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Open Access Highly Accessed Research article

Expression microarray identifies the unliganded glucocorticoid receptor as a regulator of gene expression in mammary epithelial cells

Heather D Ritter12 and Christopher R Mueller123*

Author Affiliations

1 Queen’s Cancer Research Institute, Queen’s University, Kingston, Ontario, Canada K7L 3N6

2 Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario, Canada K7L 3N6

3 Department of Pathology and Molecular Medicine, Queen’s University, Kingston, Ontario, Canada K7L 3N6

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BMC Cancer 2014, 14:275  doi:10.1186/1471-2407-14-275

Published: 22 April 2014

Abstract

Background

While glucocorticoids and the liganded glucocorticoid receptor (GR) have a well-established role in the maintenance of differentiation and suppression of apoptosis in breast tissue, the involvement of unliganded GR in cellular processes is less clear. Our previous studies implicated unliganded GR as a positive regulator of the BRCA1 tumour suppressor gene in the absence of glucocorticoid hormone, which suggested it could play a similar role in the regulation of other genes.

Methods

An shRNA vector directed against GR was used to create mouse mammary cell lines with depleted endogenous levels of this receptor in order to further characterize the role of GR in breast cells. An expression microarray screen for targets of unliganded GR was performed using our GR-depleted cell lines maintained in the absence of glucocorticoids. Candidate genes positively regulated by unliganded GR were identified, classified by Gene Ontology and Ingenuity Pathway Analysis, and validated using quantitative real-time reverse transcriptase PCR. Chromatin immunoprecipitation and dual luciferase expression assays were conducted to further investigate the mechanism through which unliganded GR regulates these genes.

Results

Expression microarray analysis revealed 260 targets negatively regulated and 343 targets positively regulated by unliganded GR. A number of the positively regulated targets were involved in pro-apoptotic networks, possibly opposing the activity of liganded GR targets. Validation and further analysis of five candidates from the microarray indicated that two of these, Hsd11b1 and Ch25h, were regulated by unliganded GR in a manner similar to Brca1 during glucocorticoid treatment. Furthermore, GR was shown to interact directly with and upregulate the Ch25h promoter in the absence, but not the presence, of hydrocortisone (HC), confirming our previously described model of gene regulation by unliganded GR.

Conclusion

This work presents the first identification of targets of unliganded GR. We propose that the balance between targets of liganded and unliganded GR signaling is responsible for controlling differentiation and apoptosis, respectively, and suggest that gene regulation by unliganded GR may represent a mechanism for reducing the risk of breast tumourigenesis by the elimination of abnormal cells.

Keywords:
Glucocorticoid receptor; Unliganded; Hydrocortisone; Expression microarray; Breast cancer; BRCA1