Connexin40 controls endothelial activation by dampening NFκB activation
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Jean-Francois Denis1,*, K.E. Ludwig Scheckenbach1,*, Anna Pfenniger1,2, Merlijn J. Meens1, Rob Krams3, Lucile Miquerol4, Steven Taffet5, Marc Chanson6, Mario Delmar7 and Brenda R. Kwak1,2
1Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland
2Department of Medical Specializations - Cardiology, University of Geneva, Geneva, Switzerland
3Department of Bioengineering, Imperial College, London, UK
4Aix-Marseille University, CNRS UMR 7288, Developmental Biology Institute of Marseille, Marseille, France
5Department of Microbiology, SUNY Upstate Medical University, Syracuse, NY, USA
6Departments of Pediatrics and of Cell Physiology and Metabolism, Geneva University Hospitals and University of Geneva, Geneva, Switzerland
7The Leon H. Charney Division of Cardiology, New York University School of Medicine, New York, NY, USA
*These authors have contributed equally to this work
Brenda R. Kwak, email: [email protected]
Keywords: atherosclerosis, endothelium, Cx40, shear stress, IκBα
Received: November 08, 2016 Accepted: February 27, 2017 Published: March 22, 2017
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, Tie2CreTgCx40fl/flApoe-/- 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.
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