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

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.5.11.2967-2974.1985 ◽

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

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1,25-Dihydroxyvitamin D3-induced upregulation of the thyrotropin-releasing hormone receptor in clonal rat pituitary GH3 cells

Journal of Endocrinology ◽

10.1677/joe.0.1470397 ◽

1995 ◽

Vol 147 (3) ◽

pp. 397-404 ◽

Cited By ~ 5

Author(s):

L M Atley ◽

N Lefroy ◽

J D Wark

Keyword(s):

Vitamin D ◽

Fold Increase ◽

Thyrotropin Releasing Hormone ◽

Gh3 Cells ◽

Pituitary Hormone ◽

Pituitary Cells ◽

Hormone Production ◽

Releasing Hormone ◽

Rat Pituitary ◽

Trh Receptor

Abstract 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is active in primary dispersed and clonal pituitary cells where it stimulates pituitary hormone production and agonist-induced hormone release. We have studied the effect of 1,25-(OH)2D3 on thyrotropin-releasing hormone (TRH) binding in clonal rat pituitary tumour (GH3) cells. Compared with vehicle-treated cells, 1,25-(OH)2D3 (10 nmol/l) increased specific [3H]MeTRH binding by 26% at 8 h, 38% at 16 h, 35% at 24 h and reached a maximum at 48 h (90%). In dose–response experiments, specific [3H]MeTRH binding increased with 1,25-(OH)2D3 concentration and reached a maximum at 10 nmol/l. Half-maximal binding occurred at 0·5 nmol 1,25-(OH)2D3/l. The vitamin D metabolite, 25-OH D3, increased [3H]MeTRH binding but was 1000-fold less potent than 1,25-(OH)2D3. In equilibrium binding assays, treatment with 10 nmol 1,25-(OH)2D3/l for 48 h increased the maximum binding from 67·4 ± 8·8 fmol/mg protein in vehicle-treated cells to 96·7 ± 12·4 fmol/mg protein in treated cells. There was no difference in apparent Kd (1·08 ± 0·10 nmol/l for 1,25-(OH)2D3-treated and 0·97 ± 0·11 nmol/l for vehicle-treated cells). Molecular investigations revealed that 10 nmol 1,25-(OH)2D3/l for 24 h caused an 8-fold increase in TRH receptor-specific mRNA. Actinomycin D (2 μg/ml, 6 h) abrogated the 1,25-(OH)2D3-induced increase in [3H]MeTRH binding. Cortisol also increased [3H]MeTRH binding but showed no additivity or synergism with 1,25-(OH)2D3. TRH-stimulated prolactin release was not enhanced by 1,25-(OH)2D3. We conclude that the active vitamin D metabolite, 1,25-(OH)2D3, caused a time- and dose-dependent increase in [3H]MeTRH binding. The effect was vitamin D metabolite-specific and resulted from an upregulation of the TRH receptor. Further studies are needed to determine the functional significance of this novel finding. Journal of Endocrinology (1995) 147, 397–404

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

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab172 ◽

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.

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Prolactin upstream factor I mediates cell-specific transcription.

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5432 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5432-5438 ◽

Cited By ~ 11

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.

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Characterization of a hyperpolarization-activated cation current in rat pituitary cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1997.272.3.e405 ◽

1997 ◽

Vol 272 (3) ◽

pp. E405-E414 ◽

Cited By ~ 9

Author(s):

S. M. Simasko ◽

S. Sankaranarayanan

Keyword(s):

Primary Cultures ◽

Gh3 Cells ◽

Pituitary Cells ◽

Sodium Permeability ◽

Cation Current ◽

Whole Cell Patch Clamp ◽

Bath Application ◽

Rat Pituitary ◽

Rat Pituitary Cells

Whole cell patch-clamp techniques were used on clonal pituitary cells (GH3) and primary cultures of somatotrophs and lactotrophs to study currents that would be active at or below voltages for the threshold for action potential generation. When GH3 cells were held at -60 mV and pulsed to -120 mV, a slow-activating sustained inward current was observed (-16.5 +/- 1.5 pA in physiological baths, n = 72; approximately 1 s to half-maximal activation, voltage for 50% activation - 101 mV). The current was insensitive to bath application of 10 mM tetraethylammonium, 10 mM 4-aminopyridine, and 1 mM barium but was completely blocked by 3 mM cesium. The current was found to be a mixed cation current with a sodium permeability of 0.29 relative to potassium. These properties indicate that the current belongs to the hyperpolarization-activated cation current (Ih), or I(f), family of currents. However, the current was not altered by the addition of adenosine 3',5'-cyclic monophosphate (cAMP) to the pipette or forskolin to the bath. A similar but smaller current was observed in 15 of 16 somatotrophs but in only 1 of 9 lactotrophs. Application of cesium to spontaneously spiking GH3 cells or somatotrophs had no effect. However, cesium did block an inward holding current observed at -80 mV. These results demonstrate that the I(h) in pituitary cells does not serve as a pacemaking current but suggest that it may influence membrane potential responses when somatotrophs become hyperpolarized.

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Tri-iodothyronine and phenytoin reduce prolactin messenger RNA levels in cultured rat pituitary cells

Journal of Endocrinology ◽

10.1677/joe.0.1090359 ◽

1986 ◽

Vol 109 (3) ◽

pp. 359-364 ◽

Cited By ~ 17

Author(s):

J. R. E. Davis ◽

T. C. Lynam ◽

J. A. Franklyn ◽

K. Docherty ◽

M. C. Sheppard

Keyword(s):

Gene Transcription ◽

Messenger Rna ◽

Pituitary Cells ◽

Mrna Levels ◽

Prolactin Release ◽

Dot Hybridization ◽

Prolactin Gene ◽

Rat Pituitary ◽

Rat Pituitary Cells ◽

Prolactin Mrna

ABSTRACT Thyroid hormones may regulate prolactin gene transcription. We have previously found that phenytoin inhibits tri-iodothyronine (T3) nuclear binding, and have suggested that phenytoin may act as a partial T3 agonist. We have therefore investigated the effects of phenytoin and T3 on prolactin release and gene transcription, using the technique of cytoplasmic dot hybridization with complementary DNA probes to estimate prolactin messenger (m) RNA concentrations in cytoplasm from cultured rat pituitary cells. Tri-iodothyronine treatment led to a small but significant fall in prolactin release by 72 h, but caused marked dose- and time-dependent reductions in prolactin mRNA levels at 48–72 h. Phenytoin, however, caused more rapid falls in both prolactin release and mRNA concentrations. Neither T3 nor phenytoin significantly altered GH mRNA levels. These studies suggest effects of phenytoin similar, but not identical, to those of T3 in the lactotroph. J. Endocr. (1986) 109, 359–364

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The effects of hypothalamic extracts and TRH on the growth and hormone production of GH3 cells in culture

Acta Endocrinologica ◽

10.1530/acta.0.1040287 ◽

1983 ◽

Vol 104 (3) ◽

pp. 287-294 ◽

Cited By ~ 5

Author(s):

Yukiko Yajima ◽

Toshikazu Saito

Keyword(s):

Dna Synthesis ◽

Gel Filtration ◽

Control Culture ◽

Gh3 Cells ◽

Thymidine Uptake ◽

Pituitary Cells ◽

Hormone Production ◽

Hormone Synthesis ◽

Rat Pituitary ◽

Rat Pituitary Cells

Abstract. Hypothalamic factors were tested for their effects on the production of hormones and the growth of GH3 cells, cloned rat pituitary cells producing prolactin (Prl) and growth hormone (GH). Hypothalamic extracts (HE) (0.05 mg/ml) and TRH (0.3 μm) stimulated the synthesis of Prl to levels of 306% and 360%, respectively, of the control culture in a medium containing 0.5% foetal bovine serum (FBS) during a 24 h incubation. They did not affect the rate of GH production. The thymidine uptake was suppressed to 57% and 46% of the control by the addition of HE and TRH, respectively. They also inhibited the growth of GH3 to 70% and 74% of the control culture during an 8-day incubation period. On the other hand, LRH affected neither the rate of hormone production nor the thymidine uptake. Somatostatin suppressed the synthesis of Prl and GH, but it did not affect the incorporation of thymidine into the cells. The gel filtration studies of HE revealed that the inhibitory effects of HE on the thymidine uptake were dependent on two substances, TRH and an unknown factor(s) of high molecular nature. The relationship between hormone synthesis and DNA synthesis will be discussed on the basis of the TRH-induced effects on Prl production and DNA synthesis in GH3 cells.

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Identification of an estrogen-responsive element from the 5'-flanking region of the rat prolactin gene.

Molecular and Cellular Biology ◽

10.1128/mcb.7.12.4247 ◽

1987 ◽

Vol 7 (12) ◽

pp. 4247-4254 ◽

Cited By ~ 134

Author(s):

R A Maurer ◽

A C Notides

Keyword(s):

Estrogen Receptor ◽

Dna Binding ◽

Gene Transcription ◽

Dna Sequences ◽

Transcription Initiation ◽

Intrinsic Property ◽

Conserved Sequence ◽

Prolactin Gene ◽

Flanking Region ◽

Estrogenic Regulation

The DNA sequences which interact with the estrogen receptor and which mediate the estrogenic regulation of prolactin gene transcription have been investigated by the use of receptor-DNA-binding experiments and gene transfer studies. Nitrocellulose filter binding assays using highly purified estrogen receptor and cloned fragments of the 5'-flanking region of the rat prolactin gene demonstrate that the receptor selectively binds to DNA sequences located between nucleotides -1713 and -1532 with respect to the transcription initiation site. The binding of the estrogen receptor to this region of the prolactin gene was strongly dependent on receptor concentration, suggesting that receptor dimers may be important in DNA binding. These data demonstrate that the selective binding of purified estrogen receptor to specific sequences of the rat prolactin gene is an intrinsic property of the receptor and is not due to the interaction of receptor with other proteins. The role of specific prolactin gene sequences in mediating the estrogenic regulation of prolactin gene transcription was confirmed by the use of prolactin-chloramphenicol acetyltransferase fusion genes. These studies demonstrated that sequences upstream of position -1532 are required for estrogen responsiveness. Furthermore, the region of the prolactin gene at -1713 to -1495 was able to confer estrogen responsiveness on the thymidine kinase promoter. Exonuclease III protection experiments further localized the receptor-binding sequences to positions -1587 to -1563. Comparison of the nucleotide sequence of the region of the prolactin gene which binds the estrogen receptor with the sequence of other estrogen-responsive genes suggested the presence of the conserved sequence [sequence in text], which shows similarity to sequences thought to mediate glucocorticoid receptor effects on transcription.

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