IDENTIFICATION OF A GONADOTROPIN-RELEASING HORMONE-RESPONSIVE REGION IN THE GLYCOPROTEIN HORMONE ALPHA-SUBUNIT PROMOTER

Transcriptional regulation of the glycoprotein hormone alpha-subunit gene has been studied extensively in placental cells, but much less is known about its regulation in the pituitary gland. In this study, transcriptional control of the human alpha-subunit gene by GnRH was analyzed using transient expression assays in primary cultures of rat pituitary cells using alpha promoter contructs linked to a luciferase reporter gene. Deletion mutants between -846 and -156 basepairs (bp) had little effect on basal expression, but further deletion to -132 bp reduced basal activity by approximately 50%. Deletion of a cAMP response element between -132 and -99 bp caused a marked loss of basal activity, reducing expression to that of background luciferase activity. The same constructs were analyzed for cAMP responsiveness in primary pituitary cells. The degree of stimulation with 1 mM 8-bromo-cAMP (3.6- to 6.0-fold) was relatively unaffected by deletions from -846 to -132 bp, whereas cAMP stimulation was decreased by further deletion to -99 bp, consistent with the presence of previously defined cAMP response elements in this region of the alpha promoter. GnRH stimulation of alpha promoter activity was highly dependent upon the time of hormone addition after transfection, being most effective when added soon after transfection. Under optimal conditions, GnRH stimulated -846alphaLUC expression by 20-fold. GnRH responsiveness was retained with deletion to -346 bp, but it was decreased by 55% after deletion to -280 bp and by 79% with deletion to -244 bp. A series of DNA sequences between -346 and -180 bp was linked to the truncated -132alpha promoter to define the GnRH-responsive region in more detail. Sequences between -346/-180 and -346/-244 conferred greater GnRH responsiveness than was seen with a -346/-280 fragment, confirming an important role for the region between -280 and -244 bp for GnRH responsiveness. These studies suggest that the transcriptional response to GnRH is mediated through one or more elements between -346 and -244 bp and that GnRH-responsive sequences are distinct from those involved in basal and cAMP-stimulated expression of the alpha promoter.