A non-genomic anti-secretory role for glucocorticoids has been described in human bronchial epithelia. Dexamethasone has been shown to cause a rapid and non-genomic reduction of intracellular calcium concentration in these epithelia, resulting in a reduction of calcium dependent chloride secretion (Urbach et al. 2002).
In this study we demonstrate rapid effects of dexamethasone on cAMP dependent protein kinase (PKA) activity (using a non-radioactive PepTag AssayTM system), MAP Kinase activity (ERK1/2 western blotting) and intracellular calcium concentration (Fura-2 fluorimetry) in cell lines from normal lung epithelia (16HBE14o-) and cystic fibrosis lung epithelia (CFTE-29o-).PKA activity was rapidly up-regulated ( < 5 min) three-fold over basal (n = 3) in response to low concentrations of dexamethasone (1-10 nM) in human bronchial epithelia. The PKA antagonist, RPcAMP (20 ÁM), was shown to inhibit this effect. Pertussis toxin, a Gi-protein inhibitor (10 ug/ml overnight), was demonstrated to inhibit the activation of PKA, while the ERK1/2 inhibitor, PD98059 (50 ÁM), had no effect. In addition, we demonstrate ERK1/2 activity was rapidly up-regulated in response to dexamethasone in human bronchial epithelia. This response was inhibited by RPcAMP, indicating PKA activates MAP Kinase.
Low concentrations of dexamethasone (1-10 nM) caused a rapid and non-genomic reduction of intracellular calcium concentration in cystic fibrosis tracheal epithelia similar to that of normal bronchial epithelia. F340/F380 relative fluorescence ratio was reduced by 0.26 ± 0.05 (mean ± S.E.M., n = 3; Student’s t test P < 0.000001). The steroid-induced reduction in intracellular calcium concentration was inhibited by pre-treatment with RPcAMP. In addition, we demonstrate PKA activity was up-regulated ( < 5 min) three-fold over basal (n = 2) by dexamethasone (1-10 nM) in cystic fibrosis tracheal epithelia and that RPcAMP inhibited this effect.
Our results show that rapid glucocorticoid activation of PKA and MAP Kinase is transduced via a Gi coupled receptor. One of the end targets of this signalling cascade is a reduced calcium mobilization in both normal and cystic fibrosis lung epithelia. Calcium is a potent regulator of secretion and MAP Kinase (ERK1/2) is anti-apoptotic in epithelia. Our results have important implications in the further development of steroid based treatments for inflammatory lung disease e.g. Cystic Fibrosis and Asthma.