A‐kinase anchoring proteins define cAMP compartments in TGF‐β1/cigarette smoke induced lung EMT

Abstract

AimsEpithelial‐to‐mesenchymal transition (EMT) plays a critical role in chronic obstructive pulmonary disease (COPD), a disorder induced by air pollution and cigarette smoke (CS). EMT is characterized by loss of epithelial and acquisition of mesenchymal marker. The phosphodiesterase (PDE)4 inhibitor rolipram inhibits TGF‐β1 mediated EMT in A549 cells. Thus, cAMP might be worthwhile to target to attenuate EMT‐associated lung diseases. A central feature of cAMP signaling is the compartmentalization via A‐kinase anchoring proteins (AKAPs). Functional studies on the role of AKAPs and their therapeutic value to target the process of EMT in the lung are still lacking.MethodsTGF‐β1 and cigarette smoke were used to induce EMT in BEAS‐2B and in primary epithelial cells. Expression (gene, protein) of E‐cadherin and collagen 1α1 were analyzed. Expression of Ezrin, AKAP95 and Yotiao was examined by qPCR (gene), western blotting and immunofluorescence (protein). β2‐Agonist fenoterol (0.01–10μM), PDE4 inhibitor rolipram, PDE3 inhibitor cilostamide, adenylyl cyclase activator forskolin (each 10 μM), dibutyryl‐cAMP (db‐cAMP, 10–100 μM) and 30μM st‐Ht31 were used 30 min before TGF‐β1. The TGF‐β1 sensitive AKAPs Ezrin, AKAP95 and Yotiao were silenced using siRNA. Migration was analyzed using both a wound healing assay and xCELLigence transwell migration. CS induced release of TGF‐β1 was measured by ELISA. CS induced reduction in E‐cadherin was blocked by an TGF‐b1 neutralizing antibody. Phosphorylation of SMAD2 and SMAD3 served as control.Results and conclusionsTGF‐β1 changed cell morphology, downregulated E‐cadherin and upregulated collagen 1α1 in BEAS‐2B cells. TGF‐β1 decreased Ezrin but increased AKAP95 and Yotiao gene expression. St‐Ht31 alone led to a strong decrease of E‐cadherin (mRNA, protein), but further augmented TGF‐β1‐induced E‐cadherin (protein) decrease and prevented collagen 1α1 (protein) upregulation. Co‐silencing of Ezrin, AKAP95, Yotiao diminished collagen upregulation, wound closure and transwell migration induced by TGF‐β1. CS exposure increased phospho‐SMAD2, TGF‐β1 release, and augmented cell migration, linking CS to TGF‐β1. Elevation of cAMP in AKAP silenced cells pointed to AKAP defined cAMP compartments.AKAP family members Ezrin, AKAP95 and Yotiao regulate EMT by TGF‐β1, a process potentially involving CS induced TGF‐β1 release. AKAPs seem to define the ability of the β2‐agonist fenoterol, and PDE inhibitors rolipram and cilostamide to modulate the EMT process. Our data point to novel strategies in COPD treatment involving members of the AKAP superfamily.Support or Funding InformationUbbo Emmius Programme, GRIP, FSE, DFG and the Gertraud und Heinz‐Rose Stiftung.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.