Abstract 857: Heat Shock Protein 90 is Critical for in Cytokine-stimulated Inducible Nitric Oxide Synthase Expression in Vascular Smooth Muscle Cells
Background: Nitric oxide (NO) production by inducible NO synthase (iNOS) plays an important role in the regulation of blood flow and vascular homeostasis. Heat shock protein 90 (Hsp90) is a chaperone protein that regulates stability and function of a number of signal proteins. Recent studies showed that Hsp90 binds to endothelial NOS and regulates its activity; however, the role of Hsp90 in iNOS expression in vascular smooth muscle cells (VSMCs) remains undefined. In the present study, we investigated the role of Hsp90 in cytokine-stimulated iNOS expression and NO production in rat VSMCs.
Methods and Results: Inhibition of Hsp90 by Hsp90-siRNA transfection or 2 types of specific Hsp90 inhibitors (geldanamycin: 0.1–5 μg/mL and radicicol: 0.1–5 μM) almost completely abrogated interleukin-1β (IL-1β)-stimulated iNOS expression (mRNA and protein levels) and NO production in VSMCs. Inhibition of iNOS expression and NO production was observed in VSMCs treated with NF-kB inhibitor CAPE (5–50 μM) and in p65-deficient fibroblasts, indicating NF-kB as an upstream molecule for IL-1β-induced iNOS expression. Luciferase reporter assay showed that IL-1β markedly stimulated NF-kB activation, and this activation was completely prevented by Hsp90 inhibition. Further, IkB degradation was also inhibited by Hsp90 inhibition. Importantly, abrogation of NO production by Hsp90 inhibition was still observed in the cells treated with IL-1β for 24 h; this suggests the modulation of iNOS protein stability by Hsp90 inhibition. Coimmunoprecipitation experiments revealed that Hsp90 directly interacted with iNOS protein induced by IL-1β Hsp90 inhibition clearly accelerated iNOS degradation and stimulated iNOS ubiquitination. Proteasome inhibition by MG-132 treatment (25 μM) significantly prevented the accelerated iNOS degradation by Hsp90 inhibition.
Conclusions: We have clarified a novel mechanism for the regulation of iNOS expression in VSMCs, and demonstrated that Hsp90 regulates iNOS expression by 2 mechanisms; iNOS mRNA expression by regulating the NF-kB activation and iNOS degradation by modulating the ubiquitin/proteasome pathway. The modulation of Hsp90 function might prove to be an efficient approach to control the accumulation of NO in the vascular wall.