OBJECTIVE: Excessive reactive oxygen species contribute to vascular dysfunction. 
We have previously shown that heat shock protein (Hsp90) inhibitors potently 
suppress Nox 1 to 3 and 5, and the goals of this study were to identify how 
molecular chaperones regulate Nox function.
METHODS AND RESULTS: In vitro, protein expression of Nox 1 to 2, 5 was decreased 
by Hsp90 inhibitors in multiple cell types (human pulmonary artery endothelial 
cells, neutrophils, macrophages, and human saphenous vein). In mice treated with 
Hsp90 inhibitors, Nox1 expression was reduced in lung along with reduced 
reactive oxygen species from leukocytes. Elevated reactive oxygen species 
production in obese (db/db) aorta was suppressed by Hsp90 inhibition. Hsp90 
inhibitors did not alter Nox5 micro RNA levels, and proteasome inhibition 
prevented Nox2 and 5 protein degradation and increased ubiquitin incorporation. 
Inhibition of Hsp90 upregulated the expression of Hsp70 and Hsp70-bound Nox2, 5 
and promoted degradation. Silencing Hsp70 prevented Hsp90 inhibitor-mediated 
degradation of Nox5. The Hsp70-regulated ubiquitin ligase, carboxyl terminus of 
Hsp70-interacting protein (CHIP), also bound Nox5 and promoted increased Nox5 
ubiquitination and degradation. The chaperone binding and ubiquitination domains 
of CHIP were required, and the silencing of CHIP blunted Hsp90 
inhibitor-mediated degradation of Nox2 and 5.
CONCLUSIONS: We conclude that Hsp90 binds to and regulates Nox protein 
stability. These actions are opposed by Hsp70 and CHIP, which promote the 
ubiquitination and degradation of Nox proteins and reduce reactive oxygen 
species production.