Many glucocorticoid (Gc) actions are of rapid onset, and therefore require acute regulation of intracellular signalling cascades. We have previously identified rapid Gc-dependent activation of the caveolae specific integral membrane protein caveolin, and the cytosolic kinase PKB in the lung epithelial cells, A549.
Immunofluorescence studies demonstrate the appearance of activated glucocorticoid receptor (P-ser211-GR) localised to focal points around the cell perimeter rapidly after Gc treatment. Subcellular fractionation studies further demonstrate co-localisation of GR to caveolin-containing membrane fractions. We propose that caveolin may serve to support an interaction between GR and PKB and that this may regulate downstream events, including the activation of PKB.
To further investigate the role of caveolae in mediating rapid Gc signalling events, A549 cells were stably transfected with an shRNA designed to target caveolin-1 and three cell lines with variant caveolin expression selected for subsequent analysis (100%, 44% and 18% wildtype A549 caveolin expression by immunoblot).
Although there is no effect on GR transactivation, downregulation of caveolin-1 significantly reduces the Gc-dependent phosphorylation of the GR and PKB in a dose dependent manner. Since the expression of the PKB isoforms is not altered following manipulation of caveolin-1, this suggests a role for caveolin in functional coupling of GR and PKB signals. Functional analysis of A549 cells with reduced caveolin-1 expression demonstrate altered rates of cellular proliferation as measured by MTT and 3H-thymidine incorporation assays. In addition, the anti-proliferative effect of Dexamethasone treatment is significantly impaired following loss of caveolin expression.
We therefore demonstrate a glucocorticoid-dependent anti-proliferative signalling pathway which requires caveolin, caveolae and PKB. Caveolin is frequently downregulated in cancer, which may explain the well documented Gc resistance of rapidly dividing cells. The physiological consequences of such pathways require further investigation.