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

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