Transcriptional activation of gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: involvement of multiple signal transduction pathways.
Previous studies have shown that GnRH activates transcriptional activity of its own receptor (GnRHR) gene in part through the cAMP signal transduction pathway. In the present study we explored the possible involvement of multiple signal transduction pathways in GnRH regulation of GnRHR gene transcription; these studies relied upon a luciferase reporter gene vector (GnRHR-pXP2) containing a 1226-bp promoter fragment (-1164 to +62, relative to the major transcription start site) of the mouse GnRHR gene in GGH3 cells (GH3 cells stably expressing rat GnRHR). Activation of protein kinase C (PKC) by phorbol myristic acid significantly stimulated GnRHR-luciferase reporter gene (GnRHR-Luc) activity, but did not potentiate the stimulation of GnRHR-Luc activity by the GnRH agonist, buserelin (GnRH-A). Inhibition of PKC by PKC inhibitor (GF 109203X) or depletion of PKC blocked phorbol myristic acid- or GnRH-A-stimulated GnRHR-Luc activity, but did not affect (Bu)2cAMP-stimulated GnRHR-Luc activity. In addition, GnRH-A-stimulated GnRHR-Luc activity was inhibited by preventing external Ca2+ influx with the external Ca2+ chelator EGTA or the Ca2+ ion channel antagonist, D600. Surprisingly, overexpression of the mitogen-activated protein kinase (MAPK) kinase kinase (Raf-1) inhibited GnRHR-Luc activity and partially blocked GnRH-A-stimulated GnRHR-Luc activity. In contrast, inhibition of MAPK activity by MAPK kinase inhibitor (PD 98059) or by overexpression of kinase-deficient MAPKs activated basal and GnRH-A-stimulated GnRHR-Luc activity. These results suggested that PKC- and Ca2+-dependent signal transduction pathways participate in the GnRH activation of GnRHR promoter activity, and that the MAPK cascade is involved in the negative regulation of basal and GnRH-stimulated GnRHR transcriptional activity conferred by the 1226-bp promoter fragment.
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Oregon Regional Primate Research Center, Beaverton 97006, USA.
Conn
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D000818:Animals; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D017930:Genes, Reporter; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D051381:Rats; D011966:Receptors, LHRH / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects*; D014162:Transfection
D004791:Enzyme Inhibitors; D005419:Flavonoids; D007211:Indoles; D008301:Maleimides; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium