Homeobox protein Gsh-1-dependent regulation of the rat GHRH gene promoter

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 dw arf 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 f ound the endogenous expression of Gsh-1 as well as GHRH mRNA, substantial t ranscriptional activity of the promoter was recognized. Promoter activity w as further enhanced by overexpression of Gsh-1 protein, whereas it was subs tantially reduced by elimination of Gsh-1 binding sites. EMSA confirmed the actual binding of Gsh-1 on the multiple binding sites of GHRH gene promote r. Finally, coexpression of CREB-binding protein significantly enhanced the Gsh-l-induced GHRH gene expression, suggesting the cooperative role of the coactivator protein. Because Gsh-1 is found to be expressed in the hypotha lamus of the adult rat, our data provide evidence that the Gsh-1 homeobox p rotein plays a key role in the expression of the GHRH gene.