Transcriptional down-regulation of human gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: Role of protein kinase C and activating protein1

Clinical applications of GnRH agonists (GnRHa) are based primarily on the d ecrease in gonadotropin release after downregulation of the GnRH receptor ( GnRHR) by continuous GnRHa administration. However, the molecular mechanism s underlying the transcriptional regulation of the human GnRHR gene after p rolonged GnRH treatment remain poorly understood. In the present study GnRH a-mediated regulation of human GnRHR gene transcription was studied by tran siently transfecting the mouse gonadotrope-derived (alpha T3-1) cells with a 2297-bp human GnRHR promoter-luciferase construct (p2300-LucF). A dose- a nd time-dependent decrease in human GnRHR promoter activity was observed af ter GnRHa treatment. An average 71% decrease in promoter activity was obser ved after 24-h treatment with 0.1 mu M GnRHa, which was blocked by cotreatm ent of the GnRH antagonist, antide. This effect was mimicked by phorbol 12- myristate 13-acetate (TPA) administration. In addition, the GnRHa- and TPA- mediated decrease in the human GnRHR promoter activity was reversed by a sp ecific protein kinase C (PKC) inhibitor, GF109203X, or depletion of PKC by TPA pretreatment. These findings indicate that the activation of the PKC pa thway is important in regulating the human GnRHR gene expression. By progressive 5'-deletion studies, we have identified a 248-bp DNA fragmen t (-1018 to -771, relative to the translation start site) at the 5'-flankin g region of the human GnRHR gene that is responsible for the GnRHa-mediated down-regulation of human GnRHR promoter activity. Analysis of this sequenc e reveals the existence of two putative activating protein-1 (AP-1) sites w ith 87% homology to the consensus sequence (5'-TGA G/C T C/A A-3'), located at -1000 to -994 (5'-TTAGACA3', in complementary orientation) and -943 to -937 (5'-TGAATAA-3'). Using competitive gel mobility shift assays, AP-1 bin ding was observed within this 248-bp region. Site-directed mutation of the putative AP-1-binding site located at - 1000 to -994 abolished the GnRHa-in duced inhibition. Further competitive GMSA and supershift experiments confi rmed the identity of AP-1 binding in this region. By the use of Western blo t analysis, a significant increase in c-Jun (100%; P < 0.05) and c-Fos (50% ; P < 0.05) protein levels was observed after GnRHa treatment in alpha T3-1 cells. In addition, our data suggested that a change in AP-1 composition, particularly c-Fos, was important in mediating GnRHa-induced inhibition of human GnRHR gene expression. We conclude that activation of the PRC pathway by GnRH is important in cont rolling human GnRHR gene expression. In addition, the putative AP-1-binding site located at - 1000 to -994 of the human GnRHR 5'-flanking region has b een functionally identified to be involved in mediating this down-regulator y effect.