Diverse mechanisms of control of aromatase gene expression

The synthesis of estrogens from androgens is catalyzed by a microsomal cytochrome P450 termed aromatase (P450arom). The expression of this enzyme is highly regulated in both a developmental and cell-type specific fashion. We have chosen to examine the molecular basis of aromatase gene regulation by studying two models of aromatase expression: the Sebright bantam chicken and the R2C rat Leydig tumor cell line. In the first model, affected (Sebright) chickens express aromatase in many extragonadal tissues, while normal Leghorn chickens express aromatase only in the ovary and hypothalamus. Our studies have demonstrated that in normal chickens the site of transcription initiation is located approx. 147 nucleotides upstream of the initiator methionine. While Sebright animals also express aromatase mRNA initiated at an analogous initiation site in the ovary, a distinctive species of aromatase mRNA is also detected and is present in ovary and extragonadal tissues. This mRNA contains an identical coding sequence, but contains an alternatively spliced 5' noncoding exon that is derived from a distinctive promoter. The second model, the R2C Leydig tumor cell line, provides ample contrast. This cell line expresses high basal levels of aromatase (150-200 pmol/h/mg protein) that is suppressed with administration of 8 bromo cAMP or forskolin but the activity is not altered by glucocorticoids or epidermal growth factor treatment. Despite this distinctive pattern of regulation, at least three species of aromatase mRNA are detected in Northern blots, each of which is also detected in rat ovary. Primer extension and S1 nuclease assays indicate that both granulosa cells and R2C cells utilize a promoter that is located approx. 97 nucleotides upstream of the initiator methionine. These studies suggest that the "ovarian" promoter is evolutionarily conserved in both rats and chickens. These results further imply that the genetic mechanisms controlling the diversity of aromatase expression among tissues and among different species are likely to fall into two groups: those that employ distinctive promoters and alternative splicing and those that effect different patterns of regulation through a common ("ovarian") promoter.