Molecular Endocrinology, Vol 10, 35-44, Copyright © 1996 by Endocrine Society

ARTICLES

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

JM Bruder, AJ Spaulding and ME Wierman
Department of Medicine, University of Colorado Health Sciences Center, Denver 80220, USA.

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.

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