Abstract 345: Inhibitor Kappa B Kinase Regulates Nitric Oxide Synthase Activity in Vascular Endothelial Cells
The generation of nitric oxide (NO) in endothelial cells is predominantly regulated by endothelial nitric oxide synthase (eNOS) activity. The role of heat shock protein 90 (Hsp-90) in the regulation of eNOS activity has been established. Earlier, we demonstrated an increased interaction of Hsp-90 with inhibitor kappa B kinase β (IKKβ) in aortic endothelial cells (ECs) under conditions of high glucose (HG). These studies implicated the possibility of a site-specific competition between eNOS and IKKβ to bind to Hsp-90. In this study, the binding site of IKKβ on Hsp-90β is demonstrated by generating 5’deletional mutants of Flag tagged Hsp-90 and full length enhanced yellow fluorescent protein tagged IKKβ (EYFP-IKKβ). Forceful co-expression of the Hsp-90 deletion constructs with EYFP-IKKβ plasmid followed by immunoprecipitation/immunoblotting revealed the binding site of IKKβ to be between 307-711aa on Hsp-90, the middle domain (440-600 aa) where eNOS has been shown to bind to Hsp-90. Co-immunoprecipitation experiments performed in lysates of human aortic endothelial cells incubated with 25mM high glucose exhibited enhanced IKK beta interaction with both Hsp-90 α and β isoforms. Independently blocking Hsp-90α or β expression by siRNA transfection significantly blocked VEGF-induced NO production implicating the possible mechanism that IKK beta regulates NO production by competing with eNOS for both the isoforms of Hsp-90. These results confirm the previously unknown participation of IKKβ in the downregulation of eNOS activity under the influence of HG and provide a novel therapeutic strategy of using IKKβ as an effective target to improve NO generation in diabetic arteries.
Author Disclosures: M. Natarajan: None. M. Krishnan: None. R. Konopinski: None. C. Gaudette: None. P. Janardhanan: None. S. Mohan: None.
- © 2014 by American Heart Association, Inc.