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 (PK A and PKC) pathways have been implicated in the regulation of GnRH syn thesis and secretion in the hypothalamus. We have investigated the rol es of these pathways in the regulation of GnRH transcription, mRNA lev els, propeptide processing, and secretion in GT1-7 cells, a mouse hypo thalamic 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-tetr adecanoylphorbol-13-acetate (TPA; 100 nm), also induced c-fos at 30 mi n, 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 i nhibitor, NPC 15437 1-(1-oxotridecyl)2-piperidinyl]methyl)hexanamide], prevented c-fos induction, but did not antagonize GnRH repression. In stead, the inhibitor itself reduced GnRH mRNA levels by 56% at 16 h (w ith 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 decr eased PKC activity, indicating a role for PKC in the maintenance of th e 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 TP A 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. Enhanc ement of GnRH secretion by TPA was robust and rapid (2.5 min), while t he response to forskolin was relatively delayed (2 h). Over a 24-h per iod, unstimulated cells released primarily unprocessed prohormone, whe reas forskolin and TPA stimulated the secretion of processed products. These data indicate that PKC and PKA may influence propeptide process ing and/or the route of GnRH secretion. These data demonstrate that th e PKA and PKC pathways regulate GnRH at the multiple levels of transcr iption, pro-GnRH processing, and GnRH secretion.