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Expression of the Transcriptional Repressor ATF3 in Gonadotrophs Is Regulated by Egr-1, CREB, and ATF2 after Gonadotropin-Releasing Hormone Receptor Stimulation

Department of Medical Biochemistry and Molecular Biology (S.I.M.,V.D., G.T.), University of Saarland Medical Center, D-66421 Homburg, Germany; Department of Cell and Developmental Biology (E.N.), Graduate School of Biostudies, Kyoto University, Kyoto 606-8507, Japan; and Department of Biochemical Genetics (S.K.), Medical Research Institute, Tokyo Medical and Dental University, Tokyo 101-0062, Japan

Address all correspondence and requests for reprints to: Gerald Thiel, Department of Medical Biochemistry and Molecular Biology, Building 44, University of Saarland Medical Center, D-66421 Homburg, Germany. E-mail: gerald.thiel{at}uniklinik-saarland.de.

Stimulation of GnRH receptors enhances expression of activating transcription factor (ATF) 3 in a pituitary gonadotroph cell line. The signaling pathway requires elevated cytosolic Ca²⁺ levels and activation of ERK and c-Jun N-terminal protein kinase. The signaling cascade was blocked by overexpression of either MAPK phosphatase (MKP)-1 or MAPK phosphatase-5 that dephosphorylate nuclear ERK and c-Jun N-terminal protein kinase. In addition, ATF3 biosynthesis was impaired after lentiviral-mediated expression of a constitutively active mutant of calcineurin A. Thus, MKP-1, MKP-5, and calcineurin may function as shut-off devices for GnRH receptor signaling. Expression of dominant-negative mutants of early growth response protein (Egr)-1, cAMP response element binding protein (CREB), and ATF2 blocked the biosynthesis of ATF3, indicating that these transcription factors connect the intracellular signaling cascade elicited by activation of GnRH receptors with transcription of the ATF3 gene. This view was corroborated by chromatin immunoprecipitation experiments revealing that Egr-1 and the phosphorylated forms of CREB and ATF2 bound to the 5'-upstream region of the ATF3 gene in buserelin-stimulated gonadotrophs. Together the data indicate that the ATF3 gene is a bona fide target gene of Egr-1, CREB, and ATF2 in gonadotrophs. Moreover, we show that in gonadotrophs ATF3 bound to its own promoter under physiological conditions. The analysis of a lentiviral-transmitted ATF3 promoter/luciferase reporter gene, embedded into the chromatin of the cells, revealed that ATF3 blocked the activity of its own promoter. We additionally identified the chromogranin B gene as bona fide target gene of ATF3 in gonadotrophs.