Regulation of PIT-1 expression by ghrelin and GHRP-6 through the GH secretagogue receptor

GH secretagogues are an expanding class of synthetic peptide and nonpeptide molecules that stimulate the pituitary gland to secrete GH through their o wn specific receptor, the GH-secretagogue receptor. The cloning of the rece ptor for these nonclassical GH releasing molecules, together with the more recent characterization of an endogenous ligand, named ghrelin, have unambi guously demonstrated the existence of a physiological system that regulates GH secretion. Somatotroph cell-specific expression of the GH gene is depen dent on a pituitary-specific transcription factor (Pit-1). This factor is t ranscribed in a highly restricted manner in the anterior pituitary gland. T he present experiments sought to determine whether the synthetic hexapeptid e GHRP-6, a reference GH secretagogue compound, as well as an endogenous li gand, ghrelin, regulate pit-1 expression. By a combination of Northern and Western blot analysis we found that GHRP-6 elicits a time- and dose-depende nt activation of pit-1 expression in monolayer cultures of infant rat anter ior pituitary cells. This effect was blocked by pretreatment with actinomyc in D, but not by cycloheximide, suggesting that this action was due to dire ct transcriptional activation of pit-1. Using an established cell line (HEK 293-GHS-R) that overexpresses the GH secretagogue receptor, we showed a mar ked stimulatory effect of GHRP-6 on the pit-1 -2,500 bp 5 ' -region driving luciferase expression. We truncated the responsive region to -231 bp, a se quence that contains two CREs, and found that both CREs are needed for GHRP -6-induced transcriptional activation in both HEK293-GHS-R cells and infant rat anterior pituitary primary cultures. The effect was dependent on PKC, MAPK kinase, and PKA activation. Increasing Pit-1 by coexpression of pCMV-p it-1 potentiated the GHRP-6 effect on the pit-1 promoter. Similarly, we sho wed that the endogenous GH secretagogue receptor ligand ghrelin exerts a si milar effect on the pit-1 promoter. These data provide the first evidence t hat ghrelin, in addition to its previously reported GH-releasing activities , is also capable of regulating pit-1 transcription through the GH secretag ogue receptor in the pituitary, thus giving new insights into the physiolog ical role of the GH secretagogue receptor on somatotroph cell differentiati on and function.