The glucocorticoid dexamethasone rapidly up regulates cAMP dependent protein kinase and MAP Kinase activity in human and cystic fibrosis lung epithelia

University of Cambridge (2004) J Physiol 555P, C145

Communications: The glucocorticoid dexamethasone rapidly up regulates cAMP dependent protein kinase and MAP Kinase activity in human and cystic fibrosis lung epithelia

J. Devaney, D. Hynes, N. Coen and B. J. Harvey

Charitable Infirmary Trust Molecular Medicine Laboratories, Royal College of Surgeons in Ireland, Beaumont Hospital, P.O.BOX 9063, Dublin 9, Republic of Ireland

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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.



Where applicable, experiments conform with Society ethical requirements.

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