PHORBOL ESTER INHIBITION OF RAT GONADOTROPIN-RELEASING-HORMONE PROMOTER ACTIVITY - ROLE OF FOS AND JUN IN THE REPRESSION OF TRANSCRIPTION

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