9-cis-Retinoic acid represses transcription of the gonadotropin-releasing hormone (GnRH) gene via proximal promoter region that is distinct from all-trans-retinoic acid response element.

We previously reported an enhancing effect of all-trans-retinoic acid (all-trans-RA) on gonadotropin-releasing hormone (GnRH) gene transcription via distal promoter elements of the rat GnRH gene. The present study examined the effects of another biologically active retinoid, 9-cis-retinoic acid (9-cis-RA), on GnRH transcription in GT1-1 cells. Similar to the action of all-trans-RA, 9-cis-RA significantly induced the luciferase activity of the strong retinoic acid response element (RARE) reporter construct, 3X beta RARE-Luc, by about 60-fold, indicating that GT1-1 cells are also responsive to 9-cis-RA. In contrast to the stimulatory effect of all-trans-RA on GnRH transcription, 9-cis-RA inhibited the GnRH promoter activity in a dose- and time-dependent manner. Significant inhibition by 9-cis-RA required at least an 18 h treatment and a further decrease of GnRH promoter-driven luciferase activity was observed up to 48 h of incubation. Accordingly, GnRH mRNA levels were decreased by 9-cis-RA treatment in a similar dose- and time-related manner, indicating that mouse GnRH expression is also negatively regulated by 9-cis-RA. Transient transfections of serial deletion constructs of the rat GnRH promoter revealed that the --230/--110 sequence of the rat GnRH promoter is responsible for 9-cis-RA-induced inhibition of GnRH transcription. Within this region, however, no consensus retinoid X receptor response element was found. To gain insights into the role of retinoid X receptors (RXRs) in GnRH expression, we examined the effects of RXR overexpression on GnRH transcriptional activity. Interestingly, co-transfection of RXR overexpression vectors significantly increased the GnRH promoter-driven luciferase activity, while treatment with 9-cis-RA not only nullified the enhancing effect of RXR overexpression but also decreased the basal GnRH promoter-driven luciferase activity by 50% compared to vehicle-treated controls. This implies that RXRs in the absence of its cognate ligand 9-cis-RA contribute to the maintenance of basal GnRH gene transcription. Northern blot analysis revealed that 9-cis-RA, but not all-trans-RA, down-regulated RXR beta expression in GT1-1 cells, suggesting that one possible mechanism of 9-cis-RA-induced repression involves down-regulation of RXR expression. In conclusion, the present study clearly demonstrates that 9-cis-RA is a negative regulator of GnRH gene expression in immortalized GnRH neurons.