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Second messenger regulation of mouse gonadotropin-releasing hormone gene expression in immortalized mouse
1Dr. Arthur M. Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY 10029.
Abstract:
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 microM), ionomycin (1 microM) 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 microM) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Activation of cellular pathways by forskolin, ionomycin, and PMA increases gonadotropin-releasing hormone (GnRH) secretion. Prolonged stimulation decreases GnRH mRNA and transcription, suggesting reduced synthesis. This reveals a feedback mechanism in GnRH regulation.
Area of Science:
- Neuroendocrinology
- Molecular Biology
- Cell Signaling
Background:
- Gonadotropin-releasing hormone (GnRH) regulates reproduction.
- Cellular signaling pathways (cAMP, Ca2+, PKC) influence GnRH secretion.
- Understanding GnRH gene regulation is crucial for reproductive health.
Purpose of the Study:
- To investigate the effects of activating cAMP, Ca2+, and PKC pathways on GnRH secretion and gene expression.
- To elucidate the mechanisms underlying GnRH regulation in GT1-3 cells.
Main Methods:
- Utilized a transgenic mouse GnRH neuronal cell line (GT1-3).
- Stimulated cells with forskolin (cAMP), ionomycin (Ca2+), and PMA (PKC).
- Measured GnRH secretion, peptide content, mRNA, and primary transcript levels.
Main Results:
- Short-term stimulation increased GnRH secretion in a dose-dependent manner.
- Prolonged stimulation (12-24h) decreased GnRH mRNA and primary transcript levels.
- GnRH secretion was attenuated over time, correlating with reduced mRNA.
- GnRH antagonist suggested autocrine negative feedback on secretion, but not mRNA inhibition.
Conclusions:
- Forskolin, ionomycin, and PMA activate GnRH secretion via distinct pathways.
- Prolonged secretagogue treatment suppresses GnRH gene transcription, leading to decreased mRNA and synthesis.
- This study reveals a transcriptional negative feedback mechanism regulating GnRH expression.