THE UNLIGANDED GLUCOCORTICOID RECEPTOR AS A TRANSCRIPTIONAL REGULATOR IN MAMMARY EPITHELIAL CELLS

Abstract

This work presents the first evidence of a ligand-independent role for the glucocorticoid receptor (GR) as a positive regulator of gene expression in mammary cells. We have demonstrated that unliganded GR interacts directly with the promoter of the tumour suppressor gene BRCA1, and upregulates its expression. The presence of the stress hormone hydrocortisone (HC) abolished this interaction and resulted in repression of BRCA1. Since low levels of BRCA1 have been implicated in the development of sporadic breast cancer, this may represent a novel mechanism through which prolonged stress signaling increases breast cancer risk. We determined that the interaction between unliganded GR and BRCA1 is mediated through the beta subunit of the Ets transcription factor GABP at the RIBS promoter element. GR and GABPβ were shown to interact in both co-immunoprecipitation and mammalian two-hybrid assays, and this interaction involved the N-terminal to central regions of both proteins. To further characterize the role of unliganded GR in breast cells, we used shRNA to generate mouse mammary cell lines with depleted endogenous GR expression. Loss of GR resulted in an impaired capacity of cells to differentiate into acini, but this effect was rescued by the addition of glucocorticoids, implicating both the liganded and unliganded forms of GR as key regulators of differentiation. We performed expression microarray to identify targets of unliganded GR using the GR-depleted cell lines. This analysis revealed 260 genes negatively regulated and 343 genes positively regulated by unliganded GR. Many of the positively regulated genes were involved in pro-apoptotic networks, and appeared to oppose the activity of liganded GR targets. Validation and further analysis of five candidates of positive regulation by unliganded GR indicated that two of these, Hsd11b1 and Ch25h, were regulated by unliganded GR in a manner similar to Brca1. The Hsd11b1 enzyme regulates intracellular glucocorticoid levels by interconverting cortisol and its inactive metabolite, cortisone. Further investigation of Hsd11b1 expression and regulation indicated that Hsd11b1 activity appears to be unidirectional in breast cells, specifically inactivating cortisol. Overall, this work suggests that gene regulation by unliganded GR represents a mechanism for protecting the breast from tumourigenesis during stress.