Endocrine Abstracts

Glucocorticoids (Gcs) act via the intracellular glucocorticoid receptor (GR) to regulate cellular homeostasis. GR is a ligand-activated transcription factor which mediates effects within the nucleus or cytoplasm to regulate genomic or non-genomic events. Localisation of the GR to a specific cellular compartment is therefore an important determinant of the cellular response to Gcs.

Live cell imaging of GR trafficking in cells transfected with fluorophore-tagged GR showed that the GR is exported from the nucleus during cell division. Studies of endogenous GR confirmed that mitotic cells have markedly greater cytoplasmic distribution of GR, even in the presence of the synthetic glucocorticoid Dexamethasone (Dex).

However, detailed immunofluorescence studies suggested a small population of hyperphosphorylated (ser211) GR apparently localised to the condensed chromatin of mitotic cells. Hyperphosphorylated GR was not evident in GR deficient U20S cells in the absence of transfected GR. GR specific knockdown (siRNA) also abolished detection of phospho-GR in HeLa cells, confirming specificity of immunoreactivity. Further analysis showed the phospho-GR to be spindle-associated during anaphase, with the same pattern of distribution as that reported for mid-zone proteins such as survivin.

As cell cycle phase regulates GR compartmentalisation, its effect on Gc signalling was also examined. The ability of GR to drive activity of a simple reporter gene (TAT3-luc) was impaired in growth arrested cells. Mitotic cells showed a marked increase in ligand-independent GR phosphorylation (ser211), and a right shift in the time course of glucocorticoid-mediated activation of MAPK.

Cell cycle phase therefore regulates ligand-independent GR localisation and activity to impact on both the rapid signalling events in the cytoplasm and also the longer acting gene regulatory events within the nucleus. We therefore correlate the sensitivity of cells to Gcs with their mitotic index which in turn may have implications for Gc action in rapidly dividing cells.