Dataset Information

CCAAT/enhancer binding protein beta regulates aromatase expression via multiple and novel cis-regulatory sequences in uterine leiomyoma.

ABSTRACT:

Context

Control of aromatase expression in uterine leiomyoma has significant clinical implications because 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 cAMP 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 five out of seven elements, including three CCAAT/enhancer binding protein (C/EBP) binding sites and two cAMP response elements, were essential for cAMP-induced promoter activity. EMSAs demonstrated that nuclear extracts from LSMCs contain complexes assembled on four of the five cis-elements, with C/EBP binding sites, including a novel -245/-231 bp sequence, clearly associating with C/EBPbeta. Chromatin immunoprecipitation assays revealed that C/EBPbeta binds specifically to the promoter I.3/II region in intact cells. Dibutyryl cAMP significantly induced nuclear C/EBPbeta protein levels in LSMCs in a time-dependent manner. Conversely, knockdown of C/EBPbeta dramatically suppressed cAMP-induced aromatase mRNA and enzyme activity.

Conclusions

C/EBPbeta, 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.

SUBMITTER: Ishikawa H

PROVIDER: S-EPMC2266947 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:High doses of genistein show an inhibitory effect on uterine leiomyoma (UtLM) cell proliferation. Using microarray analysis and Ingenuity Pathways AnalysisTM, we identified genes (up- or down-regulated, M-bM-^IM-%1.5 fold, P M-bM-^IM-$ 0.001), functions and signaling pathways that were altered following treatment with an inhibitory concentration of genistein (50 M-CM-,g/ml) in UtLM cells. Downregulation of TGF-M-CM-" signaling pathway genes, activin A, activin B, Smad3, TGF-M-CM-"2 and genes related to cell cycle regulation, with the exception of the upregulation of the CDK inhibitor P15, were identified and validated by real-time reverse transcriptase-polymerase chain reaction studies. Western blot analysis further demonstrated decreased protein expression of activin A and Smad3 in genistein-treated UtLM cells. Moreover, we found that activin A stimulated the growth of UtLM cells, and the inhibitory effect of genistein was partially abrogated in the presence of activin A. Overexpression of activin A and Smad3 were found in tissue samples of leiomyoma compared to matched myometrium, supporting the contribution of activin A and Smad3 in promoting the growth of UtLM cells. Taken together, these results suggest that downregulation of activin A and Smad3, both members of the TGF-M-CM-" pathway, may offer a mechanistic explanation for the inhibitory effect of a high-dose of genistein on UtLM cells, and might be potential therapeutic targets for treatment of clinical cases of uterine leiomyomas. Cells were treated with either genistein (50 M-NM-<g/ml) (4M-bM-^@M-^Y, 5, 7-Trihydroxyisoflavone; Sigma Chemical Company, St. Louis, MO, USA) or the vehicle control [0.3% dimethylsulfoxide (DMSO), Sigma] for 24 h. the samples from the flasks that received the same treatment were pooled, then concentrated to > 9 M-CM-,g/M-CM-,l by Microcon 30 columns (Amicon:Millipore, Bedford, MA, USA). Three replicates for the treatment, and one pooled control, were submitted for microarray analysis. Gene expression analysis was conducted using Agilent Whole Human Genome arrays (Agilent Technologies, Palo Alto, CA, USA).