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A-kinase anchoring proteins contribute to loss of E-cadherin and bronchial epithelial barrier by cigarette smoke

Significance Statement

Our studies report for the first time on the importance of A-kinase anchoring proteins (AKAPs) to support the epithelial barrier function. We show a direct interaction between AKAP9 and E-cadherin, a molecular link which is beneficial for the maintenance of the epithelial barrier and might be of interest for future drug development and targeting.

The epithelial barrier is a protective layer against unwanted particles, present in cigarette smoke, a major risk factor to develop chronic obstructive pulmonary disease. COPD is characterised by inflammation, emphysema and narrowing of the airways due to thickening of the airway smooth muscle layer and increased collagen deposition. Cyclic AMP, a central second messenger throughout the human body, increases barrier properties in endothelial cells. But the role of cAMP in the epithelial barrier is not known. The cAMP effectors, PKA and Epac, may be involved, next to compartmentalization of cAMP signalling by A-kinase anchoring proteins. Epithelial barrier is maintained by the cell-cell contact molecule E-cadherin. Interestingly, AKAPs, particularly AKAP9, are known to associate with E-cadherin. Therefore, we hypothesized that A-kinase anchoring proteins play a critical role in the maintenance of the epithelial barrier function. We studied the effect of cigarette smoke extract (CSE) on the barrier function by electrical cell-substrate impedance sensing and by analyzing E-cadherin. CSE markedly reduced the epithelial barrier function and caused delocalization of E-cadherin from cell-cell contacts. These processes were completely prevented by disruption of AKAP complexes using st-Ht31. The reduction in E-cadherin was specifically present at the cell membrane, whereas total and cytosolic expression was not significantly changed.  Interestingly, CSE reduced AKAP9 expression, while leaving the expression of AKAP5 and AKAP12 (which are not associated with E-cadherin) nearly unaffected. Indeed, silencing of AKAP9 reduces E-cadherin levels at the membrane. Taken together, our data show that CSE-induced loss of E-cadherin and AKAP9 expression at the epithelial membrane and subsequent reduction in epithelial barrier function is mediated by AKAP-PKA interactions. Since loss of AKAP9 reduces the functional barrier, we hypothesize that the observed interaction between AKAP9 and E-cadherin regulates the maintenance of cell-cell contacts. The reduction in E-cadherin and AKAP9 expression may contribute to epithelial dysfunction observed in COPD.

Next to an implication in future COPD treatment options these results indicate that AKAP based multiprotein complexes might be of interest in other obstructive pulmonary diseases. In addition these findings indicate that AKAPs are interesting drug targets and may improve drug delivery to certain areas in the cell.

 

Journal Reference

Oldenburger A, Poppinga WJ, Kos F, de Bruin HG, Rijks WF, Heijink IH, Timens W, Meurs H, Maarsingh H, Schmidt M.

Am J Physiol Cell Physiol. 2014;306(6):C585-97.

University of Groningen, Department of Molecular Pharmacology, Groningen, The Netherlands;

Abstract

 Airway epithelium, which forms the first barrier towards environmental insults, is disturbed by cigarette smoking, a major risk factor for developing chronic obstructive pulmonary disease (COPD). A-kinase anchoring proteins (AKAP) maintain endothelial barrier function and coordinate subcellular localization of protein kinase A (PKA). However, the role of A-kinase anchoring proteins in epithelial barrier function is unknown. We studied the role of A-kinase anchoring proteins in regulating human bronchial epithelial (Hogg JC, Timens W. Annu Rev Pathol 4: 435-459, 2009; HBE) barrier. Cigarette smoke extract (CSE) reducedbarrier function in 16HBE cells and the expression of the adhesion molecule E-cadherin specifically at the cell membrane. In addition, CSE reduced the protein expression of the A-kinase anchoring proteins family member AKAP9 at the cell membrane. The expression of AKAP5 and AKAP12 was unaffected by CSE. AKAP9 interacted and colocalized with E-cadherin at the cell membrane, suggesting that the reduction of both proteins may be related. Interestingly, disruption of AKAP-PKA interactions by st-Ht31 prevented the CSE-induced reduction of E-cadherin and AKAP9 protein expression and subsequent loss of barrier function. Silencing of AKAP9 reduced the functional epithelial barrier and prevented the ability of st-Ht31 to restore membrane localization of E-cadherin. Our data suggest the possibility of a specific role for AKAP9 in the maintenance of the epithelial barrier. E-cadherin, but not AKAP9, protein expression was reduced in lung tissue from COPD patients compared with controls. However, AKAP9 mRNA expression was decreased in primary bronchial epithelial cells from current smokers compared with non/ex-smokers. In conclusion, our results indicate that A-kinase anchoring proteins, most likely AKAP9, maintain the bronchial  epithelial barrier by regulating the E-cadherin expression at the cell membrane.

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