Transcriptional regulation of the human GnRH II gene is mediated by a putative cAMP response element

Human neuronal medulloblastoma cells (TE-671) were recently demonstrated to express the two forms of GnRH (GnRH-1 and GnRH-II). We have used this cell line as a model system to demonstrate regulation of the human GnRH-II gene by cAMP. RT-PCR and Southern hybridization demonstrated that GnRH-II mRNA is strongly up-regulated (similar to6-fold) by (BU)(2)cAMP. The concentrati on of GnRH-II that was released into the medium of TE-671 cells treated wit h the cAMP analog was significantly higher than that of the untreated cells . TE-671 cells that were stimulated by (Bu)(2)cAMP demonstrated morphologic al changes and strong immunoreactive GnRH-II staining in part of the cell p opulation. After screening of the GnRH-II promoter sequence, we identified a putative cAMP response element consensus site. The GnRH-I and GnRH-II pro moters were isolated by PCR using human genomic DNA and cloned into the luc iferase reporter plasmid. By measuring the basal activity of the promoters that were transfected to TE-671 cells, we found a much stronger basal activ ity of the GnRH-II promoter compared with that of GnRH-I. Treatment of tran sfected TE-671 cells with (Bu)(2)cAMP resulted in a strong activation of th e GnRH-II promoter compared with a modest activation of the GnRH-I promoter . To determine the functionality of this putative cAMP response element sit e, we mutated this site. TE-671 cells that were transfected with cAMP respo nse element mutant constructs demonstrated a diminished basal activity of t he GnRH-II promoter. Treatment of the transfected cells with the cAMP analo g demonstrated a decrease to 0.03% of the activity of the mutated promoter compared with that of the wild type. These results clearly demonstrate the importance of the putative cAMP response element site for the basal activit y as well as for induction of the GnRH-II promoter by cAMP.