Second messenger regulation of mouse gonadotropin-releasing hormone gene expression in immortalized mouse hypothalamic GT1-3 cells

Using a transgenic mouse derived GnRH expressing neuronal cell line, GT1-3, we studied the effects of activation of cAMP, Ca²⁺ and protein kinase C pathways by forskolin, ionomycin and the phorbol ester phorbol 12-myristate 13-acetate (PMA), respectively, upon gonadotropin-releasing hormone (GnRH) secretion, cellular peptide content, mRNA and RNA primary transcript levels. Forskolin, ionomycin and phorbol ester all caused an increase in GnRH secretion in GT1-3 cells in a time and dose-dependent manner during a short-term (1 h) static incubation. Prolonged treatment with forskolin (10 μM), ionomycin (1 μM) and PMA (10 nM) for 12 or 24 h resulted in significant decreases in GnRH mRNA levels. Time-course studies showed that the increases in GnRH secretion stimulated by forskolin, ionomycin and PMA were gradually attenuated over time in parallel with the decreases in mRNA expression. In contrast, there were only small and variable changes in the GnRH cellular content. Studies using a GnRH antagonist (100 μM) suggested that the released GnRH has a negative feedback effect on its own secretion. However, co-incubation with the GnRH antagonist did not alter the inhibitory effects on GnRH mRNA levels by the secretagogues. Further studies on the transcriptional effects of forskolin, ionomycin and PMA on GnRH gene expression in GT1-3 cells revealed that all three secretagogues suppressed GnRH RNA primary transcript levels, with forskolin having a slower time course of action. Thus, the inhibition of cytoplasmic GnRH mRNA, and presumably its synthesis, after 12-24 h of secretagogue treatment may be due at least in part to a suppression of GnRH gene transcription. While activation of all three types of second messenger systems lead to a decrease in GnRH gene expression suggesting some common link in the regulatory pathway, the temporal and quantitative differences between them suggest that each second messenger pathway may be functioning differently to achieve the same result.