A Composite Hormone Response Element Regulates Transcription of the Rat GHRH Receptor Gene

Abstract

To further elucidate the molecular mechanisms underlying the transcriptional regulation of the GHRH receptor (GHRH-R) gene, hormonal regulation of the promoter activity of this gene was examined. An approximately 3-kb genomic fragment spanning the promoter region of the gene was sequenced and the transcription start site was determined by RT-PCR and RNase protection assay. A major start site was localized at -105 (relative to the translation initiation codon, ATG), and a pit-1 binding sequence characteristic of pituitary specific genes was found at -155 to -146. Deletion and mutation studies demonstrated this site to be functional. In the presence of dexamethasone, the GHRH-R promoter (from -2935 to -11) directed luciferase expression in MtT-S cells, a somatotropic cell line, but not in the PC12 cells that normally do not express GHRH-R. While T(3), all trans-RA, and 9cis-RA alone weakly enhanced the reporter gene expression, each of these substances was found to act as a synergistic enhancer in the presence of dexamethasone. Additional deletion and mutation analyses demonstrated a functional RA response element at -1090 to -1074. Two functional glucocorticoid response elements and a T(3) response element were found in an 80-bp 5'-flanking sequence of the pit-1 site. Interestingly, it is suggested that the 6-bp half-site AGGACA (from -209 to -204) functions as a 3'-half-site of T(3) response element as well as a 5'-half-site of one of the glucocorticoid response elements.

The role of pit-1 in the regulation of the rat growth hormone-releasing hormone receptor gene transcription by glucocorticoids

“…2 that the transcription from dGpG with no pit-1 site responded to DEX by 4·4-fold (Fig. 2), while it coincides well with previous results that showed that the transcription of the GHRH-receptor gene requires pit-1 (Lin et al 1992, Iguchi et al 1999, Miller et al 1999, Nogami et al 2002. The only explanation possible for this discrepancy is that some DNA sequences within dGpG-p that are absent in dGpG, the pit-1 site and the 14-base pair spacer between pGRE and the pit-1 site, contain the silencer element.…”

“…The expression plasmid for the rat GR, pit-1, and thyroid hormone receptor (TR) 2 have been described previously (Nogami et al 2002). The expression plasmid for retinoic acid receptor (RXR) was a gift from Dr Goda (Department of Nutrition, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan).…”

“…The expression plasmid for CCAAT-enhancer binding protein (C/EBP) (NFIL6 (Akira et al 1990)) was obtained from Riken BRC DNA Bank, Ibaraki, Japan. The reporter construct prGHRH-R-125/+95-luc was a pGL2 (Promega, Madison, WI, USA)-based plasmid carrying a 220 base pair promoter fragment (from +95 to 125, relative to the transcription start site; Nogami et al 2002). The promoter of the herpes simplex virus thymidine kinase (from +29 to -107; tk) was PCR amplified and cloned into the BglII and HindIII sites of pGL3-basic plasmid (Promega) to generate the pGL3-tk plasmid.…”

“…Within these factors, glucocorticoids are of particular importance, because they trigger the onset of GHRH-receptor expression in the developing pituitary gland (Nogami et al 1999), which establishes functional correlation between brain and pituitary GH cells. In a previous study, we identified two GREs, a thyroid hormone response element (TRE) and a binding site for pit-1, a pituitary specific transcription factor (Bodner et al 1988, Ingraham et al 1988, in the upstream region of the rat GHRH-receptor gene (Nogami et al 2002). Pit-1 plays a principal role in the cell type specification of prolactin (PRL), growth hormone (GH) and a subset of thyroid stimulating hormone (TSH) cells, and is a prerequisite for the transcriptional activation of the genes that are specifically expressed in these cells (Bodner et al 1988, Ingraham et al 1988, including GHRH-receptor gene (Lin et al 1992, Iguchi et al 1999, Miller et al 1999, Nogami et al 2002.…”

International Union of Pharmacology. XXXV. The Glucagon Receptor Family

“…Also indicated are several of the best characterized receptor variants, although others have been reported. 186 tory element in the receptor gene promoter (Nogami et al, 2002).…”