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 d istal promoter elements of the rat GnRH gene. The present study examined th e effects of another biologically active retinoid, 9-cis-retinoic acid (9-c is-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 str ong 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 tran scription, 9-cis-RA inhibited the GnRH promoter activity in a dose- and tim e-dependent manner. Significant inhibition by 9-cis-RA required at least an 18 h treatment and a further decrease of GnRH promoter-driven luciferase a ctivity was observed up to 48 h of incubation. Accordingly, GnRH mRNA level s 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 r at GnRH promoter revealed that the -230/-110 sequence of the rat GnRH promo ter is: responsible for 9-cis-RA-induced inhibition of GnRH transcription. Within this region, however, no consensus retinoid X receptor response elem ent 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 overexpres sion vectors significantly increased the GnRH promoter-driven luciferase ac tivity, while treatment with 9-cis-RA not only nullified the enhancing effe ct of RXR overexpression but also decreased the basal GnRH promoter-driven luciferase activity by 50% compared to vehicle-treated controls. This impli es that RXRs in the absence of its cognate ligand 9-cis-RA contribute to th e maintenance of basal GnRH gene transcription. Northern blot analysis reve aled that 9-cis-RA, but not all-trans-RA, down-regulated RXR beta expressio n in GT1-1 cells, suggesting that one possible mechanism of 9-cis-RA-induce d repression involves down-regulation of RXR expression. In conclusion, the present study clearly demonstrates that 9-cis-RA is a negative regulator o f GnRH gene expression in immortalized GnRH neurons. (C) 2001 Elsevier Scie nce B.V. All rights reserved.