EVIDENCE FOR TRANSCRIPTIONAL INHIBITION OF GNRH GENE-EXPRESSION BY PHORBOL ESTER AT A PROXIMAL PROMOTER REGION

We previously showed that activation of protein kinase C (PKC) with the phorbol ester, 12-O-tetradecanoulphorbol 13-acetate (TPA) in GT1-7 hypothalamic cells decreases GnRH mRNA levels in a dose and time dependent fashion. In the present studies, we examined the mechanism of this effect. Analysis of the half-life of GnRH mRNA levels after transcriptional arrest with actinomycin-D (5 mu g/ml) estimated the half-life of GnRH mRNA to be 22 h. TPA treatment did not alter the GnRH mRNA half-life directly, suggesting that the effects of TPA occur predominantly at the level of gene transcription. Exposure of cells transiently transfected with various deletion constructs of the rat (r)GnRH promoter to TPA resulted in a decrease of 60% in luciferase reporter activity. This repression was maintained in constructs deleted to position -126 and was lost with further deletion to position -73. In conclusion, these experiments suggest that phorbol esters repress GnRH expression at the level of transcription through DNA sequences in the proximal rGnRH promoter.