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

Abstract

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

Special Issue on “New Advances in Cyclic AMP Signalling”—An Editorial Overview

“…These findings point to a central role for cyclic AMP nanodomain complexes in the development of COPD. In the current issue, this work has been expanded upon to implicate AKAPs, including ezrin, Yotiao, and AKAP95, in the mediation of epithelial-to-mesenchymal transition (EMT) induced by TGF-β1 and cigarette smoke in human bronchial epithelial cells and, therefore, plays a role in the development of COPD [ 5 ]. The authors show that disruption of PKA anchoring decreases E-cadherin but counteracts TGF-β1-induced collagen1 expression [ 5 ].…”

“…In the current issue, this work has been expanded upon to implicate AKAPs, including ezrin, Yotiao, and AKAP95, in the mediation of epithelial-to-mesenchymal transition (EMT) induced by TGF-β1 and cigarette smoke in human bronchial epithelial cells and, therefore, plays a role in the development of COPD [ 5 ]. The authors show that disruption of PKA anchoring decreases E-cadherin but counteracts TGF-β1-induced collagen1 expression [ 5 ]. TGF-β1 alters the expression of ezrin, Yotiao and AKAP95 and knock-out of these AKAPs impacts TGF-β1 inhibits collagen expression and migration of bronchial epithelial cells [ 5 ].…”

“…The authors show that disruption of PKA anchoring decreases E-cadherin but counteracts TGF-β1-induced collagen1 expression [ 5 ]. TGF-β1 alters the expression of ezrin, Yotiao and AKAP95 and knock-out of these AKAPs impacts TGF-β1 inhibits collagen expression and migration of bronchial epithelial cells [ 5 ]. They conclude, finally, that AKAPs might underlie the actions of cyclic AMP-elevating drugs, including, fenoterol, rolipram, and cilostamide, to inhibit EMT, and, therefore, might represent new drug targets for the treatment of COPD and, perhaps, the development of lung cancer.…”

“…Cyclic AMP is now recognized as a universal regulator of cellular function in a wide variety of organisms, including protozoa, plants, and animals. For example, in this Special Edition, González-Velasco et al, use proteomic and transcriptomic profiling in order to identify early developmentally regulated proteins in Dictyostelium discoideum [ 3 ], whereas, in humans, cyclic AMP has been implicated here in the control of the differentiation, proliferation, and apoptosis of stem cells [ 4 ], as well as epithelial-to-mesenchymal transition in bronchial epithelial cells, in response to cigarette smoke [ 5 ]. In addition, cyclic nucleotide metabolism has been targeted for therapy leading to the development of a series of “block busting” drugs, including beta-blockers (e.g., propranolol), anti-asthma (e.g., salbutamol) and anti-erectile dysfunction (Sildenafil) drugs.…”

“…Biological processes mediated by cyclic AMP include metabolism, gene regulation, and immune function and its deregulation is associated with many pathologies, including metabolic, psychiatric, cardiovascular, pulmonary, and inflammatory disorders, as well as certain cancers. For example, key studies in this here concentrate on regulatory roles for cyclic AMP in cardiac fibrosis [ 6 ], hepatocellular carcinoma [ 7 ], restenosis [ 8 ], and chronic obstructive pulmonary disorder (COPD) [ 5 ]. In addition, Negreiros-Lima et al described a new role for cyclic AMP in regulating important functions of monocytes/macrophages during the resolution of inflammation.…”