Context: Control of aromatase expression in uterine leiomyoma has significant clinical implications, as aromatase inhibitors reduce tumor growth and associated irregular uterine bleeding. The mechanisms that regulate aromatase expression in leiomyoma are unknown.

Objectives: We previously demonstrated that the cAMP-responsive proximal promoters I.3 and II regulate aromatase expression in vivo in uterine leiomyoma tissue. Here, we investigated the cellular and molecular mechanisms responsible for promoter I.3/II usage.

Results: In smooth muscle cells isolated from leiomyoma (LSMCs), dibutyryl cyclic AMP (Bt₂cAMP) significantly induced aromatase mRNA and enzyme activity. Reporter constructs of promoter I.3/II deletion and site-directed mutants with selective disruption of cis-regulatory elements in the -517/-16 bp region revealed that 5 out of 7 elements, including 3 CCAAT/enhancer binding protein (C/EBP) binding sites and 2 cAMP response elements, were essential for cAMP-induced promoter activity.

Electrophoretic mobility shift assays demonstrated that nuclear extracts from LSMCs contain complexes assembled on 4 of the 5 cis-elements, with C/EBP binding sites, including a novel -245/-231 bp sequence, clearly associating with C/EBP. Chromatin immunoprecipitation (ChIP) assays revealed that C/EBP binds specifically to the promoter I.3/II region in intact cells. Bt₂cAMP significantly induced nuclear C/EBP protein levels in LSMCs in a time-dependent manner. Conversely, knockdown of C/EBP dramatically suppressed cAMP-induced aromatase mRNA and enzyme activity.

Conclusion: C/EBP, which binds to multiple cis-regulatory elements in promoter I.3/II, is a key factor in the transcriptional complex controlling aromatase expression in uterine leiomyoma cells. Definition of this mechanism further may assist in designing inhibitors of aromatase specific for leiomyoma tissue.