Coupling of cell cycle progression to GR cellular distribution
LC Matthews1, DG Spiller2, CA Rivers3, MR Norman3, MRH White2 & DW Ray1
Glucocorticoids (Gcs) are important regulators of cellular homeostasis, with their effects mediated by the intracellular glucocorticoid receptor (GR). Once activated, the GR may initiate rapid effects within the cytoplasm, but also translocate to the nucleus to affect gene transcription. The subcellular localisation of the GR is therefore an important determinant in the cellular response to Gcs.
To analyse subcellular trafficking of the GR, HeLa cells were transfected with fluorophore-tagged GR and live cells imaged for 15 hours. Analyses of these images demonstrate that the GR is exported from the nucleus just prior to the onset of cell division. As evidence suggests that cell cycle position determines sensitivity of cells to the actions of Gcs, we speculate that reduced GR activity reported during G2/M may be a consequence of its nuclear exclusion.
To further correlate the localisation of the GR with its ability to activate target genes during G2/M, cells were growth arrested using Nocodazole (100 nM, confirmed by FACS analysis), assayed for GR activity (TAT3-luc reporter) and endogenous GR labeled (immunofluorescence).
Following treatment with Dexamethasone (100 nM), the ability of endogenous or fluorophore-tagged GR to increase reporter gene activity was impaired in G2 arrested cells, when compared to asynchronous cell populations. Immunofluorescence demonstrates that in G2 arrested cells, the endogenous GR is confined entirely to the cytoplasm, even in the presence of 100 nM Dexamethasone.
This indicates that the reduction in GR activity may be due to its nuclear exclusion, and since reporter gene assays conducted in cells growth arrested using Colcemid (which blocks mitosis) demonstrate no affect on the ability of the GR to activate target genes, further suggests specificity for the G2 checkpoint.
We therefore correlate the sensitivity of cells to Gcs with the cellular localisation of the GR, which may have implications for Gc treatment of rapidly dividing cells.