REGULATION OF GONADOTROPIN-RELEASING-HORMONE BY PROTEIN KINASE-A AND KINASE-C IN IMMORTALIZED HYPOTHALAMIC NEURONS

As major signal transduction cascades, the protein kinase-A and -C (PKA and PKC) pathways have been implicated in the regulation of GnRH synthesis and secretion in the hypothalamus. We have investigated the roles of these pathways in the regulation of GnRH transcription, mRNA levels, propeptide processing, and secretion in GT1-7 cells, a mouse hypothalamic GnRH neuronal cell line. Forskolin, which activates adenylate cyclase to raise cAMP levels, had no effect on GnRH mRNA levels at 10 muM, but induced c-fos mRNA at 30 min. An activator of PKC, 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nm), also induced c-fos at 30 min, but produced a progressive decline in GnRH mRNA, resulting in a 70% decrease by 16 h. Coadministration of 10 nM TPA and 20 muM of a PKC inhibitor, NPC 15437 [2,6-diamino-N-([1-(1-oxotridecyl)2-piperidinyl]methyl)hexanamide], prevented c-fos induction, but did not antagonize GnRH repression. Instead, the inhibitor itself reduced GnRH mRNA levels by 56% at 16 h (with no effect on c-fos mRNA). Thus, since extended exposure to TPA can down-regulate PKC, suppression of GnRH mRNA by TPA may be due to decreased PKC activity, indicating a role for PKC in the maintenance of the GnRH gene expression (a role that is unlikely to involve c-fos). In transient transfections, the transcriptional activity from 3 kilobases of GnRH 5'-flanking sequence was repressed 2-fold by either 100 nm TPA or 20 muM NPC 15437 at 24 h, demonstrating that suppression of GnRH mRNA is at least, in part, at the level of transcription. In contrast, both TPA (100 nm) and forskolin (10 muM) stimulated secretion. Enhancement of GnRH secretion by TPA was robust and rapid (2.5 min), while the response to forskolin was relatively delayed (2 h). Over a 24-h period, unstimulated cells released primarily unprocessed prohormone, whereas forskolin and TPA stimulated the secretion of processed products. These data indicate that PKC and PKA may influence propeptide processing and/or the route of GnRH secretion. These data demonstrate that the PKA and PKC pathways regulate GnRH at the multiple levels of transcription, pro-GnRH processing, and GnRH secretion.