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We previously demonstrated that all-trans-retinoic acid (all-trans-RA) regulates gonadotropin-releasing hormone (GnRH) release and gene expression in rat hypothalamic fragments and GT1-1 neuronal cells. Promoter analysis of rat GnRH gene revealed that the enhancing effect of all-trans-RA on GnRH transcription is mediated by cis-elements localized within −1640/−1438 of the rat GnRH promoter. In the present study, we attempted to localize functional retinoic acid response elements (RAREs) within the all-trans-RA-responsive region of the rat GnRH gene. Sequence analysis showed that there exist three putative repeats of AGGTCA-related sequences (−1637/−1617, −1579/−1562, and −1494/−1470) within this promoter sequence. Among them, only the −1494/−1470 sequence could compete the specific binding of GT1-1 nuclear extracts to the consensus RARE (direct repeat of AGGTCA with a 5-bp spacer, DR-5) and vice versa in electrophoretic mobility shift assays. In addition, like consensus RARE, the −1494/−1470 sequence could confer all-trans-RA responsiveness when inserted into the upstream region of SV40 promoter. Treatment of GT1-1 cells with all-trans- or 9-cis-RA increased the specific bindings of GT1-1 nuclear extracts to the consensus RARE and to the −1494/−1470 sequence while not affecting the specific binding to the cAMP response element (CRE). Both retinoids induced RARβ gene expression in GT1-1 cells. The −1494/−1470 sequence (5′-TCTTA G GA C TCTGTGT G AC C TAAGA) is similar to the direct repeat of T G AC C T (complementary sequence of A G GT C A) with a spacer of 5 bp (i.e. DR-5 in the reverse orientation). A mutation of the second core recognition motif of the −1494/−1470 sequence to a more divergent one from consensus RARE (from TGACCT to TTACAT) abolished the responsiveness to all-trans-RA, whereas a mutation of first core recognition motif to a more TGACCT-like sequence (from AGGACT to TGAACT) increased the responsiveness to all-trans-RA. These results indicate that the −1494/−1470 sequence is indeed a weak but functional RARE of the modified DR-5 type. Taken together, these data indicate that all-trans-RA enhances GnRH transcription via functional RARE present in the distal region of the GnRH promoter.

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.

In the present study, we report evidence that activation of RARgamma promotes cell proliferation in immortalized hippocampal progenitor cell line HiB5. We found that treatment of HiB5 cells with all-trans- (all-trans-RA) or 9-cis-retinoic acid (9-cis-RA) significantly increased the number of dead floating cells as well as viable cells in serum-free defined medium (N2). Flow cytometric analysis of DNA contents revealed that the proportion of apoptotic cells over the whole cell population was not affected by both retinoids. Instead, the proportion of S phase cells was significantly increased by retinoids. Under this condition, bcl-2 mRNA levels were significantly increased over time by retinoid treatment, whereas bax mRNA levels were not affected. This suggests that retinoids increase viable cells by enhancing proliferation rather than by suppressing apoptosis. In an attempt to dissect the molecular mechanism underlying retinoid-induced HiB5 cell proliferation, we examined the expression patterns of retinoid receptors following retinoid treatment. Retinoids induced RARgamma mRNA, which paralleled the increase in the transactivation of strong retinoic acid response element (RARE) reporter construct. Accordingly, treatment of HiB5 cells with RARgamma-selective agonist (CD666) increased HiB5 cell number in a dose-dependent manner, which was blocked by co-treatment with RARgamma-selective antagonist (CD2665). Taken together, these data clearly indicate that activation of RARgamma increases proliferation of immortalized hippocampal progenitor cells.