GONADOTROPIN-RELEASING-HORMONE CAUSES TRANSCRIPTIONAL STIMULATION FOLLOWED BY DESENSITIZATION OF THE GLYCOPROTEIN HORMONE-ALPHA PROMOTER IN TRANSFECTED ALPHA-T3 GONADOTROPE CELLS

Pulsatile GnRH regulates the biosynthesis and secretion of gonadotropins. Continuous administration of GnRH is known to desensitize gonadotropin secretion, but its effects on gonadotropin gene expression are less well characterized. We used a cell line of gonadotrope lineage (alpha T3 cells) to examine GnRH regulation of glycoprotein hormone alpha-subunit gene transcription. The Lu-subunit promoter, linked to a luciferase reporter gene (alpha LUC), was stably transfected into alpha T3 cells. Treatment with GnRH stimulated alpha LUC activity 3-fold. Stimulation of alpha LUC by GnRH was transient, with maximal activity after 6 h of treatment, followed by a return to baseline after 24 h. Stimulation of alpha-promoter activity by GnRH was inhibited entirely by a 10-fold molar excess of antide, a GnRH antagonist. Antide partially blocked GnRH stimulation even when added 4 h after GnRH, suggesting that a brief exposure to GnRH is not sufficient for maximal transcriptional stimulation. alpha LUC activity was also stimulated by treatment with 8-bromo-cAMP (3.5-fold), phorbol 12-myristate 13-acetate (TPA; 2.6-fold), or Bay K 8644 (3.3-fold). To assess whether the transient nature of GnRH stimulation was due to transcriptional desensitization, cells were pretreated with GnRH, followed by a second treatment with GnRH, cAMP, TPA, or Bay K. After pretreatment with GnRH, no further stimulation was seen after the addition of GnRH or TPA, but alpha LUC activity was further stimulated after the addition of either cAMP or Bay K. These findings indicate that the pathway for transcriptional activation by GnRH is desensitized and suggest that GnRH also desensitizes TPA-mediated stimulation. Similarly, pretreatment with TPA, but not cAMP or Bay K, prevented subsequent stimulation by GnRH. We conclude that GnRH transiently stimulates alpha gene transcription and that desensitization occurs with continuous exposure to GnRH, probably because of down-regulation of the protein kinase-C pathway.