Abstract
BackgroundWhile 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.MethodsAn 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.ResultsExpression 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.ConclusionThis 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.
Highlights
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
It is clear that glucocorticoids and liganded GR are essential for the growth and differentiation of the mammary gland, as well as the suppression of apoptosis; the role of unliganded GR in these processes has not been investigated
Since the downregulated genes represented those which are positively regulated by unliganded GR, potentially through a mechanism similar to that reported for BRCA1 [13], we examined the most significant networks comprised of this gene set via pathway analyses, and determined that several of these genes were involved in pro-apoptotic networks
Summary
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. The glucocorticoid hormone cortisol is involved in the maintenance of breast functional differentiation during the latter stages of pregnancy, where it induces the formation of the rough endoplasmic reticulum [1], and regulates the release of milk proteins [2]. GR with a point mutation in the second zinc finger of the DNA-binding domain (exon 4; A458T) cannot bind a canonical Glucocorticoid Response Element (GRE), but retains its ability to transrepress gene expression through protein-protein interactions [9] Virgin mice expressing this DNA-binding GR mutant exhibit impaired ductal development while lactating mice exhibit normally differentiated mammary glands capable of milk production, emphasizing that transcriptional regulation by protein-protein interactions, rather than DNA-binding, forms the basis of glucocorticoid action during this process [1]. It is clear that glucocorticoids and liganded GR are essential for the growth and differentiation of the mammary gland, as well as the suppression of apoptosis; the role of unliganded GR in these processes has not been investigated
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