Glucocorticoids (GCs) are steroid hormones used to treat inflammatory diseases such as rheumatoid arthritis, but their clinical efficacy is hampered by development of side effects such as impaired wound healing. GCs bind the glucocorticoid receptor (GR) to mediate cellular effects. The inactive GR is held in multi-protein complex in the cytoplasm and upon ligand binding undergoes a conformational change, interacts with cytoplasmic enzymes to mediate non-genomic effects (within minutes) and then translocates to the nucleus to regulate gene expression (over hours). GCs are known to inhibit migration of multiple cell types, although the primary mechanisms underlying this observation remain unclear.
Using a combined strategy of live cell imaging and computational analysis we show that the motion of lung epithelial (A549) cells can be modelled by an alpha stable distribution. We observe that GCs rapidly reduce the average speed of cell migration without changing the nature of the walk statistics. Interestingly, both GR agonists and antagonists ((fluticasone propionate and dexamethasone) and RU486) had a similar effect, suggesting a non-transcriptional mechanism. Using real-time imaging we could not show a rapid GC effect on dynamics of the actin cytoskeleton, but rather demonstrate that the earliest event post-GC treatment is the stabilisation of the microtubule network. Consistent with this, we observed increased acetylation of α-tubulin, a modification required for microtubule stabilisation. This suggests that GR regulates migration following either activation of an acetyltransferase or inhibition of a deacetylase enzyme that targets α-tubulin.
siRNA knockdown of the α-tubulin specific acetyltransferase (α-TAT1) did not affect GC-dependent reduction in migration. In contrast, overexpression of the α-tubulin deacetylase (HDAC6) reversed the effect of dexamethasone, suggesting that treatment with GCs rapidly stabilises the microtubule network to reduce cellular migration by inhibiting HDAC6. This shows for the first time a non-genomic, GC-dependent mechanism involving HDACs which impacts cell function. This is a clear example of how real-time imaging and computational modelling can reveal insight into biological processes.
02 Nov 2015 - 04 Nov 2015