Transcriptional activation of gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: Involvement of multiple signal transduction pathways

Previous studies have shown that GnRH activates transcriptional activity of its own receptor (GnRHR) gene in part through the cAMP signal transduction pathway. In the present study we explored the possible involvement of mult iple signal transduction pathways in GnRH regulation of GnRHR gene transcri ption; these studies relied upon a luciferase reporter gene vector (GnRHR-p XP2) containing a 1226-bp promoter fragment (-1164 to +62, relative to the major transcription start site) of the mouse GnRHR gene in GGH(3) cells (GH (3) cells stably expressing rat GnRHR). Activation of protein kinase C (PKC ) by phorbol myristic acid significantly stimulated GnRHR-luciferase report er gene (GnRHR-Luc) activity, but did not potentiate the stimulation of GnR HR-Luc activity by the GnRH agonist, buserelin (GnRH-A). Inhibition of PXC by PKC inhibitor (GF 109203X) or depletion of PKC blocked phorbol myristic acid- or GnRH-A-stimulated GnRHR-Luc activity, but did not affect (Bu)(2)cA MP-stimulated GnRHR-Luc activity. In addition, GnRH-A-stimulated GnRHR-Luc activity was inhibited by preventing external Ca2+ influx with the external Ca2+ chelator EGTA or the Ca2+ ion channel antagonist, D600. Surprisingly, overexpression of the mitogen-activated protein kinase (MAPK) kinase kinas e (Raf-l) inhibited GnRHR-Luc activity and partially blocked GnRH-A-stimula ted GnRHR-Luc activity. In contrast, inhibition of MAPK activity by MAPK ki nase inhibitor (PD 98059) or by overexpression of kinase-deficient MAPKs ac tivated basal and GnRH-A-stimulated GnRHR-Luc activity. These results sugge sted that PKC- and Ca2+-dependent. signal transduction pathways participate in the GnRH activation of GnRHR promoter activity, and that the MAPK casca de is involved in the negative regulation of basal and GnRH-stimulated GnRH R transcriptional activity conferred by the 1226-bp promoter fragment.