Dataset Information

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

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.

SUBMITTER: Ritter HD

PROVIDER: S-EPMC4021255 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Publications

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

Ritter Heather D HD Mueller Christopher R CR

BMC cancer 20140422

<h4>Background</h4>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.<h4 ...[more]

PMID: 24755251

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Project description:BACKGROUND:Inter-individual variation in normal human mammary epithelial cells in response to oxythioquinox (OTQ) is reported. Gene expression signatures resulting from chemical exposures are generally created from analysis of exposures in rat, mouse or other genetically similar animal models, limiting information about inter-individual variations. This study focused on the effect of inter-individual variation in gene expression signatures. METHODS:Gene expression was studied in primary normal human mammary epithelial cells (NHMECs) derived from four women undergoing reduction mammoplasty [Cooperative Human Tissue Network (National Cancer Institute and National Disease Research Interchange)]. Gene transcription in each cell strain was analyzed using high-density oligonucleotide DNA microarrays (HuGeneFL, Affymetrix) and changes in the expression of selected genes were verified by real-time polymerase chain reaction at extended time points (ABI). DNA microarrays were hybridized to materials prepared from total RNA that was collected after OTQ treatment for 15, 60 and 120 min. RNA was harvested from the vehicle control (DMSO) at 120 min. The gene expression profile included all genes altered by at least a signal log ratio (SLR) of +/- 0.6 and p value < or = 0.05 in three of four cell strains analyzed. RESULTS:RNA species were clustered in various patterns of expression highlighting genes with altered expression in one or more of the cell strains, including metabolic enzymes and transcription factors. Of the clustered RNA species, only 36 were found to be altered at one time point in three or more of the cell strains analyzed (13 up-regulated, 23 down-regulated). Cluster analysis examined the effects of OTQ on the cells with specific p53 polymorphisms. The two strains expressing the major variant of p53 had 83 common genes altered (35 increased, 48 decreased) at one or more time point by at least a 0.6 signal log ratio (SLR). The intermediate variant strains showed 105 common genes altered (80 increased, 25 decreased) in both strains. CONCLUSION:Differential changes in expression of these genes may yield biomarkers that provide insight into inter-individual variation in cancer risk. Further, specific individual patterns of gene expression may help to determine more susceptible populations.

Project description:To elucidate the regulation of glucocorticoid receptor (GR) signaling under pro-asthmatic conditions, cultured human airway smooth muscle (HASM) cells were treated with proinflammatory cytokines or GR ligands alone and in combination, and then examined for induced changes in ligand-dependent and -independent GR activation and downstream signaling events. Ligand stimulation with either cortisone or dexamethsone (DEX) acutely elicited GR translocation to the nucleus and, comparably, ligand-independent stimulation either with the Th2 cytokine, IL-13, or the pleiotropic cytokine combination, IL-1β/TNFα, also acutely evoked GR translocation. The latter response was potentiated by combined exposure of cells to GR ligand and cytokine. Similarly, treatment with either DEX or IL-13 alone induced GR phosphorylation at its serine-211 residue (GR(Ser211)), denoting its activated state, and combined treatment with DEX+IL-13 elicited heightened and sustained GR(Ser211) phosphorylation. Interestingly, the above ligand-independent GR responses to IL-13 alone were not associated with downstream GR binding to its consensus DNA sequence or GR transactivation, whereas both DEX-induced GR:DNA binding and transcriptional activity were significantly heightened in the presence of IL-13, coupled to increased recruitment of the transcriptional co-factor, MED14. The stimulated GR signaling responses to DEX were prevented in IL-13-exposed cells wherein GR(Ser211) phosphorylation was suppressed either by transfection with specific serine phosphorylation-deficient mutant GRs or treatment with inhibitors of the MAPKs, ERK1/2 and JNK. Collectively, these novel data highlight a heretofore-unidentified homeostatic mechanism in HASM cells that involves pro-asthmatic cytokine-driven, MAPK-mediated, non-ligand-dependent GR activation that confers heightened glucocorticoid ligand-stimulated GR signaling. These findings raise the consideration that perturbations in this homeostatic cytokine-driven GR signaling mechanism may be responsible, at least in part, for the insensirtivity to glucocorticoid therapy that is commonly seen in individuals with severe asthma.

Dataset Information

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

Background

Methods

Results

Conclusion

Publications

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

Similar Datasets

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