Triiodothyronine stimulates specifically growth hormone mRNA in rat pituitary tumor cells.

Abstract

In a cell-free protein-synthesizing system from a rabbit reticulocyte lysate, total RNA extracted from cultured rat pituitary tumor (GH3) cells directed, in a dose-related manner, the synthesis of proteins that were precipitated by antisera specific to rat growth hormone (somatotropin) and rat prolactin. A marked decrease in growth hormone secretion and growth hormone mRNA activity was observed when cells were grown in a medium deficient in thyroid hormone. Addition of triiodothyronine in physiologic amounts both prevented and completely reversed this effect within 48 hr. Thyroid hormone had no effect on prolactin secretion or prolactin mRNA activity. These data suggest that thyroid hormone may stimulate synthesis of growth hormone throug induction of transcriptional activity. The possibility of an additional effect at the posttranscriptional level has not been excluded. Although thyroid hormone is believed to have a general effect on a variety of metabolic processes, some effects, at the molecular level, may be quite selective, as indicated by the observed changes in growth hormone but not prolactin mRNA activity. The GH3 cell model is useful in the study of triiodothyronine action because of independence from secondary hormonal effects caused by hypothyroidism and because simultaneous measurement of prolactin mRNA activity serves as a unique internal control. The demonstration of triiodothyronine (T3) binding to nuclear proteins raises the possibility that thyroid hormone may regulate gene expression. Earlier work from Tata's laboratory showed that thyroid hormone-induced protein synthesis was preceded by formation of new RNA (1). Later, DeGroot et al. and Dilman et al. showed increase in the poly(A)-rich fraction of RNA (2, 3). Demonstrations of stimulation of a specific mRNA by thyroid hormones were recently provided by Kurtz et al. (4) and by Roy et al. (5) for a2u-globulin in the rat. However, because a number of hormones are known to stimulate the synthesis of this protein (6) and because thyroid hormone profoundly alters the level of such hormones (7), experiments done in the whole animal do not provide sufficient evidence for the direct induction of -d2u-globulin mRNA by thyroid hormone. Samuels et al. have reported a quantitative correlation between nuclear T3 receptor occupancy and stimulation of growth hormone (GH, somatotropin) synthesis in cultured rat pituitary cells (8). We have adopted a similar experimental model to study more directly the action of thyroid hormone on the induction of a specific mRNA. In a recent report, we have shown that GH and prolactin mRNA activities in the total RNA extracted from a rat pituitary cell line (GH3) that actively synthesizes both hormones can be quantitated using a rabbit reticulocyte lysate cell-free system (9). In this report, we show that in the normal rat serum were 5.4 ,ug/100 ml and 60 ng/100 ml, respectively, and in the Tx rat serum were 0.6 ,g/100 ml and less than 20 ng/100 ml, respectively. RNA Extraction. Each RNA preparation was obta...

Mechanism of Interference Between Influenza A/WSN and B/Kanagawa Viruses

“…The visible band was collected, resuspended in PBS, and pelleted at 20000 r.p.m, for 1 h. The virion RNA (vRNA) was extracted repeatedly with phenol--chloroform (1 : 1), precipitated from the final aqueous phase with 2 M-LiC1 for 16 h at 4 °C and dissolved in distilled water. The amount of RNA was calculated assuming that 1 A260 unit is 40 ~tg/ml (Seo et al, 1977). 125 I-labelled vRNA was prepared by the thallium chloride procedure of Tereba & McCarthy (1973).…”

“…Extraction of RNA from uninfected cells (1 x 10 s cells) was carried out according to the method of Seo et al (1977). The extracted RNA preparations were serially diluted in 30 ~tl of distilled water, mixed with 20 ~tl of 0-1 M-Tris HC1 pH 7.4, containing 0.75 M-NaC1, 5 mM-EDTA, 0.05 ~ SDS, and 125I-labelled vRNA and held at 68 °C for 96 h. Each mixture was then split into two parts, one being treated with S 1 nuclease at 40 °C for 1 h and the other left at 40 °C.…”

The synthesis and processing of a nuclear RNA precursor to rat pregrowth hormone messenger RNA

“…A number of recent studies have shown that glucocorticoid (16,17,18) and thyroid (17,18,19) hormones increase the amount of pregrowth hormone mRNA in these cultured cells, while thyrotropin-releasing hormone increases the amount of preprolactin mRNA (9,20). A long-range goal of our work is to establish whether glucocorticoid and thyroid hormones bring about an increase of pregrowth hormone mRNA (pGH mRNA) due to increased transcription or some post-transcriptional event.…”

Mechanism of Uncoating of Influenza B Virus in MDCK Cells: Action of Chloroquine

“…phenol-chloroform (1 : 1), precipitated from the final aqueous phase with 2 M-LiCI for 16 h at 4 °C and dissolved in distilled water. The amount of RNA was calculated assuming that one A_,60 unit is 40 ~tg/ml (Seo et al, 1977).…”

“…Mock-infected or influenza B virus-infected cells were harvested 3-5 h after infection. The extraction of cellular RNA was carried out according to the method of Seo et al (1977).…”