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

Pulsatile GnRH regulates the biosynthesis and secretion of gonadotropi ns. Continuous administration of GnRH is known to desensitize gonadotr opin secretion, but its effects on gonadotropin gene expression are le ss well characterized. We used a cell line of gonadotrope lineage (alp ha T3 cells) to examine GnRH regulation of glycoprotein hormone alpha- subunit gene transcription. The Lu-subunit promoter, linked to a lucif erase reporter gene (alpha LUC), was stably transfected into alpha T3 cells. Treatment with GnRH stimulated alpha LUC activity 3-fold. Stimu lation of alpha LUC by GnRH was transient, with maximal activity after 6 h of treatment, followed by a return to baseline after 24 h. Stimul ation of alpha-promoter activity by GnRH was inhibited entirely by a 1 0-fold molar excess of antide, a GnRH antagonist. Antide partially blo cked 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-fo ld), 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, c AMP, TPA, or Bay K. After pretreatment with GnRH, no further stimulati on 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. The se findings indicate that the pathway for transcriptional activation b y GnRH is desensitized and suggest that GnRH also desensitizes TPA-med iated stimulation. Similarly, pretreatment with TPA, but not cAMP or B ay K, prevented subsequent stimulation by GnRH. We conclude that GnRH transiently stimulates alpha gene transcription and that desensitizati on occurs with continuous exposure to GnRH, probably because of down-r egulation of the protein kinase-C pathway.