Soto J L, Castrillo J L, Dominguez F, Dieguez C
Department of Physiology, Faculty of Medicine, University of Santiago de Compostela, Spain.
Endocrinology. 1995 Sep;136(9):3863-70. doi: 10.1210/endo.136.9.7649093.
Pituitary-specific expression of the GH gene is dependent on a pituitary-specific transcription factor GH factor-1 (GHF-1), a homeodomain protein also known as pituitary-specific transcription factor-1 (Pit-1). The aim of this study was to investigate the regulation of GHF-1 messenger RNA (mRNA) levels in primary monolayer cultures of rat anterior pituitary cells. Specifically, in addition to direct activators of second messenger signaling systems, we studied the effects of different hormones, all of which are known to be involved in the regulation of somatotroph cell function. We found that GH-releasing hormone (GHRH) increased GHF-1 mRNA levels in a time- and dose-dependent fashion. GHF-1 mRNA levels were increased 2.5-fold (P < 0.01) after incubation for 2 h with 10(-8) M GHRH. Longer incubations (6, 12, or 24 h) with GHRH failed to show a similar stimulatory effect. A significant increase in GHF-1 mRNA concentration (1.7-fold, P < 0.01) was observed after a 2-h treatment with physiological concentrations (10(-11) M) of GHRH. The action of GHRH seems to occur at the transcriptional level without the need of protein synthesis. Thus, treatment of cells with actinomycin D (5 micrograms/ml) completely abolished GHRH-induced increase in GHF-1 mRNA levels. Cycloheximide (23 micrograms/ml) alone increased GHF-1 mRNA levels (6-fold increase after treatment for 12 h, P < 0.01), as well as potentiating GHRH-induced increase in GHF-1 mRNA concentration (9-fold increase after treatment with GHRH plus cycloheximide for 12 h, P < 0.01). The effect of GHRH on GHF-1 mRNA levels could be mimicked by direct activators of second messenger signaling systems such as forskolin (10(-5) M) or the phorbol ester tumor promoter tetradecanoyl phorbol acetate (TPA) (10(-6) M). Other peptides such as pituitary adenylate cyclase activating polypeptide-38 (10(-7) M) but not GHRP-6 (10(-10) to 10(-5) M), were also able to increase GHF-1 mRNA levels. Treatment of the cells with somatostatin (10(-6) M) for either 2 or 48 h failed to modify basal or GHRH-induced GHF-1 mRNA levels. In contrast, pretreatment of the cells with insulin-like growth factor-1 (5 nM) inhibited basal GHF-1 mRNA concentration as well as completely blunting the subsequent response to cells exposed to GHRH for 2 h. These data demonstrate that GHRH, acting at the transcriptional level and through a mechanism not dependent on protein synthesis, plays a stimulatory role on GHF-1 mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)
生长激素（GH）基因在垂体中的特异性表达依赖于一种垂体特异性转录因子GH因子-1（GHF-1），它是一种同源结构域蛋白，也被称为垂体特异性转录因子-1（Pit-1）。本研究的目的是调查大鼠垂体前叶细胞原代单层培养物中GHF-1信使核糖核酸（mRNA）水平的调控。具体而言，除了第二信使信号系统的直接激活剂外，我们还研究了不同激素的作用，所有这些激素都已知参与生长激素细胞功能的调节。我们发现，生长激素释放激素（GHRH）以时间和剂量依赖的方式增加GHF-1 mRNA水平。与10⁻⁸ M GHRH孵育2小时后，GHF-1 mRNA水平增加了2.5倍（P < 0.01）。与GHRH更长时间的孵育（6、12或24小时）未显示出类似的刺激作用。用生理浓度（10⁻¹¹ M）的GHRH处理2小时后，观察到GHF-1 mRNA浓度显著增加（1.7倍，P < 0.01）。GHRH的作用似乎发生在转录水平，无需蛋白质合成。因此，用放线菌素D（5微克/毫升）处理细胞完全消除了GHRH诱导的GHF-1 mRNA水平的增加。单独使用环己酰亚胺（23微克/毫升）增加了GHF-1 mRNA水平（处理12小时后增加6倍，P < 0.01），同时增强了GHRH诱导的GHF-1 mRNA浓度的增加（用GHRH加环己酰亚胺处理12小时后增加9倍，P < 0.01）。GHRH对GHF-1 mRNA水平的作用可被第二信使信号系统的直接激活剂如福司可林（10⁻⁵ M）或佛波酯肿瘤促进剂十四烷酰佛波醇乙酸酯（TPA）（10⁻⁶ M）模拟。其他肽类如垂体腺苷酸环化酶激活多肽-38（10⁻⁷ M）而非生长激素释放肽-6（10⁻¹⁰至10⁻⁵ M）也能够增加GHF-1 mRNA水平。用生长抑素（10⁻⁶ M）处理细胞2小时或48小时均未能改变基础或GHRH诱导的GHF-1 mRNA水平。相反，用胰岛素样生长因子-1（5 nM）预处理细胞抑制了基础GHF-1 mRNA浓度，并完全减弱了随后对暴露于GHRH 2小时的细胞的反应。这些数据表明，GHRH在转录水平起作用，通过一种不依赖蛋白质合成的机制，对GHF-1 mRNA水平起刺激作用。（摘要截短至400字）
