Opposite Effect of Hsp90α and Hsp90β on eNOS Ability to Produce Nitric Oxide or Superoxide Anion in Human Embryonic Kidney Cells

Abstract

Heat shock protein 90 subfamily is composed by two cytosolic isoforms known as Hsp90α and Hsp90β. Endothelial nitric oxide synthase (eNOS) is regulated by Hsp90, however the specific role of each Hsp90 isoform on NO production has not been established. This study was designed to evaluate the effect of Hsp90α and Hsp90β over-expression on eNOS/NO pathway. Rat Hsp90α and Hsp90β were cloned into pcDNA3.1(+) and transfected in human embryonic kidney cells (HEK-293). Hsp90α and Hsp90β transfection was corroborated by Western blot analysis and their effect on NO production (NO₂/NO₃), eNOS protein and its phosphorylation at Ser1177 and Thr495, as well as Akt/PKB Ser473 phosphorylation was determined. The interaction of Hsp90α and Hsp90β with eNOS and the dimer/monomer ratio of Hsp90, as well as O₂^- generation were also assessed. After transfection, Hsp90α and Hsp90β levels were significantly increased in HEK-293 cells. The Hsp90α over-expression induced a significant increase in NO₂/NO₃ levels, an effect that was associated with increased phosphorylation of eNOS Ser 1177 and Akt/PKB Ser473, as well as with a greater Hsp90α dimerization. Noteworthy, pcHsp90β transfection reduced significantly NO₂/NO₃ and increased O₂^- generation. These effects were associated with a reduction of eNOS dimeric conformation, increased eNOS Thr495 phosphorylation, reduced Akt/PKB phosphorylation, and by a greater amount of monomeric Hsp90β conformation. These data show for first time that Hsp90α and Hsp90β differentially modulate NO and O₂^- generation by eNOS through promoting changes in eNOS conformation and phosphorylation state.