REGULATION OF THE RAT AROMATASE GENE IN OVARIAN GRANULOSA-CELLS AND R2C LEYDIG-CELLS

Aromatase cytochrome P450 is regulated in granulosa cells of ovarian follicles by the synergistic action of FSH and steroids. The effect of FSH can be mimicked by forskolin suggesting that transcription of the aromatase gene is regulated by cAMP. In contrast, aromatase is constitutively expressed in the rat R2C Leydig cells. To characterize the functional regions of the promoter in these two cell types, a fragment containing 534 bp of the aromatase promoter sequence and various deletion mutants were ligated to a reporter gene, chloramphenicol acetyl transferase and used in transient transfection assays. The results suggest that the region between -176 and -31 bp is essential both for cAMP regulation in granulosa cells and constitutive expression in R2C cells. Nuclear proteins from granulosa and R2C cells specifically bind the -176 fragment in an electrophoretic mobility shift assay. Binding was completed by an oligonucleotide (-90/-66 bp) containing a hexameric sequence, AGGTCA, which has been found in the promoters of other steroidogenic genes. These results suggest that cAMP regulation and constitutive expression of the rat aromatase promoter requires sequences between -176 and -31 bp, particularly the sequence AGGTCA at -82/-77 and nuclear proteins binding to these sequences.