Transcriptional Regulation of the Human GnRH II Gene Is Mediated by a Putative cAMP Response Element
Autor: | Orly Laskar-Levy, Nurit Ben-Aroya, Yitzhak Koch, Alon Chen |
---|---|
Rok vydání: | 2001 |
Předmět: |
endocrine system
Transcription Genetic Molecular Sequence Data Population Response element Gonadotropin-Releasing Hormone Endocrinology Genes Reporter Cyclic AMP Tumor Cells Cultured Transcriptional regulation Humans Protein Isoforms RNA Messenger Cyclic AMP Response Element-Binding Protein Luciferases Promoter Regions Genetic education Southern blot Neurons education.field_of_study Base Sequence biology Promoter Transfection Molecular biology Up-Regulation Bucladesine Gene Expression Regulation Mutagenesis Cell culture biology.protein CREB1 hormones hormone substitutes and hormone antagonists Plasmids |
Zdroj: | Endocrinology. 142:3483-3492 |
ISSN: | 1945-7170 0013-7227 |
Popis: | 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. |
Databáze: | OpenAIRE |
Externí odkaz: |