A-Kinase Anchoring Proteins Diminish TGF-β1 / Cigarette Smoke-Induced Epithelial-to-Mesenchymal Transition

Epithelial-to-mesenchymal transition (EMT) plays a role in chronic obstructive29 pulmonary diseases (COPD). Cyclic adenosine monophosphate (cAMP) can inhibit transforming30 growth factor-β1 (TGF-β1) mediated EMT. Although compartmentalization via A-kinase anchoring31 proteins (AKAPs) is central to cAMP signaling, functional studies on their therapeutic value in the32 lung EMT process are lacking. Bronchial epithelial (BEAS-2B, primary HAE cells) were exposed to33 TGF-β1. Epithelial (E-cadherin, ZO-1) and mesenchymal markers collagen Ӏ (mRNA, protein) were34 analyzed. St-Ht31 disrupted AKAP-PKA interactions. TGF-β1 release was measured by ELISA. TGF35 β1-sensitive AKAPs Ezrin, AKAP95 and Yotiao were silenced using siRNA. Cell migration was36 analyzed by wound healing assay, xCELLigence, Incucyte. Prior to TGF-β1, dibutyryl-cAMP37 (dbcAMP), fenoterol, rolipram, cilostamide, forskolin were used to elevate intracellular cAMP. TGF38 β1 induced morphological changes, decreased E-cadherin but increased collagen Ӏ and cell migration,39 a process reversed by PF-670462. TGF-β1 altered (mRNA, protein) expression of Ezrin, AKAP95 and40 Yotiao. St-Ht31 decreased E-cadherin (mRNA, protein), but counteracted TGF-β1-induced collagen Ӏ41 upregulation. Cigarette smoke (CS) increased TGF-β1 release, activated TGF signaling, augmented42 cell migration and reduced E-cadherin expression, a process blocked by TGF-β1 neutralizing antibody. Silencing of Ezrin, AKAP95 and Yotiao diminished TGF-β1-induced collagen Ӏ expression,44 as well as TGF-β1-induced cell migration. Fenoterol, rolipram, and cilostamide, in AKAP silenced45 cells pointed to distinct cAMP compartments. We conclude that Ezrin, AKAP95 and Yotiao promote46 TGF-β1-mediated EMT, linked to a TGF-β1 release by CS. AKAP members define the ability of47 fenoterol, rolipram and cilostamide to modulate the EMT process, and are potential relevant targets48 in the treatment of COPD.