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-I 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 ( approximately 6-fold) by (Bu)(2)cAMP. The concentration of GnRH-II that was released into the medium of TE-671 cells treated with the cAMP analog was significantly higher than that of the untreated cells. TE-671 cells that were stimulated by (Bu)(2)cAMP demonstrated morphological changes and strong immunoreactive GnRH-II staining in part of the cell population. After screening of the GnRH-II promoter sequence, we identified a putative cAMP response element consensus site. The GnRH-I and GnRH-II promoters were isolated by PCR using human genomic DNA and cloned into the luciferase reporter plasmid. By measuring the basal activity of the promoters that were transfected to TE-671 cells, we found a much stronger basal activity of the GnRH-II promoter compared with that of GnRH-I. Treatment of transfected TE-671 cells with (Bu)(2)cAMP resulted in a strong activation of the GnRH-II promoter compared with a modest activation of the GnRH-I promoter. To determine the functionality of this putative cAMP response element site, we mutated this site. TE-671 cells that were transfected with cAMP response element mutant constructs demonstrated a diminished basal activity of the GnRH-II promoter. Treatment of the transfected cells with the cAMP analog 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 activity as well as for induction of the GnRH-II promoter by cAMP.
Chen
A
A
Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel.
Laskar-Levy
O
O
Ben-Aroya
N
N
Koch
Y
Y
D001483:Base Sequence / Q000235:genetics; D003994:Bucladesine / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000502:physiology*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009474:Neurons / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D017362:Cyclic AMP Response Element-Binding Protein; D020033:Protein Isoforms; D012333:RNA, Messenger; D007987:Gonadotropin-Releasing Hormone; D003994:Bucladesine; D000242:Cyclic AMP; D008156:Luciferases