High glucose-induced NF-κB activation occurs via tyrosine phosphorylation of IκBα in human glomerular endothelial cells: involvement of Syk tyrosine kinase

Abstract

Activation of nuclear factor-κB (NF-κB) occurs by dissociation from IκB after serine or tyrosine phosphorylation of IκBα, but the way of NF-κB activation by high glucose has not been defined. High glucose is known to activate NF-κB via protein kinase C and reactive oxygen species (ROS). In this study, we investigated how high glucose activates NF-κB for CC chemokine ligand 2 production in cultured human glomerular endothelial cells. High glucose increased nuclear translocation of p65 and also increased NF-κB DNA binding activity. High glucose-induced NF-κB activation occurred without degradation of IκBα. In agreement with this, there was no increase in serine phosphorylation of IκBα, while tyrosine phosphorylation of IκBα was increased by high glucose. High glucose increased the generation of ROS, whereas both α-lipoic acid and N-acetylcysteine scavenged the ROS and decreased high glucose-induced tyrosine phosphorylation of IκBα, nuclear translocation of p65, and NF-κB DNA binding activity. Protein kinase C pseudosubstrate inhibited high glucose-induced ROS production, tyrosine phosphorylation of IκBα, and nuclear translocation of p65. Both BAY 61-3606, a specific inhibitor of Syk protein-tyrosine kinase, and small interfering RNA directed against Syk inhibited high glucose-induced tyrosine phosphorylation of IκBα as well as p65 nuclear translocation. High glucose increased tyrosine phosphorylation of Syk, while it was inhibited by α-lipoic acid and protein kinase C pseudosubstrate. In summary, high glucose-induced NF-κB activation occurred not by serine phosphorylation of IκBα. Our data suggest that ROS-mediated tyrosine phosphorylation of IκBα is the mechanism for high glucose-induced NF-κB activation, and Syk may play a role in tyrosine phosphorylation of IκBα.