Connexin40 controls endothelial activation by dampening NFκB activation

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, 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.

Endothelial connexins in vascular function

“…Moreover, Cx40 has been shown to interact with IκBα. Their binding dampens NFκB nuclear translocation and, in consequence, endothelial activation (6). Many more players of the endothelial Cx interactome remain to be identified in the years to come.…”

“…In general, Cx37 and Cx40 are abundantly expressed in ECs of large arteries, whereas Cx43 and Cx45 are found in their SMCs (2,3). Detailed en face immunofluorescence studies revealed that Cx37 and Cx40 are highly expressed in ECs of straight parts of arteries that are exposed to high laminar shear stress and that these Cxs are downregulated in ECs at arterial branch points or bifurcations subjected to a disturbed blood flow (6,7,8). In contrast, the expression of Cx43 is induced in ECs exposed to disturbed flow (3,7), as illustrated in Fig.…”

Biological Functions of Connexin43 Beyond Intercellular Communication

“…Moreover, the sequestration of the transcription factor specificity protein (Sp)-1 in the cytoplasm of bone marrow stromal cells after expression of Cx43 and Cx45, was demonstrated to impact C-X-C motif chemokine (CXCL)-12 synthesis [30]. Along these lines, hijack of the nuclear factor kappa B (NF-κB) transcription factor by Cx40 has been shown to inhibit its nuclear translocation and initiation of proinflammatory signaling, thus ascribing to Cx40 a vital role upon endothelial homeostasis [31]. In addition, interaction of Cx32 with the scaffolding protein Discs Large homolog 1 (Dlgh1) at the plasma membrane was suggested to block cell cycle progression in hepatocytes [32].…”

Connexins and Pannexins in Vascular Function and Disease

“…In contrast to Cx43, Cx40 expression has been reported to protect against atherosclerosis in mice by synchronizing endothelial anti-inflammatory signaling thus inhibiting leukocyte recruitment to the atherosclerotic lesion [ 42 ]. Interestingly, Cx40 expression is induced in arterial ECs by high laminar shear stress, as normally observed in straight parts of arteries that are known to be protected from atherosclerosis [ 43 ]. IκBα, a member of a protein complex inhibiting the activation of the transcription factor NFκB, was recently identified as a binding partner of Cx40-CT.…”

“…IκBα, a member of a protein complex inhibiting the activation of the transcription factor NFκB, was recently identified as a binding partner of Cx40-CT. The Cx40 interactome may be relevant for the control of NFκB activation in arterial ECs and the initiation of atherogenesis [ 43 ].…”