Induction of expression and functional analyses of glucocorticoid receptors in the growth hormone cells of the fetal rat pituitary gland

为了阐明生长激素（GH）细胞功能成熟的分子机制，利用原代培养的胎鼠垂体细胞研究了诱导糖皮质激素（GC）受体表达的机制。妊娠第16天，未检测到GH表达的大鼠胎儿垂体细胞强迫表达糖皮质激素受体，导致GH表达显著增加。在单独的实验中，在含有血清的培养基中培养数天后，类似的培养物中出现免疫反应性GH细胞。这些结果提示GC受体的表达是未成熟GH细胞启动GH表达的重要步骤，胎儿GH细胞中诱导GC受体的因子可能存在于血清中。然而，该因素的确切性质在本研究中无法阐明。本研究的另一个结果是阐明了GC调控ghrh受体基因表达的分子基础。克隆了一个横跨该基因启动子区约3kb的基因组片段，对其进行了测序，并通过RT-PCR和RNase保护实验确定了转录起始位点。在地塞米松（DEX）存在下，GHRH-R启动子（从11到2935）在MtT-S细胞（一种促生长细胞系）中指导荧光素酶的表达，而在PC12细胞中没有。虽然甲状腺激素、所有反式维甲酸和9顺式维甲酸单独微弱地增强了报告基因的表达，但在DEX存在时，这些物质都被发现具有协同增强作用。对启动子截断的报告子结构的进一步分析表明，在-1167 -664区域有一个功能性维甲酸反应元件，在pit-1位点的80 bp 5'侧序列中有三个糖皮质激素反应元件和甲状腺激素反应元件的候选序列。综上所述，GC是胎儿大鼠垂体GH细胞功能成熟的关键分子，诱导GH和ghrh受体两种GH细胞特异性分子的表达。然而，目前的研究结果表明，每种分子的诱导机制完全不同。GC诱导GH表达是由GC诱导的未知蛋白介导的，在这种情况下，GC受体与GH启动子之间不需要直接相互作用，而GC作为转录因子与5'上游区域的特定序列相互作用，诱导ghrh受体表达。少

In order to elucidate the molecular mechanisms underlying functional maturation of growth hormone (GH) cells, the mechanisms responsible for the induction of glucocorticoid (GC) receptor expression was investigated using fetal rat pituitary cells in primary culture. The forced expression of glucocorticoid receptor in the pituitary cells from rat fetuses on day 16 of gestation, where no GH expression was detected, resulted in a marked increase in GH expression. In the separate experiment, the immunoreactive GH cells appeared in the similar culture when they were cultured for several days in the medium containing serum. These results suggest that the expression of GC receptor is an important step for the immature GH cells to initiate GH expression and that the factor required for the induction of GC receptor in the fetal GH cells may reside in serum. However, the exact nature of the factor could not be elucidated in this study.Another result of this study was the elucidation of the molec … More ular basis for the GC regulation of the GHRH-receptor gene expression. An approximately 3 kb genomic fragment spanning the promoter region of the gene was cloned, sequenced and the transcription start site was determined by RT-PCR and RNase protection assay. In the presence of dexamethasone (DEX), the GHRH-R promoter (from-11 to-2935) directed luciferase expression in MtT-S cells, a somatotropic cell line, but not in the PC12 cells. While thyroid hormone, all trans-retinoic acid and 9cis-retinoic acid alone weakly enhanced the reporter gene expression, each of these substances was found to act as a synergistic enhancer in the presence of DEX.Additional analyses of promoter-truncated reporter constructs demonstrated a functional retinoic acid response element in the region between-1167 and-664 and three candidate sequences for a glucocorticoid response element and a thyroid hormone response element in an 80 bp 5'-flanking sequence of the pit-1 site.These results taken as a whole indicate that GC is an key molecule for the functional maturation of GH cells in the fetal rat pituitary gland, that induces expressions of two GH cell specific molecules, GH and GHRH-receptor. However, the present results showed that the mechanisms for the induction of each molecule are totally different. GC-induction of GH expression is mediated by unknown protein that is induced by GC, no direct interaction between GC receptors and GH promoter being required in this case, while GC acts as the transcription factor to interact with particular sequences in the 5'-upstream region to induce GHRH-receptor expression. Less