A-kinase anchoring proteins diminish lung EMT induced by TGF-ß1/cigarette smoke

Abstract

Epithelial-to-mesenchymal transition (EMT) - characterized typical epithelial-mesenchymal phenotypes - is critical in COPD, a disease induced by air pollution and cigarette smoke (CS). The cAMP elevator phosphodiesterase (PDE)4 inhibitor rolipram inhibits TGF-β1 mediated EMT in A549 cells. Despite that A-kinase anchoring proteins (AKAPs) are central to cAMP signaling, functional studies on the role of AKAPs in the process of lung EMT are still lacking. TGF-β1 (3ng/ml, 24 hours) and cigarette smoke induce EMT in BEAS-2B cells and primary human airway epithelial cells. Next to studies (gene, protein, immunofluorescence) on typical EMT markers, functional impact of AKAP expression was analyzed in cells silenced for the TGF-β1 sensitive AKAPs Ezrin, AKAP95 and Yotiao using readouts for cell migration (wound healing assay, xCELLigence, IncuCyte); partly in the presence of cAMP elevators to define cAMP compartments. TGF-β1 release induced by CS was measured by ELISA, TGF-b1 signaling was blocked by a neutralizing antibody. The casein kinase inhibitor PF-670462 served as control. TGF-β1 induced morphological changes, downregulated E-cadherin and upregulated collagen 1α1 in BEAS-2B cells. Silencing of Ezrin, AKAP95, Yotiao primarily diminished collagen upregulation by TGF-β1, and reduced cell migration (measured as wound closure, transwell migration, IncuCyte); processes induced by TGF-β1 and mimicked by CS. Elevation of cAMP by fenoterol, rolipram, cilostamide, forskolin and db-cAMP in AKAP silenced cells differentially reduced the EMT process pointing to AKAP defined cAMP compartments. Our data point to novel strategies in COPD treatment involving members of the AKAP superfamily.