Repression of the inhibin α-subunit gene by the transcription factor CCAAT/enhancer-binding protein-β

Inhibin is a dimeric peptide hormone produced in ovarian granulosa cells that suppresses FSH synthesis and secretion in the pituitary. Expression of inhibin α- and β-subunit genes in the rodent ovary is positively regulated by FSH and negatively regulated after the preovulatory LH surge. We have investigated the role of the transcription factor CCAAT/ enhancer-binding protein-β (C/EBPβ) in repressing the inhibin α-subunit gene. C/EBPβ knockout mice fail to appropriately down-regelate inhibin α-subunit mRNA levels after treatment with human chorionic gonadotropin, indicating that C/EBPβ may function to repress inhibin gene expression. The expression and regulation of C/EBPβ were examined in rodent ovary, and these studies show that C/EBPβ is expressed in ovary and granulosa cells and is induced in response to human chorionic gonadotropin. Transient cotransfections with an inhibin promoter-luciferase reporter in a mouse granulosa cell line, GRMO2 cells, show that C/EBPβ is capable of repressing both basal and forskolin-stimulated inhibin gene promoter activities. An upstream binding site for C/EBPβ in the inhibin α-subunit promoter was identified by electrophoretic mobility shift assays, which, when mutated, results in elevated inhibin promoter activity. However, C/EBPβ also represses shorter promoter constructs lacking this site, and this component of repression is dependent on the more proximal promoter cAMP response element (CBE). Electrophoretic mobility shift assays show that C/EBPβ effectively competes with CRE-binding protein for binding to this atypical CBE. Thus, there are two distinct mechanisms by which C/EBPβ represses inhibin α-subunit gene expression in ovarian granulosa cells.