CFTR regulation in human airway epithelial cells requires integrity of the actin cytoskeleton and compartmentalized cAMP and PKA activity

Monterisi, S., Favia, M., Guerra, L., Cardone, R.A., Marzulli, D., Reshkin, S.J., Casavola, V. and Zaccolo, M. (2012) CFTR regulation in human airway epithelial cells requires integrity of the actin cytoskeleton and compartmentalized cAMP and PKA activity. Journal of Cell Science, 125(5), pp. 1106-1117. (doi:10.1242/jcs.089086)

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Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) mutation ΔF508CFTR still causes regulatory defects when rescued to the apical membrane, suggesting that the intracellular milieu might affect its ability to respond to cAMP regulation. We recently reported that overexpression of the Na+/H+ exchanger regulatory factor NHERF1 in the cystic fibrosis (CF) airway cell line CFBE41o-rescues the functional expression of ΔF508CFTR by promoting F-actin organization and formation of the NHERF1–ezrin–actin complex. Here, using real-time FRET reporters of both PKA activity and cAMP levels, we find that lack of an organized subcortical cytoskeleton in CFBE41o-cells causes both defective accumulation of cAMP in the subcortical compartment and excessive cytosolic accumulation of cAMP. This results in reduced subcortical levels and increased cytosolic levels of PKA activity. NHERF1 overexpression in CFBE41o-cells restores chloride secretion, subcortical cAMP compartmentalization and local PKA activity, indicating that regulation of ΔF508CFTR function requires not only stable expression of the mutant CFTR at the cell surface but also depends on both generation of local cAMP signals of adequate amplitude and activation of PKA in proximity of its target. Moreover, we found that the knockdown of wild-type CFTR in the non-CF 16HBE14o-cells results in both altered cytoskeletal organization and loss of cAMP compartmentalization, whereas stable overexpression of wt CFTR in CF cells restores cytoskeleton organization and re-establishes the compartmentalization of cAMP at the plasma membrane. This suggests that the presence of CFTR on the plasma membrane influences the cytoskeletal organizational state and, consequently, cAMP distribution. Our data show that a sufficiently high concentration of cAMP in the subcortical compartment is required to achieve PKA-mediated regulation of CFTR activity.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Zaccolo, Professor Manuela
Authors: Monterisi, S., Favia, M., Guerra, L., Cardone, R.A., Marzulli, D., Reshkin, S.J., Casavola, V., and Zaccolo, M.
College/School:College of Medical Veterinary and Life Sciences > Institute of Neuroscience and Psychology
Journal Name:Journal of Cell Science
ISSN:0021-9533
Published Online:02 February 2012

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
432501Transatlantic networks of excellence in cardiovascular diseaseMiles HouslayFoundation Leducq (LEDUCQ-VIL)06 CVD 02Institute of Neuroscience and Psychology
465341Role of PDE2 in the control of cardiac myocyte hypertrophy.Manuela ZaccoloBritish Heart Foundation (BHF)PG/07/091/23698Institute of Neuroscience and Psychology
479533Collaborative Research: Spatial and temporal aspects of cAMP/PKA signaling underlying information processing in neuronsManuela ZaccoloNational Institute of Health (NIH-GA)1R01AA 018060-0Institute of Neuroscience and Psychology