK gene transcription during exercise

1994 ◽  
Vol 266 (4) ◽  
pp. E560-E566 ◽  
Author(s):  
J. E. Friedman
Keyword(s):  
Transgenic Mice ◽  
Gene Transcription ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Mrna Levels ◽  
Camp Response Element ◽  
Carbohydrate Diet ◽  
High Carbohydrate ◽  
Enhancer Binding Protein ◽  
Response To Exercise

The objective of these studies was to determine the molecular basis for the activation of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription during prolonged submaximal exercise. Mice were fed a high-carbohydrate diet for 1 wk and exercised continuously by swimming for up to 120 min. The level of hepatic PEPCK mRNA increased progressively during exercise, reaching 510% above control, whereas transcription of the PEPCK gene increased 1,000%, before decreasing to control levels within 60 min of recovery. In transgenic mice carrying a chimeric gene consisting of the PEPCK promoter linked to a reporter gene for bovine growth hormone (bGH), PEPCK(-460)-bGH, the level of hepatic bGH mRNA increased by 490% in response to exercise, similar to the increase in the expression of the native PEPCK gene. However, in transgenic mice with a deletion of the glucocorticoid regulatory unit, PEPCK(-355)-bGH, bGH mRNA did not increase above control values. In transgenic mice with a block mutation in adenosine 3',5'-cyclic monophosphate (cAMP) regulatory regions -90/-82 and -250/-234, PEPCK cAMP response element 1 (CRE-1)/P3(1)-bGH, exercise increased bGH mRNA 260% above controls. Adrenalectomy (Adx) had no effect on PEPCK mRNA levels in nonexercised mice, whereas in adrenalectomized (Adx)-exercised mice, PEPCK mRNA increased only 80% above basal, and, in Adx mice injected with dexamethasone, PEPCK mRNA increased with exercise 570% above controls. Exercise was also associated with a large increase in transcription of the gene for the transcription factor CCAAT/enhancer-binding protein beta (C/EBP-beta) and a smaller rise in transcription of c-jun gene, both of which returned to control levels during recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of expression of a sheep metallothionein 1a-sheep growth hormone fusion gene in transgenic mice

1989 ◽  
Vol 9 (12) ◽  
pp. 5473-5479
Author(s):  
C M Shanahan ◽  
N W Rigby ◽  
J D Murray ◽  
J T Marshall ◽  
C A Townrow ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Transgenic Mice ◽  
Mrna Expression ◽  
Transgene Expression ◽  
Fusion Gene ◽  
Developmental Expression ◽  
Regulation Of Expression ◽  
Tissue Specific ◽  
Transgenic Livestock ◽  
Main Regulator

Transgenic mice containing a sheep metallothionein 1a-sheep growth hormone fusion gene exhibited low, tissue-specific basal levels of transgene mRNA expression, resulting in slightly elevated levels of circulating growth hormone that did not lead to a detectable increase in growth. After zinc stimulation, high levels of transgene mRNA expression were induced in a number of tissues; these levels correlated with increased levels of circulating growth hormone, resulting in growth increases of up to 1.5 times the levels of controls and unstimulated transgenic mice. After removal of the zinc stimulus, transgene expression and circulating growth hormone concentrations returned to basal levels. Additional evidence from the pattern of developmental expression of the transgene suggests that zinc is the main regulator of this promoter in mice. The demonstrated regulation and low basal level of expression of the sheep metallothionein 1a promoter make it a candidate for use in other mouse transgenic studies and for use in transgenic livestock, in which regulation of expression is essential.

249. Fibroblast growth factor receptor-1 (FGFR-1) is essential for spermiogenesis, capacitation and male fertility

2005 ◽  
Vol 17 (9) ◽  
pp. 99
Author(s):  
L. M. Cotton ◽  
G. M. Gibbs ◽  
D. M. De Kretser ◽  
M. K. O'Bryan
Keyword(s):  
Transgenic Mice ◽  
Male Fertility ◽  
Transgene Expression ◽  
Surrogate Marker ◽  
Dominant Negative ◽  
Mrna Levels ◽  
Sperm Tail ◽  
Expression Levels ◽  
Over Expression ◽  
Sperm Development

Male infertility is often a result of irregular sperm development/function. The identification of snt-2 (Suc-1 associated Neurotrophic Factor Target 2) and Fgfr-1 to the sperm tail, lead to the hypothesis that Fgf signalling through snt-2 is involved in sperm tail development/function. To test this hypothesis, transgenic mice carrying a dominant-negative variant of Fgfr-1, driven by the protamine 1 promoter (haploid specific) were created. Breeding experiments confirmed male fertility; however, one line was significantly sub-fertile and demonstrated a significantly reduced daily sperm production (DSP, 30%↓). Transgene expression levels were up to 70 times above native mRNA levels in wt mice; however, there was a concurrent upregulation of the native receptor in transgenic mice, resulting in only a 6× over-expression in transgenic:native mRNA. To increase transgene expression, independent lines were crossed (double heterozygous, DH). DH transgene expression levels were up to 120 times above the native mRNA in wild type mice, resulting in a 20× over-expression in transgenic:native mRNA. Breeding experiments showed males from 1 cross were significantly subfertile with DSPs further reduced (41%↓). Collectively this data shows Fgfr-1 signalling is required for quantitatively normal spermiogenesis. Given the millions of sperm that mice produce, a 40%↓ in DSP is unlikely to be responsible for the sub-fertility observed i.e. 2 v. 9 pups/litter. Therefore, a disruption of Fgfr-1 signalling may also induce a post-testicular phenotype. Western blot analysis, using tyrosine phosphorylation as a surrogate marker of sperm capacitation, showed transgenic mice had a significantly attenuated ability to initiate capacitation. As capacitation is an absolute requirement for fertilisation, the absence of capacitating capability is probably the major contributor to the sub-fertility seen in the transgenic mice. This research demonstrates for the first time that the Fgfr-1 signalling cascade is one of several pathways associated with sperm development and function.

scholarly journals Tumor Necrosis Factor-α Stimulates Lactate Dehydrogenase A Expression in Porcine Cultured Sertoli Cells: Mechanisms of Action

Endocrinology ◽  
1999 ◽  
Vol 140 (7) ◽  
pp. 3054-3062 ◽  
Author(s):  
Fayçal Boussouar ◽  
Renée Grataroli ◽  
Jingwei Ji ◽  
Mohamed Benahmed
Keyword(s):  
Mrna Expression ◽  
Gene Transcription ◽  
Sertoli Cells ◽  
Tumor Necrosis ◽  
Actinomycin D ◽  
Lactate Production ◽  
Mrna Levels ◽  
Kinase C ◽  
Factor Α ◽  
Necrosis Factor

Abstract In the present study, we investigated the regulatory action of tumor necrosis factor-α (TNFα) on lactate dehydrogenase A (LDH A), a key enzyme involved in lactate production. To this end, use was made of a primary culture system of porcine testicular Sertoli cells. TNFα stimulated LDH A messenger RNA (mRNA) expression in a dose (ED50 = 2.5 ng/ml; 0.1 nm TNFα)-dependent manner. This stimulatory effect was time dependent, with an effect detected after 6 h of TNFα treatment and maximal after 48 h of exposition (5-fold; P < 0.001). The direct effect of TNFα on LDH A mRNA could not be accounted for by an increase in mRNA stability (half-life = 9 h), but was probably due to an increase in LDH A gene transcription. Inhibitors of protein synthesis (cycloheximide), gene transcription (actinomycin D and dichlorobenzimidazole riboside), tyrosine kinase (genistein), and protein kinase C (bisindolylmaleimide) abrogated completely (actinomycin D, dichlorobenzimidazole riboside, cycloheximide, and genistein) or partially (bisindolylmaleimide) TNFα-induced LDH A mRNA expression. These observations suggest that the stimulatory effect of TNFα on LDH A mRNA expression requires protein synthesis and may involve a protein tyrosine kinase and protein kinase C. In addition, we report that LDH A mRNA levels were increased in Sertoli cells treated with FSH. However, although the cytokine enhances LDH A mRNA levels through increased gene transcription, the hormone exerts its stimulatory action through an increase in LDH A mRNA stability. The regulatory actions of the cytokine and the hormone on LDH A mRNA levels and therefore on lactate production may operate in the context of the metabolic cooperation between Sertoli and postmeiotic germ cells in the seminiferous tubules.

scholarly journals LPA stimulates intestinal DRA gene transcription via LPA2 receptor, PI3K/AKT, and c-Fos-dependent pathway

2012 ◽  
Vol 302 (6) ◽  
pp. G618-G627 ◽  
Author(s):  
Amika Singla ◽  
Anoop Kumar ◽  
Shubha Priyamvada ◽  
Maliha Tahniyath ◽  
Seema Saksena ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Gene Transcription ◽  
Promoter Activity ◽  
Intestinal Epithelial Cells ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Short Term ◽  
Exchange Activity

DRA (downregulated in adenoma) or SLC26A3 is the major apical anion exchanger mediating Cl− absorption in intestinal epithelial cells. Disturbances in DRA function and expression have been implicated in diarrheal conditions such as congenital chloride diarrhea and inflammatory bowel diseases. Previous studies have shown that DRA is subject to regulation by short-term and transcriptional mechanisms. In this regard, we have recently shown that short-term treatment by lysophosphatidic acid (LPA), an important bioactive phospholipid, stimulates Cl−/HCO3−(OH−) exchange activity via an increase in DRA surface levels in human intestinal epithelial cells. However, the long-term effects of LPA on DRA at the level of gene transcription have not been examined. The present studies were aimed at investigating the effects of LPA on DRA function and expression as well as elucidating the mechanisms underlying its transcriptional regulation. Long-term LPA treatment increased the Cl−/HCO3− exchange activity in Caco-2 cells. LPA treatment (50–100 μM) of Caco-2 cells significantly stimulated DRA mRNA levels and DRA promoter activity (−1183/+114). This increase in DRA promoter activity involved the LPA2 receptor and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. Progressive deletions from −1183/+114 to −790/+114 abrogated the stimulatory effects of LPA, indicating that the −1183/−790 promoter region harbors LPA response elements. Utilizing EMSA and mutational studies, our results showed that LPA induced the DRA promoter activity in a c-Fos-dependent manner. LPA also increased the protein expression of c-Fos and c-Jun in Caco-2 cells. Furthermore, overexpression of c-Fos but not c-Jun enhanced the DRA promoter activity. This increase in DRA transcription in response to LPA indicates that LPA may act as an antidiarrheal agent and could be exploited for the treatment of diarrhea associated with inflammatory or infectious diseases of the gut.

Tumor necrosis factor inhibits growth hormone-mediated gene expression in hepatocytes

2006 ◽  
Vol 291 (1) ◽  
pp. G35-G44 ◽  
Author(s):  
Tamer Ahmed ◽  
Gladys Yumet ◽  
Margaret Shumate ◽  
Charles H. Lang ◽  
Peter Rotwein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Mrna Expression ◽  
Gene Transcription ◽  
Protein Tyrosine Phosphatases ◽  
Mrna Levels ◽  
Inhibitory Effects ◽  
Protein Levels ◽  
Mrna Induction ◽  
Igf I

Growth hormone (GH) stimulates STAT5 phosphorylation by JAK2, which activates IGF-I and serine protease inhibitor 2.1 (Spi 2.1) transcription, whereas STAT5 dephosphorylation by protein tyrosine phosphatases (PTPs) terminates this signal. We hypothesized that the inhibitory effects of TNF on GH signaling and gene transcription were responsible for hepatic GH resistance. CWSV-1 hepatocytes were treated with TNF, pervanadate (a PTP inhibitor), or both, before GH stimulation. Total and tyrosine-phosphorylated JAK2, STAT5, ERK1/2, SHP-1 and SHP-2, IGF-I, and Spi 2.1 mRNA levels were measured. GH stimulated STAT5 and ERK1/2 phosphorylation, IGF-I, and Spi 2.1 mRNA expression. TNF attenuated JAK2/STAT5 and ERK1/2 phosphorylation and IGF-I and Spi 2.1 mRNA expression following GH stimulation. SHP-1 and SHP-2 protein levels were unaltered by TNF or GH, and the GH-induced increase in SHP-1 PTP activity was not further increased by TNF. In TNF-treated cells, pervanadate restored STAT5 and ERK1/2 phosphorylation to control levels following GH stimulation but did not restore IGF-I or Spi 2.1 mRNA induction. Cells transfected with a Spi 2.1 promoter-luciferase vector demonstrate a 50-fold induction in luciferase activity following GH stimulation or cotransfection with a constitutively active STAT5 vector. TNF prevented the induction of Spi 2.1 promoter activity by GH and the STAT5 construct. We conclude that TNF does not inhibit GH activity by inducing SHP-1 or -2 expression and that correction of GH signaling defects in TNF-treated cells by pervanadate does not restore GH-induced gene expression. The inhibitory effects of TNF on GH-mediated gene transcription appear independent of STAT5 activity and previously identified abnormalities in JAK2/STAT5 signaling.

scholarly journals Enhanced AP-1 and NF-κB activities and stability of interleukin 8 (IL-8) transcripts are implicated in IL-8 mRNA superinduction in lung epithelial H292 cells

1998 ◽  
Vol 330 (1) ◽  
pp. 429-435 ◽  
Author(s):  
Thierry ROGER ◽  
A. Theo OUT ◽  
Naofumi MUKAIDA ◽  
Kouji MATSUSHIMA ◽  
M. Henk JANSEN ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Dna Binding ◽  
Mrna Expression ◽  
Gene Transcription ◽  
Molecular Mechanisms ◽  
Interleukin 8 ◽  
Mrna Levels ◽  
Tnf Α ◽  
Lung Epithelial ◽  
Inhibitor Of Protein Synthesis

Inhibition of protein synthesis may result in superinduction of short-lived transcripts and has been attributed variably to stabilization of transcripts and/or increased gene transcription. Little is known about the kinetics of these processes and relevant transcriptional elements have not been identified. In this study, we describe superinduction of interleukin 8 (IL-8) mRNA, an important inflammatory mediator, in lung epithelial-like H292 cells and identify the underlying molecular mechanisms and their kinetics. Cycloheximide (CHI, 10 μg/ml), an inhibitor of protein synthesis, maximally increased IL-8 mRNA levels 30-fold in H292 cells. Tumour necrosis factor α (TNF-α), which induced IL-8 mRNA 3-fold, synergized with CHI causing a 150-fold increase at 6 h. CHI early on increased the stability of IL-8 mRNA (from 40 min in cells cultured with medium to more than 4 h with CHI). CHI also increased transcription as shown by transfection with IL-8 promoter constructs. Truncated and mutated constructs identified NF-κB and AP-1 binding sites as primary cis-acting elements in IL-8 gene transcription and IL-8 mRNA superinduction. Electrophoretic mobility shift assays indicated that CHI increased NF-κB and prolonged AP-1 DNA-binding activities and that the synergism of TNF-α and CHI on IL-8 mRNA expression was paralleled by a further increase of AP-1 DNA-binding activity. This synergism was still noticed when 4 h elapsed between the addition of CHI and that of TNF-α. Taken together, our results indicate that CHI interferes with both post-transcriptional and transcriptional repressive mechanisms of IL-8 mRNA expression.

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