A STEROIDOGENIC FACTOR-1-BINDING SITE AND CYCLIC ADENOSINE 3,5-MONOPHOSPHATE RESPONSE ELEMENT-LIKE ELEMENTS ARE REQUIRED FOR THE ACTIVITY OF THE RAT AROMATASE PROMOTER IN RAT LEYDIG TUMOR-CELL LINES

Although transcription initiation within CYP19 (cytochrome P450 aromat ase) occurs immediately 5' to the initiator methionine (proximal promo ter) in two rat Leydig tumor cell lines (R2C and H540) that express hi gh aromatase activity and in rat ovary, the patterns of aromatase expr ession in the two cell types are distinctive. To define mechanisms con trolling different patterns of expression of the rat aromatase proxima l promoter, we performed transient transfection and gel mobility shift assays. Transfection experiments using different sized promoter fragm ents fused to a reporter gene were used to identify regions that are f unctionally important for transcriptional regulation in steroidogenic cell lines [R2C, H540, and Y1 (mouse adrenocortical cells that express low aromatase activity)]. These experiments indicate that the cAMP re sponse element (CRE) al; -231 and the steroidogenic factor-1 (SF1) mot if are both required for expression of the reporter gene in each stero idogenic cell line and that the CRE at -169 is similarly required in R 2C cells. Gel mobility shift assays confirm binding of nuclear protein s from the steroidogenic cell lines to the SF1 motif and to CRE (-231) . Leydig tumor cells also contain nuclear proteins that bind to the CR E (-169), but nuclear extracts from R2C cells produce a uniquely shift ed band compared with H540 cells. These results suggest that differenc es in proteins that bind to distinct elements within the rat aromatase promoter may be responsible for different patterns and levels of arom atase expression in these steroidogenic cell lines.