Phorbol ester inhibition of rat gonadotropin-releasing hormone promoter activity: role of Fos and Jun in the repression of transcription

• Published in: Molecular endocrinology (Baltimore, Md.) Vol. 10; no. 1; pp. 35 - 44
• Main Authors: Bruder, J M, Spaulding, A J, Wierman, M E
• Format: Journal Article
• Language: English
• Published: United States 01.01.1996
• Subjects: Animals; Base Sequence; Binding Sites; DNA - metabolism; Gene Expression; Genes, fos - genetics; Genes, jun - genetics; Gonadotropin-Releasing Hormone - genetics; Leucine Zippers; Luciferases - genetics; Molecular Sequence Data; Promoter Regions, Genetic; Rats; RNA, Messenger - metabolism; Tetradecanoylphorbol Acetate - pharmacology; Transcription Factor AP-1 - pharmacology; Transcription, Genetic - drug effects; Transfection
• ISSN: 0888-8809
• DOI: 10.1210/me.10.1.35

Treatment of GT1-7 neuronal cells with the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA), inhibits GnRH gene transcription. The present studies investigated the role of AP-1 (Fos and Jun) in this repression. Treatment of cells with TPA increased c-fos mRNA 20-fold with only a 2-fold increase in c-jun mRNA levels. In transient transfection studies, a luciferase expression vector containing fragments of the 5'-flanking DNA of the rat GnRH (rGnRH) promoter was cotransfected with Fos and Jun expression vectors to mimic the effects of TPA. A dose-dependent decrease in reporter activity was noted with increasing amounts of Fos but not with Jun overexpression. Deletion analysis mapped the region that mediates repression by AP-1 to the area between -126 and -73 base pairs (bp) of the rGnRH 5'-flanking region: the same area that mediates TPA-induced repression and contains an imperfect TPA response element sequence at -99. Gel retardation assays, however, showed that a DNA fragment from -111 to -73 of the rGnRH promoter does not directly interact with Fos in GT1-7 extracts. Coexpression of Fos proteins with mutations in the DNA-binding region, the dimerization domain, or carboxy terminus partially blocked inhibition of rGnRH promoter activity. These data support a novel mechanism of AP-1 repression of GnRH transcription that is mediated by Fos interaction with other protein(s) that directly bind to the proximal rGnRH promoter.