Pituitary-Specific Expression and Pit-1 Regulation of the Rat Growth Hormone-Releasing Hormone Receptor Gene

Abstract

The GHRH receptor is expressed in the somatotroph cell of the anterior pituitary, where it functions to mediate GHRH-stimulated GH release. To study pituitary and somatotroph cell-specific expression of this gene, a transgenic mouse model and complementary cell culture experiments were developed. The activity of the 1.6-kb proximal rat GHRH receptor promoter was examined in vivo by generating transgenic mice with the promoter directing expression of a luciferase reporter. The promoter directs tissue-specific expression; luciferase is highly expressed in the pituitary but absent from 14 other tissues. Immunocytochemistry experiments show that transgene expression is targeted to GH-expressing somatotroph cells. The transgene is 5-fold more highly expressed in males than females, and there is an increase in transgene expression leading up to the onset of puberty. The 1.6-kb promoter was further examined in cell culture experiments, which revealed that the promoter is selectively activated in pituitary cells and that promoter-reporter expression in nonpituitary cells can be enhanced by the pituitary-specific transcription factor Pit-1. EMSAs identified 10 short regions that specifically bind Pit-1 with highly variable relative affinities. The highest affinity site was previously identified and is required for Pit-1 activation of the promoter. Four additional sites contribute to Pit-1 regulation of the promoter and are important to achieving full activation of the gene. The results show that the 1.6-kb promoter is sufficient to direct tissue- and cell-specific expression in vivo and is regulated by Pit-1.

Development of a pituitary‐specific cre line targeted to the Pit‐1 lineage

“…After confirming good expression in the target tissue, a panel of 14 tissues of the mouse, including all major organs (heart, lung, liver kidney, intestine, brain) and a subset of endocrine glands (adrenals, pancreas, gonads, thyroid, mammary) were tested. Consistent with endogenous rGhrhr expression (Mayo, 1992) and the finding of the rGhrhr-luc transgenic line (McElvaine et al, 2007), the blue staining was seen exclusively in the mouse pituitary gland, except for some scant staining of hair follicles in some parts of the skin (data not shown). To determine which cells within the pituitary gland expressed Cre, lacZ-stained sections were subject to immunohistochemical analysis for anterior pituitary hormones, and examples of this data are shown in Figure 2.…”

“…Although the promoter used for the generation of these transgenic mice is of rat origin, the Ghrh receptor itself is 100% conserved between mouse and rat (Toogood et al, 2006), and the promoter regions exhibit 83% nucleotide identity (data not shown). Moreover, transgenic mice containing a rGhrhr-luciferase transgene expressed luciferase solely in the pituitary gland (McElvaine et al, 2007), suggesting that this promoter could function in a highly tissue-specific fashion.…”

Cellular in vivo imaging reveals coordinated regulation of pituitary microcirculation and GH cell network function

“…Previous studies have described that GHRH receptors are restrictively expressed in GH cells (27)(28)(29), although we cannot exclude the possibility that low levels of receptor may be present in some other pituitary cell types. If GHRH receptors are completely restricted to GH cells, then local alterations in flow are most likely a consequence of secretory activity from stimulated GH cells.…”

Antagonist of GH-releasing hormone receptors alleviates experimental ocular inflammation

“…However, the pituitary-type GHRH-R rather than SV-1 is the predominant form of receptor expressed in normal ocular tissues (26). Moreover, the presence of Pit-1, an important activator of the GHRH-R and GH promoters (21,27), supports the involvement of GHRH signaling in these tissues. The ocular tissues may respond to GHRH and augment synthesis of GH, which binds to GH receptor (GH-R) on the ciliary epithelium, for production of molecules downstream of GH signaling, including IGF1.…”