Interplay of pituitary adenylate cyclase-activating polypeptide with a silencer element to regulate the upstream promoter of the human gonadotropin-releasing hormone receptor gene.

Multiple transcription start sites were identified in the human gonadotropin releasing hormone receptor (hGnRHR) gene. Recently, an upstream promoter residing at -1727/-1674, in vicinity of a CAP site at -1673, was characterized. In this report, we elucidated the underlying mechanisms for the regulation of this promoter. Functionally, this promoter was constitutively suppressed by a silencer element (-1673/-1351) situated immediately downstream to it. On the other hand, pituitary adenylate cyclase-activating polypeptide (PACAP), via the cAMP pathway, was found to be the extracellular cue to control the upstream promoter. Following PACAP-27, PACAP-38 (30 nM) and forskolin (25 microM) treatment, there were significant increases in the reporter gene activities. By deletion analysis, the region residing at -1727 to -1577, containing the distal promoter and 97 bp of the silencer was subsequently found to be responsible for PACAP/cAMP induction. To localize the PACAP-dependent cis-acting element(s) within the silencer, block replacement scanning mutation was performed and a hGnRHR gene PACAP-responsive element (GPRE) was identified at -1676/-1648. The actions of PACAPs and forskolin on the GPRE were further evidenced by gel mobility shift assays. There was an increase in protein binding onto this element only after peptide treatment. As GnRH receptor number on gonadotrope cell surface is a key factor in regulating gonadotropin release, the present study provides an insight into the interplay between PACAP and GnRH receptors on pituitary gonadotropes to control human reproductive functions.