Opposite Effect of Hsp90 alpha and Hsp90 beta on eNOS Ability to Produce Nitric Oxide or Superoxide Anion in Human Embryonic Kidney Cells

Heat shock protein 90 subfamily is composed by two cytosolic isoforms known as Hsp90 alpha and Hsp90 beta. 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 Hsp90 alpha and Hsp90 beta over-expression on eNOS/NO pathway. Rat Hsp90 alpha and Hsp90 beta were cloned into pcDNA3.1(+) and transfected in human embryonic kidney cells (HEK-293). Hsp90 alpha and Hsp90 beta 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 Hsp90 alpha and Hsp90 beta with eNOS and the dimer/monomer ratio of Hsp90, as well as O-2(-) generation were also assessed. After transfection, Hsp90 alpha and Hsp90 beta levels were significantly increased in HEK-293 cells. The Hsp90 alpha 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 Hsp90 alpha dimerization. Noteworthy, pcHsp90 beta transfection reduced significantly NO2/NO3 and increased O-2(-) 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 Hsp90 beta conformation. These data show for first time that Hsp90 alpha and Hsp90 beta differentially modulate NO and O-2(-) generation by eNOS through promoting changes in eNOS conformation and phosphorylation state. Copyright (C) 2010 S. Karger AG, Basel