Cortes-González Cesar, Barrera-Chimal Jonatan, Ibarra-Sánchez María, Gilbert Mark, Gamba Gerardo, Zentella Alejandro, Flores María Elena, Bobadilla Norma A
Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City.
Cell Physiol Biochem. 2010;26(4-5):657-68. doi: 10.1159/000322333. Epub 2010 Oct 29.
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 (NO(2)/NO(3)), 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 O(2)(-) 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 NO(2)/NO(3) 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 NO(2)/NO(3) 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 Hsp90β conformation. These data show for first time that Hsp90α and Hsp90β differentially modulate NO and O(2)(-) generation by eNOS through promoting changes in eNOS conformation and phosphorylation state.
热休克蛋白90亚家族由两种胞质异构体组成，即热休克蛋白90α（Hsp90α）和热休克蛋白90β（Hsp90β）。内皮型一氧化氮合酶（eNOS）受热休克蛋白90调控，然而，每种热休克蛋白90异构体对一氧化氮生成的具体作用尚未明确。本研究旨在评估热休克蛋白90α和热休克蛋白90β过表达对eNOS/NO途径的影响。将大鼠热休克蛋白90α和热休克蛋白90β克隆到pcDNA3.1(+)中，并转染到人胚肾细胞（HEK-293）中。通过蛋白质免疫印迹分析证实热休克蛋白90α和热休克蛋白90β的转染，并测定它们对一氧化氮生成（NO₂/NO₃）、eNOS蛋白及其在Ser1177和Thr495位点的磷酸化，以及Akt/PKB Ser473磷酸化的影响。还评估了热休克蛋白90α和热休克蛋白90β与eNOS的相互作用、热休克蛋白90的二聚体/单体比例以及超氧阴离子（O₂⁻）的生成。转染后，HEK-293细胞中热休克蛋白90α和热休克蛋白90β的水平显著升高。热休克蛋白90α过表达导致NO₂/NO₃水平显著升高，这一效应与eNOS Ser 1177和Akt/PKB Ser473磷酸化增加以及热休克蛋白90α二聚化增强有关。值得注意的是，pcHsp90β转染显著降低了NO₂/NO₃水平并增加了O₂⁻的生成。这些效应与eNOS二聚体构象减少、eNOS Thr495磷酸化增加、Akt/PKB磷酸化减少以及热休克蛋白90β单体构象增加有关。这些数据首次表明，热休克蛋白90α和热休克蛋白90β通过促进eNOS构象和磷酸化状态的变化，对eNOS产生的一氧化氮和O₂⁻生成进行差异调节。
