We have examined the effects of nuclear receptor hormones such as glucocorticoid, gonadal steroid hormones, thyroid hormone and retinoids on the transcriptional regulation of the 5′-promoter activity of growth hormone (GH) gene using the MtT/S rat pure somatotrope cell line or MtT/SGL, a subclone of MtT/S in which the rat GH gene 5′-promoter (1.7 Kb)-luciferase fusion gene was stably incorporated. RT-PCR analyses revealed that receptors for all the hormones except androgen receptor were expressed in the cell line. Triiodothyronine (T₃, 10 nM) transiently but significantly stimulated the promoter activity of GH gene, whereas retinoic acids (9-cis and all-trans, both 1 µM) showed sustained stimulation. There were no additive effects among the T₃, all-trans, and9-cis retinoic acids. Synthetic glucocorticoid hormone dexamethasone (100 nM) showed an inhibitory effect but, interestingly, significantly enhanced T₃-stimulated GH promoter activity during long-term incubation. Among the gonadal steroid hormones tested, estradiol and estriol had significant stimulatory effects, and deletion analysis showed that the estrogen effect was maintained with the shortest construct examined (–150 to +6, +1 denotes the transcription start site). These results suggest that thyroid hormone and retinoids stimulate the transcription of GH gene, probably through a common response element, whereas glucocorticoid has both negative and positive effects on GH expression, depending on the combination with other hormones and the time of exposure. Estrogens also have direct stimulatory effects through the proximal promoter region of GH gene.

The two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin (SRIF), are known to regulate GH secretion. However, the effects of these hormones on GH gene expression are not completely clear, partly because of the lack of appropriate host cells maintaining the original characteristics of the somatotroph. Since MtT/S, a pure somatotroph cell line, has become available, the effects of GHRH and SRIF on GH gene transcription have been studied using a subclone of MtT/S (MtT/SGL), in which the GH gene 5′-promoter-luciferase fusion gene was stably incorporated. The expression of GHRH receptor and SRIF receptor subtypes was also studied by RT-PCR. The results showed that MtT/SGL cells intrinsically expressed the functional GHRH receptor and all of the SRIF receptor subtypes. The expression of GHRH receptor was markedly enhanced by glucocorticoid pretreatment and, in the presence of corticosterone and 3-isobutyl-1-methylxanthine, GHRH (at or above 100 pM) stimulated GH gene 5′-promoter activity in a dose-dependent manner. On the other hand, SRIF (100 nM) significantly antagonized the effect of GHRH, which was completely reversed by pretreatment with pertussis toxin (50 ng/ml). Taken together, the present data indicated that both GHRH and SRIF are involved in the transcriptional regulation of the GH gene, and that the effect of SRIF is mediated through pertussis toxin-sensitive G protein. The MtT/SGL cell line is a good in vitro model for studying the molecular mechanisms of GH gene transcription by GHRH and/or SRIF.

The V(1b) vasopressin receptor, expressed mainly in the corticotroph of the anterior pituitary, mediates the stimulatory effect of vasopressin on ACTH release. To clarify the regulation of receptor expression, we cloned, sequenced (up to approximately 5 kb from the translation start site), and characterized the 5'-flanking region of the rat V(1b) receptor gene. We identified the transcription start site by amplification of cDNA ends and found a new intron within the 5'-untranslated region (5'-UTR) by comparing the sequence with that of cDNA. We then confirmed that the obtained promoter indeed has transcriptional activity by use of the luciferase reporter in AtT-20 mouse corticotroph cells. Interestingly, there were five short upstream open reading frames (uORFs) located within the 5'-UTR that were found to suppress V(1b) expression. Subsequent mutational analyses showed that the two downstream uORFs have an inhibitory effect on expression in both homologous and heterologous contexts. Furthermore, the inhibition did not accompany a parallel decrease in mRNA, suggesting that the suppressive effect occurs at a level downstream of transcription. Taken together, our data strongly suggest that the expression of the V(1b) receptor is regulated at the posttranscriptional as well as transcriptional level through uORFs within the 5'-UTR region of the mRNA. Whether the uORF-mediated regulation of V(1b) expression is functionally linked to any intracellular and/or extracellular factor(s) awaits further research.

Although GHRH is known to play a pivotal role in the regulation of the GHRH-GH-IGF-I axis, the molecular mechanism of GHRH gene expression has not yet been examined. Here we studied the transcriptional regulation of the GHRH gene 5'promoter using an in vitro experimental model system. We especially focused on the role of homeobox transcriptional factor Gsh-1, because a dwarf phenotype and abolished GHRH expression was observed in Gsh-1 knockout mice. First, we cloned human Gsh-1, which showed 87.3% homology with mouse Gsh-1 at the nucleotide level. When the 5'-promoter region of the rat GHRH gene was introduced into the human placental cell line JEG-3, in which we found the endogenous expression of Gsh-1 as well as GHRH mRNA, substantial transcriptional activity of the promoter was recognized. Promoter activity was further enhanced by overexpression of Gsh-1 protein, whereas it was substantially reduced by elimination of Gsh-1 binding sites. EMSA confirmed the actual binding of Gsh-1 on the multiple binding sites of GHRH gene promoter. Finally, coexpression of CREB-binding protein significantly enhanced the Gsh-1-induced GHRH gene expression, suggesting the cooperative role of the coactivator protein. Because Gsh-1 is found to be expressed in the hypothalamus of the adult rat, our data provide evidence that the Gsh-1 homeobox protein plays a key role in the expression of the GHRH gene.

Polyamines are a ubiquitous group of amines that play diverse biological roles. In the anterior pituitary, intracellular polyamine levels are reported to show diurnal changes, although the biological significance remains to be elucidated. In this study, we examined the effects of polyamines on the transcriptional activity of the rat pro-opiomelanocortin (POMC) gene using AtT20PL, a clone of the AtT20 cell line in which an approximately 0.7 kb of the rat POMC 5' promoter-luciferase fusion gene was stably incorporated. The results showed that three representative polyamines (putrescine, spermidine and spermine) all stimulated POMC promoter activity in a time- and dose-related manner, spermine showing the most potent effect (maximum approximate three-fold increase). This effect was not observed under treatment with actinomycin D, suggesting the effect of polyamine at the transcriptional level. On the other hand, methylglyoxal bis (guanylhydrazone), an inhibitor of polyamine synthesis, showed the opposite effect, further supporting the positive role of intracellular polyamines. Taken together, our findings suggest that polyamines are involved in the regulation of POMC gene expression (especially in terms of diurnal changes) in corticotroph cells. The precise molecular mechanisms of polyamine effects await further research.