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 alpha- and beta- 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- beta ( C/ EBP beta) in repressing the inhibin alpha- subunit gene. C/ EBP beta knockout mice fail to appropriately down- regulate inhibin alpha- subunit mRNA levels after treatment with human chorionic gonadotropin, indicating that C/ EBP beta may function to repress inhibin gene expression. The expression and regulation of C/ EBP beta were examined in rodent ovary, and these studies show that C/ EBP beta 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/ EBP beta is capable of repressing both basal and forskolin- stimulated inhibin gene promoter activities. An upstream binding site for C/ EBP beta in the inhibin alpha- subunit promoter was identified by electrophoretic mobility shift assays, which, when mutated, results in elevated inhibin promoter activity. However, C/ EBP beta also represses shorter promoter constructs lacking this site, and this component of repression is dependent on the more proximal promoter cAMP response element ( CRE). Electrophoretic mobility shift assays show that C/ EBP beta effectively competes with CRE- binding protein for binding to this atypical CRE. Thus, there are two distinct mechanisms by which C/ EBP beta represses inhibin alpha- subunit gene expression in ovarian granulosa cells.