High glucose-induced IKK-Hsp-90 interaction contributes to endothelial dysfunction.

A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hallmark of diabetes. The availability of NO to the vasculature is regulated by endothelial nitric oxide synthase (eNOS) activity and the involvement of heat shock protein-90 (Hsp-90) in the regulation of eNOS activity has been demonstrated. Hsp-90 has been shown to interact with upstream kinases [inhibitor κB kinases (IKK)α, β, and γ] in nonvascular cells. In this study, we have investigated the interaction of Hsp-90-IKKβ in endothelial cells under conditions of high glucose (HG) as a possible mechanism that diminishes Hsp-90-eNOS interaction, which could contribute to reduced bioavailability of NO. We report for the first time that IKKβ interacts with Hsp-90, and this interaction is augmented by HG in vascular endothelial cells. HG also augments transcriptional (3.5 ± 0.65-fold) and translational (1.97 ± 0.17-fold) expression as well as the catalytic activity of IKKβ (2.45 ± 0.4-fold). Both IKKβ and eNOS could be coimmunoprecipitated with Hsp-90. Inhibition of Hsp-90 with geldanamycin (2 μM) or Radicicol (20 μM) mitigated (0.45 ± 0.04-fold and 0.93 ± 0.16-fold, respectively) HG induced-IKKβ activity (2.5 ± 0.42-fold). Blocking of IKKβ expression by IKK inhibitor II (15 μM wedelolactone) or small interferring RNA (siRNA) improved Hsp-90-eNOS interaction and NO production under conditions of HG. These results illuminate a possible mechanism for the declining eNOS activity reported under conditions of HG.