Opposite Effect of Hsp90αand 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.

Endothelial Nitric Oxide Synthase Dimerization Is Regulated by Heat Shock Protein 90 Rather than by Phosphorylation

“…It is established that heat shock protein 90 (Hsp90) plays an important role in the regulation of eNOS function [38]. However, it is unknown whether Hsp90 specifically targets either eNOS dimers or monomers and how it affects the phosphorylation status of eNOS.…”

CXCL1 Inhibits Airway Smooth Muscle Cell Migration through the Decoy Receptor Duffy Antigen Receptor for Chemokines

“…6A), supporting a possible role for HSP90 in CXCL1-mediated ASMC migration inhibition. However, there are two isoforms of HSP90, HSP90a and HSP90b (47,48), which are known to differentially regulate cellular processes (49). When we examined the mRNA expression of HSP90a and HSP90b in ASMCs in response to 4 ng/ml of CXCL1, we found a significant increase in the mRNA expression of HSP90a, but not HSP90b, after 30 and 60 min of stimulation (Supplemental Fig.…”

Regulation and function of the human HSP90AA1 gene

“…For example, Hsp90α is required for peptide-loaded MHC1 complex processing in spermatocyte maturation and hERG ion channel maturation (Kunisawa and Shastri 2006; Grad et al 2010; Peterson et al 2012). Hsp90α and Hsp90β also have opposing effects on the biogenesis of KCNQ4 channels and on endothelial nitric oxide (eNOS) activity (Cortes-Conzalez et al 2010). Hsp90 contains three conserved domains: the amino-terminal ATP-binding domain, the middle domain, and carboxy-terminal dimerization domain.…”