Connexin40 controls endothelial activation by dampening NFκB activation

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 NF kappa B pathway in ECs. In this study, we investigated the relation between shear stress, Cx40 and NF kappa B. 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 I kappa B alpha, a member of the protein complex inhibiting NF kappa B activation. Binding of I kappa B alpha (peptide) and Cx40 was confirmed by crosslinking and en face proximity ligation assay on carotid arteries. TNF alpha-induced nuclear translocation of NF kappa B 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 TNF alpha-induced NF kappa B phosphorylation. Finally, Tie2Cre(Tg)Cx40(fl/fl)Apoe(-/-) mice showed exaggerated shear stress-induced atherosclerosis and enhanced NF kappa B nuclear translocation. Our data show a novel functional I kappa B alpha-Cx40 interaction that may be relevant for the control of NF kappa B activation by shear stress in atherogenesis.