Connexin40 controls endothelial activation by dampening NFκB activation

// Jean-Francois Denis 1, * , K.E. Ludwig Scheckenbach 1, * , Anna Pfenniger 1, 2 , Merlijn J. Meens 1 , Rob Krams 3 , Lucile Miquerol 4 , Steven Taffet 5 , Marc Chanson 6 , Mario Delmar 7 and Brenda R. Kwak 1, 2 1 Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland 2 Department of Medical Specializations - Cardiology, University of Geneva, Geneva, Switzerland 3 Department of Bioengineering, Imperial College, London, UK 4 Aix-Marseille University, CNRS UMR 7288, Developmental Biology Institute of Marseille, Marseille, France 5 Department of Microbiology, SUNY Upstate Medical University, Syracuse, NY, USA 6 Departments of Pediatrics and of Cell Physiology and Metabolism, Geneva University Hospitals and University of Geneva, Geneva, Switzerland 7 The Leon H. Charney Division of Cardiology, New York University School of Medicine, New York, NY, USA * These authors have contributed equally to this work Correspondence to: Brenda R. Kwak, email: Brenda.KwakChanson@unige.ch Keywords: atherosclerosis, endothelium, Cx40, shear stress, IκBα Received: November 08, 2016      Accepted: February 27, 2017      Published: March 22, 2017 ABSTRACT Connexins are proteins forming gap junction channels for intercellular communication. Connexin40 (Cx40) is highly expressed by endothelial cells (ECs) of healthy arteries but this expression is lost in ECs overlying atherosclerotic plaques. Low/oscillatory shear stress observed in bends and bifurcations of arteries is atherogenic partly through activation of the pro-inflammatory NFκB pathway in ECs. In this study, we investigated the relation between shear stress, Cx40 and NFκB. Shear stress-modifying casts were placed around carotid arteries of mice expressing eGFP under the Cx40 promoter ( Cx40 +/eGFP ). We found that Cx40 expression is decreased in carotid regions of oscillatory shear stress but conserved in high and low laminar shear stress regions. These results were confirmed in vitro . Using phage display, we retrieved a binding motif for the intracellular regulatory Cx40 C-terminus (Cx40CT), i.e. HS[I, L, V][K, R]. One of the retrieved peptides (HSLRPEWRMPGP) showed a 58.3% homology with amino acids 5-to-16 of IκBα, a member of the protein complex inhibiting NFκB activation. Binding of IκBα (peptide) and Cx40 was confirmed by crosslinking and en face proximity ligation assay on carotid arteries. TNFα-induced nuclear translocation of NFκB in ECs was enhanced after reducing Cx40 with siRNA. Transfection of HeLa cells with either full-length Cx40 or Cx40CT demonstrated that Cx40CT was sufficient for inhibition of TNFα-induced NFκB phosphorylation. Finally, Tie2Cre Tg Cx40 fl/fl Apoe -/- mice showed exaggerated shear stress-induced atherosclerosis and enhanced NFκB nuclear translocation. Our data show a novel functional IκBα-Cx40 interaction that may be relevant for the control of NFκB activation by shear stress in atherogenesis.