Gonadotropin-releasing hormone (GnRH) gene regulation by N-methyl-D-aspartic acid in GT1-1 neuronal cells: differential involvement of c-fos and c-jun protooncogenes.

The present study examined the regulatory mechanisms of GnRH gene expression by N-methyl-d-aspartic acid (NMDA) in immortalized hypothalamic GnRH neurons (GT1-1 cells). NMDA (100 microM) stimulated GnRH mRNA levels transiently at 2 h after treatment. Dose-response experiment showed that there was a biphasic action of NMDA on GnRH mRNA levels: GnRH mRNA levels were increased by NMDA at lower concentrations (10 and 100 microM), but not at higher concentrations (1 and 10 mM). NMDA (100 microM)-induced GnRH mRNA levels were efficiently blocked by pre-treatment with NMDA receptor antagonists, MK-801 and AP-5. We next examined the signal transduction pathways involved in NMDA-induced GnRH gene expression based on previous findings that NMDA signal propagates into the cell through Ca2+ and nitric oxide (NO) pathways in many neurons. While ionomycin, a Ca2+ ionopore, application failed to alter GnRH gene expression, treatment of GT1-1 cells with sodium nitroprusside (SNP), an NO donor, increased GnRH gene expression with a similar time course to NMDA treatment. Moreover, application of GT1-1 cells with nitric oxide synthase (NOS) inhibitors (l-NAME, d-NAME, and NA) prior to NMDA treatment, inhibited NMDA-induced GnRH gene expression. These results indicate that the effect of NMDA is mediated by the NO signalling cascade. The mouse GnRH promoter activity was also increased by NMDA at low concentration (100 microM), but not at high concentration (1 microM), confirming the biphasic action of NMDA on GnRH mRNA levels. Since NMDA (100 microM) and SNP (1 microM) markedly induced c-jun expression, but not c-fos expression, we hypothesized that Jun activation is responsible for the transcriptional activation of GnRH gene expression. To examine this, we performed two different experiments. Treatment of NMDA greatly increased the activity of heterologous promoter of Fos/Jun responsive sequence (-187/-69) from the mouse GnRH promoter fused to hsv-tk minimal promoter. Moreover, overexpression of c-jun induced GnRH promoter activity, while c-fos overexpression decreased GnRH promoter activity. Taken together, this study indicates that NMDA regulates GnRH gene expression in GT1-1 cells through the NO-Jun signal transduction pathway.