Preprint Article Version 1 This version is not peer-reviewed

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

Version 1 : Received: 29 October 2019 / Approved: 31 October 2019 / Online: 31 October 2019 (02:15:56 CET)

How to cite: Zuo, H.; Heijink, I.H.; van der Veen, C.H.; Hesse, L.; Faber, K.N.; Poppinga, W.J.; Maarsingh, H.; Nikolaev, V.O.; Schmidt, M. A-Kinase Anchoring Proteins Diminish TGF-β1 / Cigarette Smoke-Induced Epithelial-to-Mesenchymal Transition. Preprints 2019, 2019100358 (doi: 10.20944/preprints201910.0358.v1). Zuo, H.; Heijink, I.H.; van der Veen, C.H.; Hesse, L.; Faber, K.N.; Poppinga, W.J.; Maarsingh, H.; Nikolaev, V.O.; Schmidt, M. A-Kinase Anchoring Proteins Diminish TGF-β1 / Cigarette Smoke-Induced Epithelial-to-Mesenchymal Transition. Preprints 2019, 2019100358 (doi: 10.20944/preprints201910.0358.v1).

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.

Subject Areas

epithelial-to-mesenchymal transition; TGF-β1; cAMP; A-kinase anchoring protein; Ezrin; AKAP95; Yotiao; cigarette smoke; COPD

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