Induction of mPer1 expression by GnRH in pituitary gonadotrope cells involves EGR-1.
We reported earlier that gonadotropin-releasing hormone (GnRH) activates period1 (mPer1) gene expression in immortalized gonadotropes through protein kinase C and p42/44 mitogen-activated protein kinase pathways. GnRH stimulation also leads to the upregulation of early growth response protein 1 (EGR-1), a critical transcription factor for GnRH-induced luteinizing hormone beta (LHbeta) synthesis. The parallels between the GnRH-LHbeta and the GnRH-mPer1 pathways led us to explore whether EGR-1 is involved in the regulation of mPer1 expression in gonadotropes. Of particular interest was the presence of an EGR-1 binding site in the proximal promoter of the mPer1 gene. Stimulation of LbetaT2 gonadotrope cells with a GnRH agonist caused the rapid induction of Egr-1 mRNA, which was rapidly followed by mPer1 expression. Chromatin immunoprecipitation revealed that the mPer1 promoter can bind EGR-1, while site-directed mutagenesis experiments confirmed the involvement of Egr-1 sequences in maintaining basal and allowing GnRH-stimulated mPer1 transcription. By means of RNA interference experiments, it could also be demonstrated that silencing of Egr-1 expression resulted in markedly lower mPer1 transcript levels. This silencing effect of the Egr-1 siRNA could be rescued by transfecting the cells with an EGR-1 overexpression vector. In summary, these results all point to a role for the EGR-1 protein in transactivating both the LHbeta as well as the mPer1 gene in pituitary gonadotrope cells.
Resuehr
H E Sikes
HE
Florida State University College of Medicine, Department of Biomedical Sciences, 1115 West Call Street, Tallahassee, FL 32306-4300, USA.
Resuehr
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D
Olcese
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D000818:Animals; D002064:Buserelin / Q000494:pharmacology; D056926:CLOCK Proteins / Q000235:genetics / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D051766:Early Growth Response Protein 1 / Q000235:genetics / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D017930:Genes, Reporter; D052681:Gonadotrophs / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D007987:Gonadotropin-Releasing Hormone / Q000819:agonists / Q000494:pharmacology*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors; D016297:Mutagenesis, Site-Directed; D056950:Period Circadian Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D013997:Time Factors
D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D005419:Flavonoids; D056950:Period Circadian Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D007987:Gonadotropin-Releasing Hormone; D008156:Luciferases; D056926:CLOCK Proteins; D019950:Mitogen-Activated Protein Kinase 1; D002064:Buserelin; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one