Abstract
The glucocorticoid receptor (GR) is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors. In contrast to many other nuclear receptors, GR is thought to be exclusively cytoplasmic in quiescent cells, and only translocate to the nucleus on ligand binding. We now demonstrate significant nuclear GR in the absence of ligand, which requires nuclear localisation signal 1 (NLS1). Live cell imaging reveals dramatic GR import into the nucleus through interphase and rapid exclusion of the GR from the nucleus at the onset of mitosis, which persists into early G1. This suggests that the heterogeneity in GR distribution is reflective of cell cycle phase.The impact of cell cycle–driven GR trafficking on a panel of glucocorticoid actions was profiled. In G2/M-enriched cells there was marked prolongation of glucocorticoid-induced ERK activation. This was accompanied by DNA template-specific, ligand-independent GR transactivation. Using chimeric and domain-deleted receptors we demonstrate that this transactivation effect is mediated by the AF1 transactivation domain. AF-1 harbours multiple phosphorylation sites, which are consensus sequences for kinases including CDKs, whose activity changes during the cell cycle.In G2/M there was clear ligand independent induction of GR phosphorylation on residues 203 and 211, both of which are phosphorylated after ligand activation. Ligand-independent transactivation required induction of phospho-S211GR but not S203GR, thereby directly linking cell cycle driven GR modification with altered GR function. Cell cycle phase therefore regulates GR localisation and post-translational modification which selectively impacts GR activity. This suggests that cell cycle phase is an important determinant in the cellular response to Gc, and that mitotic index contributes to tissue Gc sensitivity.
Highlights
Glucocorticoids (Gc) are essential for life, mediating a diverse array of effects to regulate bone and glucose homeostasis, tissue remodeling and repair, and the immune response [1]
High content analysis of fixed cells labelled with a glucocorticoid receptor (GR) antibody and DNA counterstained with DAPI confirms that enriching for mitotic cells by gating with nocodazole significantly reduces the proportion of cells where GR colocalises with DNA (Fig. 2D), suggesting exclusion of GR from DNA
While treatment of normally cycling or Go/G1 cells with 100 nM dex for 1 hour increases the proportion of cells that have nuclear GR, cells sychronised with nocodazole appear refractory to Gc treatment (Fig. 2D)
Summary
Glucocorticoids (Gc) are essential for life, mediating a diverse array of effects to regulate bone and glucose homeostasis, tissue remodeling and repair, and the immune response [1]. Ligand activated GR is released from this complex and is free to initiate non-genomic effects within the cytoplasm, and translocate to the cell nucleus where it dimerises and binds palindromic Gc-response elements (GREs). Other investigators have shown that GR can move between cytoplasm and nucleus when unliganded and bound to the heat shock protein complex [15]. This aberration has been attributed to low-level ligand activation, but other explanations have not been explored [16,17,18,19]
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