Suen C S, Chin W W
Department of Medicine, Brigham and Women's Hospital, Howard Hughes Medical Institute, Boston, Massachusetts.
Mol Cell Biol. 1993 Mar;13(3):1719-27. doi: 10.1128/mcb.13.3.1719-1727.1993.
The expression of the rat growth hormone (rGH) gene in the anterior pituitary gland is modulated by Pit-1/GHF-1, a pituitary-specific transcription factor, and by other more widely distributed factors, such as the thyroid hormone receptors (TRs), Sp1, and the glucocorticoid receptor. Thyroid hormone (T3)-mediated transcriptional stimulation of rGH gene expression has been extensively studied in vivo and in vitro including the measurements of (i) rGH mRNA by blot hybridization, (ii) transcriptional rate of rGH gene by nuclear run-on, and (iii) reporter gene expression in which a chimeric plasmid containing 5'-flanking sequences of the rGH gene linked to a reporter gene has been transfected either stably or transiently into pituitary and/or nonpituitary cells. From these studies, it has been suggested that the Pit-1/GHF-1 binding site is necessary for full T3 action. We developed a cell-free in vitro transcription system to examine further the roles of the TRs and Pit-1/GHF-1 in rGH gene activation. Using GH3 nuclear extract as a source of TRs and Pit-1/GHF-1, this in vitro transcription assay showed that T3 stimulation of rGH promoter activity is dependent on the addition of T3 to the GH3 nuclear extract. This transcriptional stimulation was augmented with increasing concentrations of ligand and was T3, but not T4 or reverse T3, specific. T3-mediated stimulation of rGH promoter activity was completely abolished by preincubation of the nuclear extract with rGH-thyroid hormone response element (-200 to -160) but not with Pit-1/GHF-1 (-137 to -65) oligonucleotides. Further, neither deletion of both Pit-1/GHF-1 binding sites nor mutation of the proximal Pit-1/GHF-1 binding site from the rGH promoter abrogated the T3 effect. These results provide evidence that T3-stimulated rGH promoter activity is independent of Pit-1/GHF-1 and raise the possibility that the stimulation of rGH gene expression by T3 might involve direct interaction of TRs with the general transcriptional apparatus.
大鼠生长激素（rGH）基因在前脑垂体中的表达受垂体特异性转录因子Pit-1/GHF-1以及其他分布更为广泛的因子调控，如甲状腺激素受体（TRs）、Sp1和糖皮质激素受体。甲状腺激素（T3）介导的rGH基因表达转录刺激作用已在体内和体外得到广泛研究，包括通过以下方法进行测定：（i）通过印迹杂交检测rGH mRNA；（ii）通过核转录分析rGH基因的转录速率；（iii）报告基因表达，即将含有与报告基因相连的rGH基因5'侧翼序列的嵌合质粒稳定或瞬时转染到垂体和/或非垂体细胞中。从这些研究中可以看出，Pit-1/GHF-1结合位点对于T3的完全作用是必需的。我们开发了一种无细胞体外转录系统，以进一步研究TRs和Pit-1/GHF-1在rGH基因激活中的作用。使用GH3核提取物作为TRs和Pit-1/GHF-1的来源，这种体外转录分析表明，T3对rGH启动子活性的刺激取决于向GH3核提取物中添加T3。这种转录刺激随着配体浓度的增加而增强，并且具有T3特异性，而非T4或反式T3。用rGH-甲状腺激素反应元件（-200至-160）对核提取物进行预孵育可完全消除T3介导的rGH启动子活性刺激，但用Pit-1/GHF-1（-137至-65）寡核苷酸预孵育则不会。此外，从rGH启动子中删除两个Pit-1/GHF-1结合位点或使近端Pit-1/GHF-1结合位点发生突变均不会消除T3的作用。这些结果证明，T3刺激的rGH启动子活性独立于Pit-1/GHF-1，并增加了T3刺激rGH基因表达可能涉及TRs与一般转录装置直接相互作用的可能性。
