Prolactin upstream factor I mediates cell-specific transcription

1988 ◽  
Vol 8 (12) ◽  
pp. 5432-5438
Author(s):  
Z D Cao ◽  
E A Barron ◽  
Z D Sharp
Keyword(s):  
Gene Transcription ◽  
Rna Synthesis ◽  
Transcriptional Activity ◽  
In Vitro Transcription ◽  
Gh3 Cells ◽  
In Vitro Mutagenesis ◽  
Pituitary Cells ◽  
Factor I ◽  
Prolactin Gene

DNA sequence-specific chromatography was used to purify prolactin upstream factor I (PUF-I) approximately 10,000- to 20,000-fold from rat GH3 cells. The purified transcription factor reconstituted enhanced pituitary-specific prolactin RNA synthesis in nonpituitary in vitro transcription assays. In vitro mutagenesis demonstrated that the capacity to stimulate prolactin gene transcription was directly correlated with PUF-I binding to an A+T-rich region located from -63 to -36 in the prolactin 5'-flanking DNA. We propose that PUF-I is a critical modulator of transcriptional activity in pituitary cells and has a central role in the stimulation of prolactin gene transcription in the mammalian pituitary lactotroph.

scholarly journals Prolactin upstream factor I mediates cell-specific transcription.

1988 ◽  
Vol 8 (12) ◽  
pp. 5432-5438 ◽  
Author(s):  
Z D Cao ◽  
E A Barron ◽  
Z D Sharp
Keyword(s):  
Gene Transcription ◽  
Rna Synthesis ◽  
Transcriptional Activity ◽  
In Vitro Transcription ◽  
Gh3 Cells ◽  
In Vitro Mutagenesis ◽  
Pituitary Cells ◽  
Factor I ◽  
Prolactin Gene

DNA sequence-specific chromatography was used to purify prolactin upstream factor I (PUF-I) approximately 10,000- to 20,000-fold from rat GH3 cells. The purified transcription factor reconstituted enhanced pituitary-specific prolactin RNA synthesis in nonpituitary in vitro transcription assays. In vitro mutagenesis demonstrated that the capacity to stimulate prolactin gene transcription was directly correlated with PUF-I binding to an A+T-rich region located from -63 to -36 in the prolactin 5'-flanking DNA. We propose that PUF-I is a critical modulator of transcriptional activity in pituitary cells and has a central role in the stimulation of prolactin gene transcription in the mammalian pituitary lactotroph.

172 IN VITRO EXPOSURE TO XENOESTROGENS INDUCES GROWTH HORMONE TRANSCRIPTION AND RELEASE VIA AN ESTROGEN RECEPTOR-DEPENDENT PATHWAY IN RAT PITUITARY GH3 CELLS

2008 ◽  
Vol 20 (1) ◽  
pp. 166
Author(s):  
V.-H. Dang ◽  
E.-B. Jeung
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Estrogen Receptor ◽  
Gene Transcription ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Rat Pituitary ◽  
In Vitro Exposure ◽  
Gh Gene

The term endocrine disruptor (ED) has been used widely to characterize natural and synthetic environmental compounds that may interfere with the endocrine system(s) of humans and wildlife. In previous studies, we demonstrated that in vitro single exposure to EDs induces CaBP-9k expression, a useful biomarker for detecting the estrogenic activities of EDs in rat pituitary GH3 cells. Here we employ the identical model to examine the effects of EDs in the regulation of growth hormone (GH) gene expression, an important hormone in growth, development, and body composition. We measured levels of GH mRNA transcription and GH release using semi-quantitative RT-PCR and EIA kit, respectively. GH3 cells were treated with alkyphenols (APs), i.e., octyl-phenol (OP), nonyl-phenol (NP), and bisphenol A (BPA), in a dose-dependent manner (10–5, 10–6, and 10–7 M) and harvested following 24 h of treatment. Cells were also exposed to a high concentration (10–5 M) of OP, NP, or BPA and harvested at various time points (1, 3, 6, 12, and 24 h). An anti-estrogen, ICI 182780 (10–7 M) was used to examine the potential involvement of estrogen receptor (ER) in the induction of GH by EDs through an ER-mediated pathway. The data were analyzed by one-way ANOVA, followed by Tukey's multiple comparison. OP, NP, and BPA induced a significant increase in GH gene expression at high (10–5 M) and medium (10–6 M) doses at 24 h. ED-exposure induced a marked increase in GH gene transcription as early as 6 h and peaked at 12 h. Co-treatment with ICI 182780 significantly attenuated ED-induced GH expression in GH3 cells. Interestingly, the level of in vitro GH release was increased significantly at 24 h in response to OP, NP, or BPA, whereas co-treatment with ICI 182780 significantly diminished ED-induced GH secretion in GH3 cells, indicating that ER may play a part in both GH gene transcription and GH release in these cells. Here we demonstrate for the first time that single in vitro exposure to OP, NP, or BPA results in an increase in GH expression at 24 h in GH3 rat pituitary cells. These results may provide new insight into the mode of ED action in GH gene regulation as well as the biological pathway underlying these molecular events. Furthermore, data showing GH responsiveness evoked by EDs supports the aim to develop an assay for use in predicting adverse health effects of EDs in humans and wildlife.

scholarly journals Transcriptional and posttranscriptional regulation of the rat prolactin gene by calcium.

1990 ◽  
Vol 10 (2) ◽  
pp. 442-448 ◽  
Author(s):  
G M Preston ◽  
W M Billis ◽  
B A White
Keyword(s):  
Posttranscriptional Regulation ◽  
In Vitro Transcription ◽  
Gh3 Cells ◽  
Mrna Levels ◽  
Nuclear Extracts ◽  
Nuclear Site ◽  
Gene Treatment ◽  
Prolactin Gene ◽  
Prolactin Mrna

The rat prolactin gene is expressed at a high basal level in the pituitary tumor GH3 cell line. Culturing GH3 cells in a low-Ca2+, serum-free medium (SFM) depresses prolactin mRNA levels, and subsequent addition of Ca2+ to the SFM results in a specific, gradual, and sustained increase in prolactin mRNA levels. We have now examined whether the observed increase in prolactin mRNA levels can be attributed solely to an increase in the transcriptional rate of the prolactin gene. Treatment of GH3 cells in SFM with 0.4 mM CaCl2 for 24 to 48 h increased cytoplasmic prolactin mRNA levels by 5- to 10-fold, whereas the transcriptional rate of the prolactin gene was increased by less than twofold over values for SFM controls. Prolactin mRNA levels increased progressively during the 24-h period after Ca2+ addition, whereas prolactin gene transcription never exceeded a twofold increase over values for SFM controls. The activities of nuclear extracts from control and Ca2(+)-induced cells were examined in an in vitro transcription assay. The two extracts directed transcription from the prolactin promoter and the adenovirus major late promoter equally well. Cycloheximide had no effect on the ability of Ca2+ to increase or maintain prolactin mRNA levels. In dactinomycin mRNA clearance experiments, prolactin mRNA was cleared at the same rate in the absence and presence of Ca2+. These results demonstrate that although Ca2+ has a small effect on the transcriptional rate of the prolactin gene, Ca2+ produces a significant increase in prolactin mRNA levels by acting at a posttranscriptional site(s). Furthermore, Ca(2+) appears to increase prolactin mRNA levels by posttranslational modification of a stable protein, probably at a nuclear site.

scholarly journals Reconstitution of cell-type-specific transcription of the rat prolactin gene in vitro.

1987 ◽  
Vol 7 (10) ◽  
pp. 3402-3408 ◽  
Author(s):  
Z D Cao ◽  
E A Barron ◽  
A J Carillo ◽  
Z D Sharp
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Expression System ◽  
In Vitro Transcription ◽  
Gh3 Cells ◽  
Nuclear Extracts ◽  
Prolactin Gene ◽  
Flanking Region ◽  
Imperfect Palindrome

We present evidence for the existence of prolactin upstream factor 1 (PUF-1) in rat pituitary-derived cells and demonstrate its interaction with a symmetrical DNA element located in the 5' flanking region of the gene. An in vitro expression system developed from pituitary-derived GH3 cells was used to determine that 420 base pairs (bp) of 5' flanking DNA was sufficient for cell-specific, accurate, and efficient RNA polymerase II transcription of the rat prolactin gene. Reconstitution of in vitro transcription with pituitary and nonpituitary nuclear extracts suggested that the presence of GH3 cell-specific factors mediated the activation of prolactin gene expression. We also demonstrated that a functionally stable transcription complex assembled on the prolactin promoter. Using DNase I protection procedures, we have identified the DNA-protein binding area in the prolactin 5' flanking region. GH3 nuclear extracts contain a cell-specific protein (PUF-I) that binds to a 28-bp region (-63 to -36)which contains an 18-bp imperfect palindrome (-63 to -46). The role that the interaction between PUF-I and the imperfect palindrome plays in in vitro pituitary-specific prolactin gene expression is discussed.

Reconstitution of cell-type-specific transcription of the rat prolactin gene in vitro

1987 ◽  
Vol 7 (10) ◽  
pp. 3402-3408
Author(s):  
Z D Cao ◽  
E A Barron ◽  
A J Carillo ◽  
Z D Sharp
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Expression System ◽  
In Vitro Transcription ◽  
Gh3 Cells ◽  
Nuclear Extracts ◽  
Prolactin Gene ◽  
Flanking Region ◽  
Imperfect Palindrome

We present evidence for the existence of prolactin upstream factor 1 (PUF-1) in rat pituitary-derived cells and demonstrate its interaction with a symmetrical DNA element located in the 5' flanking region of the gene. An in vitro expression system developed from pituitary-derived GH3 cells was used to determine that 420 base pairs (bp) of 5' flanking DNA was sufficient for cell-specific, accurate, and efficient RNA polymerase II transcription of the rat prolactin gene. Reconstitution of in vitro transcription with pituitary and nonpituitary nuclear extracts suggested that the presence of GH3 cell-specific factors mediated the activation of prolactin gene expression. We also demonstrated that a functionally stable transcription complex assembled on the prolactin promoter. Using DNase I protection procedures, we have identified the DNA-protein binding area in the prolactin 5' flanking region. GH3 nuclear extracts contain a cell-specific protein (PUF-I) that binds to a 28-bp region (-63 to -36)which contains an 18-bp imperfect palindrome (-63 to -46). The role that the interaction between PUF-I and the imperfect palindrome plays in in vitro pituitary-specific prolactin gene expression is discussed.

Transcriptional and posttranscriptional regulation of the rat prolactin gene by calcium

1990 ◽  
Vol 10 (2) ◽  
pp. 442-448
Author(s):  
G M Preston ◽  
W M Billis ◽  
B A White
Keyword(s):  
Posttranscriptional Regulation ◽  
In Vitro Transcription ◽  
Gh3 Cells ◽  
Mrna Levels ◽  
Nuclear Extracts ◽  
Nuclear Site ◽  
Gene Treatment ◽  
Prolactin Gene ◽  
Prolactin Mrna

The rat prolactin gene is expressed at a high basal level in the pituitary tumor GH3 cell line. Culturing GH3 cells in a low-Ca2+, serum-free medium (SFM) depresses prolactin mRNA levels, and subsequent addition of Ca2+ to the SFM results in a specific, gradual, and sustained increase in prolactin mRNA levels. We have now examined whether the observed increase in prolactin mRNA levels can be attributed solely to an increase in the transcriptional rate of the prolactin gene. Treatment of GH3 cells in SFM with 0.4 mM CaCl2 for 24 to 48 h increased cytoplasmic prolactin mRNA levels by 5- to 10-fold, whereas the transcriptional rate of the prolactin gene was increased by less than twofold over values for SFM controls. Prolactin mRNA levels increased progressively during the 24-h period after Ca2+ addition, whereas prolactin gene transcription never exceeded a twofold increase over values for SFM controls. The activities of nuclear extracts from control and Ca2(+)-induced cells were examined in an in vitro transcription assay. The two extracts directed transcription from the prolactin promoter and the adenovirus major late promoter equally well. Cycloheximide had no effect on the ability of Ca2+ to increase or maintain prolactin mRNA levels. In dactinomycin mRNA clearance experiments, prolactin mRNA was cleared at the same rate in the absence and presence of Ca2+. These results demonstrate that although Ca2+ has a small effect on the transcriptional rate of the prolactin gene, Ca2+ produces a significant increase in prolactin mRNA levels by acting at a posttranscriptional site(s). Furthermore, Ca(2+) appears to increase prolactin mRNA levels by posttranslational modification of a stable protein, probably at a nuclear site.

scholarly journals Detection of two chromatin proteins which bind specifically to the 5'-flanking region of the rat prolactin gene.

1985 ◽  
Vol 5 (11) ◽  
pp. 2967-2974 ◽  
Author(s):  
B A White ◽  
G M Preston ◽  
T C Lufkin ◽  
C Bancroft
Keyword(s):  
Transcription Initiation ◽  
Genomic Clone ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Sequence Specificity ◽  
Strong Binding ◽  
Prolactin Gene ◽  
Rat Pituitary ◽  
Chromatin Proteins ◽  
Flanking Region

We employed a protein gel blotting procedure to search for nuclear proteins from rat pituitary cells that bind preferentially to the 5'-flanking region of the rat prolactin gene. By gel blots of chromatin proteins from GH3 rat pituitary tumor cells with a 32P-labeled prolactin genomic clone, we detected two major binding proteins with molecular weights of approximately 44,000 and 48,000, designated NP44 and NP48, respectively. Both NP44 and NP48 are minor chromatin proteins which are extracted at low salt concentrations (0.4 M NaCl) and exhibit a range of slightly acidic isoelectric variants. NP44 and NP48 were detected at similar levels in chromatin extracts of GH3 cells, the prolactin-negative GC cell variant of the GH3 cells, and normal rat pituitary tissue. Considerably lower levels of these two proteins were found in chromatin extracts from rat liver and rat C6 glial cells. NP44 and NP48 exhibit DNA sequence specificity, as evidenced by their strong binding to the upstream flanking region of the prolactin gene, but only very weak binding to plasmid DNA, rat prolactin or growth hormone cDNAs, or upstream flanking regions of two other rat genes. By analyzing subclones of a rat prolactin genomic clone, we established that NP44 and NP48 bind to at least two sites, which are located between 0.4 and 2.0 kilobases (region I) and between 2.0 and 4.8 kilobases (region II) upstream of the transcription initiation site. These findings are discussed in the context of a possible functional association between the strong binding of NP44 and NP48 to the prolactin 5'-flanking region and pituitary-specific expression of the prolactin gene.

Glucocorticoids regulate NHE-3 transcription in OKP cells

1996 ◽  
Vol 270 (1) ◽  
pp. F164-F169 ◽  
Author(s):  
M. Baum ◽  
M. Amemiya ◽  
V. Dwarakanath ◽  
R. J. Alpern ◽  
O. W. Moe
Keyword(s):  
Gene Transcription ◽  
Half Life ◽  
Time Course ◽  
In Vitro Transcription ◽  
Mrna Abundance ◽  
Proton Secretion ◽  
Threefold Increase ◽  
Mrna Half Life ◽  
Synthetic Glucocorticoid

OKP cells express NHE-3, an amiloride-resistant Na+/H+ antiporter, which is likely an isoform responsible for apical proton secretion by the proximal tubule. We have previously shown that an amiloride-resistant Na+/H+ antiporter in OKP cells is regulated by dexamethasone, a synthetic glucocorticoid. The purpose of the present study was to examine the mechanism for the glucocorticoid-mediated increase in Na+/H+ antiporter activity. Incubation of OKP cells with 10(-6) M dexamethasone resulted in a two- to threefold increase in NHE-3 mRNA abundance. This increase was seen after 4 h of incubation with dexamethasone, a time course similar to that found for Na+/H+ antiporter activity. To examine the mechanism for the increase in NHE-3 mRNA abundance, mRNA half-life and in vitro transcription experiments were performed. NHE-3 mRNA had a half-life of 8 h in control and dexamethasone-treated cells. The rate of in vitro transcription was 1.8-fold greater when OKP cells were treated with dexamethasone. These data suggest that the glucocorticoid-mediated increase in Na+/H+ antiporter activity is due to an increase in NHE-3 gene transcription.

scholarly journals Transcriptionally inactive oocyte-type 5S RNA genes of Xenopus laevis are complexed with TFIIIA in vitro.

1987 ◽  
Vol 7 (10) ◽  
pp. 3503-3510 ◽  
Author(s):  
L J Peck ◽  
L Millstein ◽  
P Eversole-Cire ◽  
J M Gottesfeld ◽  
A Varshavsky
Keyword(s):  
Xenopus Laevis ◽  
Differential Expression ◽  
Gene Transcription ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
5S Rna ◽  
Differential Binding ◽  
5S Rna Genes ◽  
Rna Genes

An extract from whole oocytes of Xenopus laevis was shown to transcribe somatic-type 5S RNA genes approximately 100-fold more efficiently than oocyte-type 5S RNA genes. This preference was at least 10-fold greater than the preference seen upon microinjection of 5S RNA genes into oocyte nuclei or upon in vitro transcription in an oocyte nuclear extract. The approximately 100-fold transcriptional bias in favor of the somatic-type 5S RNA genes observed in vitro in the whole oocyte extract was similar to the transcriptional bias observed in developing Xenopus embryos. We also showed that in the whole oocyte extract, a promoter-binding protein required for 5S RNA gene transcription, TFIIIA, was bound both to the actively transcribed somatic-type 5S RNA gene and to the largely inactive oocyte-type 5S RNA genes. These findings suggest that the mechanism for the differential expression of 5S RNA genes during Xenopus development does not involve differential binding of TFIIIA to 5S RNA genes.

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