CCAAT enhancer binding protein beta (C/EBPβ) regulates aromatase P450 expression VIA new cis-acting element in uterine leiomyoma cells

Abstract

We investigated the regulation of aromatase expression by the PGE2-cAMP signaling pathway in uterine leiomyoma. Uterine leiomyoma is the most common tumor in reproductive age women, and causes recurrent pregnancy loss and excessive uterine bleeding. Sex steroids play important roles in leiomyoma growth. Aromatase is the key enzyme that catalyzes the conversion of C19 steroids to estrogens. Aromatase expression in leiomyoma is clinically significant since an aromatase inhibitor could shrink leiomyoma in a perimenopausal woman. We previously demonstrated that the PGE2/cAMP-responsive proximal promoter region I.3/II regulates aromatase expression in leiomyoma smooth muscle cells (LSMCs). However, the mechanism responsible for activation of this promoter is unknown. Prospective experimental study. We isolated and cultured primary LSMCs and myometrial smooth muscle cells (MSMCs) from adjacent normal-appearing tissue in subjects (n = 30). We used radiolabeled [3H]-release assay and real-time PCR to measure aromatase enzyme activity and mRNA levels. Deletion and site-directed mutants of the promoter I.3/II fused to the luciferase reporter gene were used for analyzing cis-regulatory sequences. We used EMSA and ChIP-PCR to analyze binding of transcription factors to these regions, and checked protein levels of transcription factors by western blot. PGE2, and the cAMP analog dibutyryl (Bt2) cAMP significantly induced aromatase mRNA and enzyme activity in MSMCs and LSMCs. Transient transfections with promoter I.3/II deletion mutants revealed that the −517/−278 bp region conferred induction by Bt2 cAMP. Selective disruption of 7 cis-regulatory elements in the −517/−124 region showed that 2 out of 3 CAATT/enhancer binding protein (C/EBP) sites and both cAMP response elements (CREs) were essential for cAMP induction of aromatase promoter I.3/II. The role of −245/−231 C/EBP binding site and −292/−285 CRE have not been published and thus is novel. Bt2 cAMP treatment of LSMCs significantly increased the occupation of all 4 sites by nuclear extracts, and induced binding of C/EBPβ to both −317/−304 and −245/−231 sequences. Moreover, Bt2 cAMP strikingly increased C/EBPβ levels in LSMCs. Cyclic AMP, downstream of PGE2 or other hormones appears to be the most potent inducer of aromatase expression in uterine leiomyoma cells. This cAMP effect is mediated by induction of C/EBPβ protein that binds to multiple sites in the proximal promoter I.3/II of the aromatase gene.